BIOL 109L Laboratory Four Fall 2018 Milk, Metabolism, and Lactose Intolerance

BIOL 109L Laboratory Four Fall 2018 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 disaccharide composed of the monosaccharides glucose and fructose. Lactose is milk sugar. It is a disaccharide composed of the monosaccharides galactose and glucose. 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 1

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. 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, 2

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. Table 1. Primary Lactose Intolerance and Ethnicity Ethnicity % Adults with Primary Lactose Intolerance American Indians 80 to 100 % Asians 90-95% African Americans 60 80 % Ashkenazi Jews 60 80% Hispanics 50 80% Northern European 1-2% Origin 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. 3

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 lactosefree 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 -galactosidase which breaks the 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. 4

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 Carbon Dioxide or Glycolysis Pyruvic Acid Ethanol Fructose Figure 3. Chemical Reaction Equation for Alcohol Fermentation. Alcohol fermentation is the breakdown of glucose or fructose in the absence of oxygen. Materials 1. Active Dry Yeast or Rapid Rise Yeast (2 21 g packages per group of students) 2. White Milk 3. Chocolate Milk 4. Lactaid Milk 5. Lactaid Tablets 6. Mortar and Pestle (1 per group) 7. Serological Pipettes or graduate cylinders 8. 50 ml Erlenmeyer Flasks (5 per group) 9. 250 ml beakers (3 per group) 10. 100 ml beakers (3 per group) 11. 100 ml graduated cylinder (1 per group) 12. 10 ml serological pipettes (3 per group) 13. Water Bath (1 per group 37 C) 5

14. Timer 15. 18 inch piece of string 16. 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. 6

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 15 30 45 60 White Milk 0 White Milk Treated with Lactaid 0 Chocolate Milk 0 Chocolate Milk Treated with Lactaid 0 Lactaid Milk 0 For the follow week - Data Analysis: This is an individual write up assignment. A title page must have your full name and a title for your work. Graphs are to be made in Excel and pasted into word. Label all graphs and give each one a descriptive title. Answer all nine questions in full, providing appreciate references. 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 7

Average Balloon Circumference at 60 minutes vs Sugar Source b. Two using the classes data i. Line Graph Chocolate milk treated with lactaid tablet 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 Lactose Intolerance: Information for Health Care Providers. U.S. Department of Health and Human Services. NIH Publication Number 05-5305B. 2006 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 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. 8