BIOL 109
Laboratory Three
Milk, Metabolism, and Lactose Intolerance
Introduction
In the hierarchy of human energy metabolism, carbohydrates are the first energy
source used to meet our metabolic needs. These carbohydrates enter glycolysis, the first
step in the energy metabolism pathways as monosaccharides. Thus, whether we ingest
carbohydrates as starches, table sugar, or milk sugar these energy sources need to be
broken down into monosaccharides to enter glycolysis.
A one cup serving of whole milk (244 grams) contains 8 grams of fat, 8 grams of protein,
and 13 grams of sugar. The primary sugar found in milk and dairy products is Lactose.
Lactose is a disaccharide formed from galactose and glucose. This can be compared to
common table sugar which is sucrose, a disaccharide comprised of glucose and fructose
(Figure 1).
Figure 1. The Structures of Sucrose and Lactose. Sucrose is common table sugar. It is a
dissacharide composed of the monosaccharides glucose and fructose. Lactose is milk sugar.
It is a dissacharide composed of the monosaccharides galactose and gluose.
Lactose Intolerance is the inability or insufficient ability to digest lactose. Lactose
intolerance is caused by reductions in the production of the enzyme lactase by the cells of
the small intestine. Lactase deficiency develops as a person ages. Lactase production is at
its highest in infants at birth regardless of ethnicity. This allows infants to digest breast
milk. Primary lactase deficiency occurs when a person’s body decreases the formation of
lactase. This typically begins between 2 and 5 years of age, but most individuals will not
experience symptoms until early adulthood. Secondary lactase deficiency occurs following
injury to the small intestine which can occur at any age. It is estimated that as many as
50 million American adults are lactose intolerant (1). Primary lactose intolerance appears
to have a genetic component with specific populations showing high levels of intolerance.
A. Lactose
B. Sucrase
Figure 2. Lactase and Sucrase Activity. To supply energy for metabolism, disaccharides must
be broken down into their respective monosachharides. These reactions are catalyzed by
specific enzymes. A) Lactase is the enzyme that catalyzes the breakdown of lactose. B)
Sucrase is the enzyme that breaks down sucrose. All enzymes have an active site where the
sugar substrate binds to the enzyme. Lactase cannot break down sucrose and sucrase cannot
break down lactose.
Table 1. Primary Lactose Intolerance and Ethnicity
Ethnicity
American Indians
Asians
African Americans
Ashkenazi Jews
Hispanics
Northern European
Origin
% Adults with Primary Lactose
Intolerance
80 to 100 %
90 - 95%
60 – 80 %
60 – 80%
50 – 80%
1 - 2%
Many people with ancestry in Europe, West Asia, India, and parts of East Africa maintain
lactase production into adulthood. In many of these areas, milk from mammals such as
cattle, goats, and sheep is used as a large source of food. Hence, it was in these regions
that genes for lifelong lactase production first evolved. The genes of lactose tolerance
have evolved independently in various ethnic groups. By descent, more than 70% of
western Europeans can drink milk as adults, compared with less than 30% of people from
areas of Africa, eastern and south-eastern Asia and Oceania.
What are the symptoms?
Symptoms of lactose intolerance can be mild to severe, depending on how much
lactase your body makes. Symptoms usually begin 30 minutes to 2 hours after you eat or
drink milk products. If you have lactose intolerance, your symptoms may include:
•
•
•
•
•
•
Bloating.
Pain or cramps.
Gurgling or rumbling sounds in your belly.
Gas.
Loose stools or diarrhea.
Throwing up.
Many people who have gas, belly pain, bloating, and diarrhea suspect they may be
lactose-intolerant. The best way to check this is to avoid eating all milk and dairy products
to see if your symptoms go away. If they do, then you can try adding small amounts of milk
products to see if your symptoms come back.
If you feel sick after drinking a glass of milk one time, you probably do not have
lactose intolerance. But if you feel sick every time you have milk, ice cream, or another
dairy product, you may have lactose intolerance. Sometimes people who have never had
problems with milk or dairy products suddenly have lactose intolerance. This is more
common as you get older.
How is it treated?
There is no cure for lactose intolerance, but you can treat your symptoms by either
using dietary supplemental digestive enzymes that help digest lactose, or by limiting or
avoiding milk products. Some people use milk with reduced lactose, or they substitute soy
milk and soy cheese for milk and milk products. Some people who are lactose-intolerant can
eat yogurt without problems, especially yogurt with live cultures.. In time, most people
with lactose intolerance get to know their bodies well enough to avoid symptoms.
One of the biggest concerns for people who are lactose-intolerant is making sure
they get enough of the nutrients found in milk products, especially calcium. Calcium is most
important for children, teens, pregnant women, and women after menopause. There are
many nondairy foods that contain calcium, including:
•
•
•
•
•
Broccoli, okra, kale, collards, and turnip greens.
Canned sardines, tuna, and salmon.
Calcium-fortified juices and cereals.
Calcium-fortified soy products such as soy milk, tofu, and soybeans.
Almonds.
For people who are lactose intolerant and want to enjoy dairy foods, there are a
variety of products on the market that allow them to do so. These products include
lactose-free milk and the lactase enzyme in tablet form. Lactose-free milk products have
the lactose pre-digested into its two monosaccharides glucose and galactose. Lactase
tablets are taken immediately prior to drinking milk or eating dairy products giving the
person the enzyme it lacks to breakdown the lactose.
Experimental Plan:
This experiment, derived from the work of Reinking et al. (1994) and Preszler
(2000), uses an enzyme found in dietary supplements to evaluate the lock and key model of
enzyme specificity. This dietary supplement, Lactaid® contains β-galactosidase which
breaks the β−linkage in lactose to produce glucose and galactose
One way to gain a better understanding of the role of lactase in the energy
metabolism of milk is to do a simple fermentation experiment using common baker’s yeast
(Saccharomyces cerevisiae). Fermentation is the anaerobic breakdown of sugars to obtain
energy, but not all sugars support fermentation. Baker’s yeast does not produce lactase
and thus cannot use lactose for fermentation, functionally making Baker’s yeast lactose
intolerant. In this simple experiment you can use different milk products or milk products
treated in different ways as an energy source for fermentation.
Alcohol fermentation is the breakdown of sugar in the absence of oxygen to
produce energy. The absence of oxygen functionally turns off the energy metabolism in
the mitochondria of cells, leaving on the process of glycolysis in the cytoplasm to convert
energy in sugar to energy in the form of ATP. The other products of alcohol fermentation
are ethanol and carbon dioxide.
In this experiment, we will use Baker’s yeast as a model organism and investigate
their ability carry out alcohol fermentation to investigate the metabolism of milk sugar
and lactose intolerance.
Yeast Cytoplasm
Glucose
or
Fructose
Carbon Dioxide
Glycolysis
Pyruvic Acid
Figure 3. Chemical Reaction Equation for Alcohol Fermentation.
breakdown of glucose or fructose in the absence of oxygen.
Ethanol
Alcohol fermentation is the
Materials
1.
2.
3.
4.
5.
6.
7.
8.
9.
Active Dry Yeast or Rapid Rise Yeast (2 21 g packages per group of students)
White Milk
Chocolate Milk
Lactaid Milk
Lactaid Tablets
Mortar and Pestle (1 per group)
Serological Pipettes or graduate cylinders
50 mL Erlenmeyer Flasks (5 per group)
250 mL beakers (3 per group)
10.
11.
12.
13.
14.
15.
16.
100 mL beakers (3 per group)
100 mL graduated cylinder (1 per group)
10 mL serological pipettes (3 per group)
Water Bath (1 per group 37°C)
Timer
18 inch piece of string
Ruler or Meter Stick
Protocol
1. Rehydrate 2 packets of active dry yeast (21 g each) yeast in 100 mL tap water.
(Incubate for a minimum of 15 minutes)
2. Crush 1 Lactaid tablet and add to 50 mL White Milk. (Incubate 15 minutes)
3. Crush 1 Lactaid tablet and add to 50 mL Chocolate Milk (Incubate 15 minutes)
4. Label Erlenmeyer Flasks 1 to 5
5. Add
10
mL
yeast
solution
to
each
of
the
5
Flasks.
Make sure that the yeast is mixed and fully suspended prior to pipetting it from
the stock beaker.
6. Add 10 mL white milk to Flask 1
7. Add 10 mL white milk treated with the Lactaid tablet to Flask 2
8. Add 10 mL chocolate milk to Flask 3
9. Add 10 mL chocolate milk treated with the Lactaid tablet to Flask 4
10. Add 10 mL Lactaid Mile to Flask 5
11. Mix each of the flasks thoroughly.
12. Attach a 9 inch balloon to the top of each Flask
13. Place all 5 flasks into the 37° C water bath
14. Make sure there is enough room between the flasks and balloons so that the
balloons can stand once carbon dioxide production begins.
15. Use a piece of string to measure changes in balloon circumference at each time
interval for each experimental condition.
16. For ease of data collection and analysis consider the balloon circumference zero
until the balloon stands on its own.
17. Record the circumference of the balloons in millimeters every 15 minutes for one
hour.
18. Share the data with the class so that you can work with both your group’s individual
data and the combined data from all laboratory groups.
Table 2. Data Table for Milk Metabolism Experiment. Determine the circumference of the
balloon every 15 minutes during the experiment. Record the data in the table below. Enter 0
for an empty balloon.
Energy Source / Time
0
White Milk
0
White Milk Treated
with Lactaid
0
Chocolate Milk
0
Chocolate Milk
Treated with Lactaid
0
Lactaid Milk
0
15
30
45
Data Analysis
1. Create Two Data Tables
a. Your Groups Data
b. Class Data using Average Balloon Circumference and Sugar Source
2. Create Four Graphs
a. Two using your own data
i. Line Graph
Best data from your group
mm Balloon Circumference vs Time
ii. Bar Graph
All data from your group
Average Balloon Circumference at 60 minutes vs Sugar Source
b. Two using the classes data
i. Line Graph
Chocolate milk treated with lactaid tablet
60
Average Balloon Circumference vs time
ii. Bar Graph
All of the classes data
Average Balloon Circumference at 60 minutes vs Sugar Source
3. Create a Table providing the amount of sugar in the different milk products
Discussion Questions
1. Describe the amount of sugar and types of sugars present in the different milk
products
2. Based upon the amount of sugars in the different milk products predict the outcome of
the experiment.
3. Which experimental energy source produced carbon dioxide first?
4. Which experimental energy source produced the most carbon dioxide in 60 minutes?
5. Which experimental energy source produced the least carbon dioxide in 60 minutes?
6. Were your results what you predicted they would be? Explain why or why not.
7. Were the class results what you expected they would be? Explain why or why not.
8. Were your results similar to the class results? If not what do you think happened that
made them different?
9. What does this experiment demonstrate in terms of enzyme production and enzyme
specificity?
References
1. Lactose Intolerance: Information for Health Care Providers. U.S. Department of
Health and Human Services. NIH Publication Number 05-5305B. 2006
2. Preszler, R., W. (2000). The use of writing in investigative biology laboratories. In
tested studies for laboratory teaching (ed, Karcher, S. J.), 21, 492-496
3. Reinking, L.N., J.L. Reinking, and K.G. Miller. (1994). Fermentation, respiration and
enzyme specificity: a simple device and key experiments with yeast. American Biology
Teacher, 56: 164-168.