Name: Organic Molecule Composition of Milk: Lab Investigation Introduction & Background Milk & milk products have been a major food source from earliest recorded history. Milk is a natural, nutritionally complete food source. It contains all of the essential classes of biological molecules proteins, carbohydrates, and fats that are important in nutrition. The principal components of whole milk are proteins (3.5%), carbohydrates (5%) and fat (4%). In addition, milk is also an important source of a variety of essential minerals and vitamins. In this lab activity, the biochemical nature of proteins and carbohydrates is explored by analyzing the nutritional components of skim milk. Students will separate the protein and carbohydrate components of skim milk and verify their identity. Proteins Proteins are essential constituents of all living cells, and are vital for proper cell structure and function. Although individual protein structure and function varies, all proteins share a common basic composition they are polymers composited of amino acids joined by peptide linkages into long-chain molecules. The presence of proteins in solution is determined via the Biuret test. In the Biuret test, copper (II) sulfate (CuSO 4 ), a blue liquid, is added to the solution being tested. The copper ions bind to the nitrogen atoms and carbonyl groups in the amino acids forming the protein. This forms a purple coordination complex the purple color indicates a positive test result, and the presence of protein in the solution. There are three main proteins in milk: casein, lactalbumin, and lactoglobulins. Casein is the chief milk protein, forming about 80% of total protein content. It is a phosphoprotein, meaning that it contains many negatively-charged phosphate (PO 4 3- ) groups. Positively-charged calcium ions (Ca 2+ ) bind to the phosphate groups in the casein protein, giving milk its high calcium content. Casein is almost completely insoluble in water, meaning that it can easily be precipitated out of solution with the addition of acid at 40 C. On the other hand, both lactalbumin and lactoglobulin are water-soluble proteins. Lactalbumin is involved in the synthesis of lactose. Lactoglobulins are responsible for the immunological properties of milk, giving milk its protective role. Both lactalbumin and lactoglobulin can be precipitated out of solution by heating milk to higher temperatures (80 to 90 C). Carbohydrates Lactose is the principal carbohydrate component in milk. It is a disaccharide and a reducing sugar, and is composed of the two monosaccharides glucose and galactose. Lactose can be separated from milk by heating the protein filtrate (the solution that is left over once the protein is precipitated out) at 90 C until the liquid has evaporated. Presence of lactose can be determined by Benedict s test. A positive result for Benedict s test occurs when the blue color of Benedict s solution fades and a yellow, orange, or red precipitate is formed. Fats Fats are water-insoluble substances found in plant and animal cells. They are members of a class of nonpolar molecules called lipids, and are essential to the structure and function of cell membranes. Whole milk is an emulsion, meaning that molecules of fat are suspended in solution in tiny globules. Skim milk is made by separating out the fat content via centrifuging. The milk is spun rapidly and the lipids, which are less dense that the water component, float to the top where they can be skimmed off.
Pre-Lab Questions Use the background information and your Chromebooks to answer the following questions. 1. List the three principal nutritional components of milk, and list their average percentage compositions in whole milk. Principal Nutritional Component Average Composition (%) 2. The three principal nutritional components only make up 12.5% of whole milk on average. What do you think the majority of the remainder of milk is composed of? 3. Name the principal proteins present in milk, and give their functions. 4. Consult the nutritional labels for whole milk, low-fat (2%) milk, and non-fat skim milk. Compare their protein, carbohydrate, and fat contents. Whole Milk 2% Milk Skim Milk Protein Carbohydrate Fat 5. What is the difference between whole milk, 2% milk, and skim milk? 6. Vitamins A & D are fat-soluble vitamins. What does this mean? Why do these vitamins have to be added to skim milk? 7. Name & describe the qualitative color test that will be used to identify the proteins in milk. 8. Name & describe the qualitative color test that will be used to identify the carbohydrates in milk. 9. What conditions will be used to precipitate casein? 10. What conditions will be used to precipitate lactalbumin and lactoglobulin? 11. What conditions will be used to precipitate lactose?
Safety Precautions Acetic acid and sodium hydroxide solutions are corrosive liquids. Ethyl alcohol is a flammable organic solvent and a dangerous fire risk. Benedict s solution contains copper tartrate and is an alkaline solution. Biuret solution is a highly alkaline solution and is corrosive to eyes and body tissue. Avoid exposure of all chemicals to eyes and skin. Wear chemical splash goggles, chemical-resistant gloves, and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. You will be using heat plates and handling heated glassware. Handle all glassware with caution, and use protective gloves or hot hands when handling all hot glassware. Procedure Part A: Isolation of Casein 1. Use a graduated cylinder to measure out 20 ml of skim milk. Record the volume of skim milk (to the nearest 0.1 ml) in Data Table 1. Transfer the milk to an Erlenmeyer flask. 2. Heat the milk to 40 C on the lowest setting of the hot plate. 3. In a gradual, drop-wise manner, add 2 ml of acetic acid to the milk, stirring constantly. 4. Continue heating until a while precipitate appears and the liquid portion is pale yellow (~5 minutes). 5. Moisten your filter paper with tap water and fold fit it to the funnel. Place the filter-lined funnel in a clean Erlenmeyer flask. 6. Slowly filter the milk/acetic acid solution through the filter-lined funnel. 7. Remove the Erlenmeyer flask full of filtrate & save it for Part B. 8. Place a clean test tube under the filter funnel, and rinse the filtrate with 1-2 ml of ethyl alcohol. Continue the gravity filtration until all of the liquid has been removed. 9. Weigh the empty Petri dish labeled casein and record the mass in Data Table 1. 10. Gently scrape the precipitate into the Petri dish. 11. Let the precipitate air dry overnight. 12. Weigh the Petri dish containing the precipitate, and record the mass in Data Table 1. Part B: Isolation of Lactalbumin & Lactoglobulin 1. Place the Erlenmeyer flask containing the clear filtrate from Part A (Step 7) on the hot plate. Heat it to 80-90 C for 10 minutes. The filtrate will gradually turn cloudy, and a granular white solid will precipitate out of solution. 2. Weigh a dry piece of filter paper and record the mass in Data Table 1. 3. Prepare a gravity filtration setup (see Part A Step 5), using a clean Erlenmeyer flask as receiving vessel. 4. Use hot hands to pour the heated solution into the gravity filtration apparatus. 5. Once the filtration is complete, save the filtrate for Parts C & D. 6. Place precipitate & filter paper on a clean Petri dish and set it aside to air dry overnight. 7. Weigh the dried filter paper & precipitate, and record the information in Data Table 1. Part C: Isolation of Lactose 1. Pour the filtrate from Part B (Step 5) into a clean graduated cylinder. Use a pipette to add distilled water to dilute the filtrate solution to the original milk volume. 2. Weigh the empty watch glass labeled lactose and record the mass in Data Table 1. 3. Remove 1 ml of the diluted filtrate solution and add it to the watch glass. Save the remaining filtrate for Part D. 4. Place the watch glass on a heat plate and heat it until medium until the liquid evaporates. 5. Once the watch glass has cooled, measure its mass and record it in Data Table 1.
Part D: Quantitative Tests for Protein and Carbohydrate 1. Prepare casein solution by dissolving <0.1 g of dry casein (from Part A) into 5 ml of sodium hydroxide solution in a test tube. 2. Prepare lactalbumin/lactoglobulin solution by dissolving <0.1 g of dry lactalbumin/lactoglobulin (from Part B) into 5 ml of distilled water in a test tube. 3. Obtain six clean test tubes and label them 1-6. Fill the test tubes with 1 ml of solution according to Data Table 2 Biuret Test column. 4. Add 2 ml of Biuret solution to each test tube. 5. After 3 minutes, record the color of each test sample in Data Table 2. Note if the reaction for presence of protein is positive or negative. 6. Obtain six more clean test tubes and label them 1-6. Fill the test tubes with 1 ml of solution according to the Data Table 2 Benedict s Test column. 7. Ad 2 ml of Benedict s solution to each test tube and place them in a hot water bath. 8. After 3-5 minutes, record the color and appearance of each sample in Data Table 2. Note if the reaction for presence of a reducing sugar is positive or negative. 9. Dispose of all solutions as instructed, and rinse all glassware. Data & Observations Data Table 1. Protein & Carbohydrate Content of Skim Milk Volume of skim milk used (ml) Mass of skim milk used (assume density=1.0 g/ml) Mass of empty precipitate dish part A (g) Mass of part A dish + casein (g) Mass of dry casein (g) Mass of filter paper part B (g) Mass of filter paper + lactalbumin & lactoglobulin (g) Mass of lactalbumin & lactoglobulin (g) Combined mass of total proteins from milk (g) Mass of empty precipitate dish part C (g) Mass of part C dish + lactose (g) Mass of lactose from 1 ml milk (g)
Data Table 2. Qualitative Tests for Protein & Carbohydrate Test Biuret Test Benedict s Test Tube Solution Color Result (+/-) Solution Color Result (+/-) 1 Distilled water (control) Distilled water (control) 2 Skim milk Skim milk 3 Casein solution Casein solution 4 Lactalbumin solution Lactalbumin solution 5 Filtrate (lactose solution) Filtrate (lactose solution) 6 Protein reference Carbohydrate reference Post-Lab Calculations & Analysis Be sure to show all work and units for calculations. 1. Based on the combined mass of protein isolated from 20 ml of skim milk, calculate the percent composition of total protein in skim milk. What percent of total protein is due to the casein fraction? (Assume the density of milk is equal to 1 g/ml) 2. Using your experimental value for the percent protein in milk, calculate the mass of protein (in grams) that would be present in 1 serving size (1 cup, or 240 ml) of skim milk. Compare this result with the information provided on the nutritional label for the protein content in 1 serving of skim milk. 3. Based on the amount of lactose isolated from 1 ml of skim milk filtrate, calculate the percent composition of lactose in skim milk. 4. Using your experimental value for the percent lactose in milk, calculate the mass of lactose (in grams) that would be present in 1 service size (1 cup, or 240 ml) of skim milk. Compare this result with the information provided on the nutritional label for the carbohydrate content in 1 serving of skim milk. 5. Based on your experimental data, do you think the experimental procedures for the separation of the protein and carbohydrate fractions of skim milk were effective? Use your data to justify your answer. If you do not think procedures were effective, identify the sources of error that may have resulted in the differences. Use the back of this sheet if you do not have enough room to answer. Adapted from the Flinn Scientific, Inc. Milk is a Natural laboratory kit (Publication No. 7582).