Austin Peay State University Department of Chemistry CHEM 1021 ISOLATION OF CASEIN FROM MILK Purpose: In this lab you will be isolating the proteins casein and lactalbumin from a sample of milk. You will use these values to determine the percent protein in milk compared to the listed value on the box. Caution: Wear eye protection because of the possible shattering of dropped glassware and because 10% acetic acid is used in this experiment. If any of this acid comes in contact with your skin, wash it off immediately with plenty of water. Also, avoid breathing vapors of this solution and of course, do not drink any of the solutions in this experiment. Introduction: Milk is a complex biological mixture of chemicals that serves as the primary food source for infant mammals. Milk contains most of the biological molecules necessary to sustain life including water, a variety of vitamins, minerals, proteins, sugars, and lipids (DNA, although necessary for life, is made by your body and therefore is not needed on a dietary level). Although most mammals stop drinking milk upon maturity, many (but not all) human cultures continue drinking milk and consuming milk products (for example, cheese, butter, and cream) throughout their entire life. It would be quite challenging to isolate every component of milk because some of the chemicals are present in very small quantities. You can very easily, however, isolate the most abundant components from each other. A common example of this Table 1. Milk components (% composition) is removing the fat from cow’s milk to make skim milk or Cow Human Goat removing the sugar from milk to make lactose‐free milk. Water 87.8 87.4 87.0 Table 1 shows the average composition of milk from Protein 3.0 1.4 3.3 Lipids 3.9 4.0 4.2 several mammals consumed by humans. A small Sugars 4.6 7.0 4.8 discussion of the components of milk is warranted and Minerals 0.7 0.2 0.7 described below. Lipids: If you were asked if fat mixes with water, chances are you would say “no”. However, milk contains around 4% fat in water. This means that in 100 mL of water, about 4 mL of fats would be present. This is possible because milk contains, in addition to fats, a number of lipids called phospholipids. These molecules are similar to the fatty acids you studied in class, but have a negatively‐ charged phosphate group attached to one end (Figure 1). Figure 1. Example phospholipid O O O P O hydrophobic ( water hating) area O hydrophilic ( water loving) area The negatively charged phosphate group, like other charged, polar groups, is quite soluble in water. The non‐polar, uncharged hydrocarbon area is completely insoluble in water. The dual Revision SP11 Page 1 of 7 Austin Peay State University Department of Chemistry CHEM 1021 ISOLATION OF CASEIN FROM MILK personality of the phospholipid (chemists would call it amphipathic) lets the milk fat be soluble in the milk water by aligning a number of its hydrophobic areas with the other fats, with the phosphates facing out toward the water (Figure 2). This makes a mixture called an emulsion, meaning a fat dissolved in water by means of an amphipathic molecule, called an emulsifying agent (soaps are emulsifying agents that work to dissolve grease). In this lab, you will be using powdered milk that has already had the fat removed. Proteins: There are three main proteins in milk; casein (case‐in), lactalbumin (lact‐al‐bumin), and lactoglobulin (lact‐o‐glob‐ulin). In this lab you will be isolating the casein, and a mixture of the lactalbumin and lactogobulin proteins (they isolate under the same conditions and are difficult to separate from each other). Chances are you have consumed casein without knowing it. Casein can precipitate from water when its normal structure is destroyed. We call this process protein denaturation. The curds in cottage cheese are precipitated casein protein (the enzyme rennin is used in this case to precipitate the protein) and the Indian cheese paneer is made by adding an acid to milk to precipitate the curds. Many cheeses are made by precipitating casein and removing it from the left over liquid, the whey. The lactalbumin proteins are easily precipitated by heating. After casein proteins are removed, heating the whey solution can provide the solid protein. Although a minor component, the lactoglobulin proteins are the immune proteins present in milk that protect a baby from illness until it can develop its own immune system. Sugars: The main carbohydrate present in milk is the sugar lactose. Lactose is a disaccharide containing the monosaccharides galactose and glucose. Mammals produce an enzyme called lactase that breaks the disaccharide into its monomers during digestion. Many mammals stop producing lactase after maturity leading them to be lactose intolerant—they lack the ability to digest the sugar in milk. Although most people can digest lactose upon maturity, there are many who can’t. These people can buy lactose‐free products, or buy the enzyme supplement lactase to aid in digestion of the sugar. On a practical note, when bacteria get into milk, they digest the lactose and form the acid lactic acid. This causes a precipitation of the protein casein. This is what happens when old milk “sours”. Revision SP11 Page 2 of 7 Austin Peay State University Department of Chemistry CHEM 1021 ISOLATION OF CASEIN FROM MILK Equipment: 10% acetic acid solution 10‐mL Graduated cylinder Powdered milk spatula disposable pipettes nylon hose filter flask filter paper Buchner funnel Procedure: Casein isolation 1. Place about 4 g of powdered nonfat milk and 42 mL of water into a beaker and record the mass. Heat the milk to 40 oC. 2. When the milk has reached 40 oC, start adding the acetic acid solution 5 drops at a time. After every 5 drops, stir the solution with a spatula. You will see a white solid forming. This is the casein. Push the casein to the side of the beaker above the solution and push the liquid out and to compress the protein. Transfer to a watch glass as you go. Stop after 20 drops of the acetic acid (1 mL). If you see any liquid on the watch glass, pipette it back into the beaker. 3. Add 0.2 g of calcium carbonate to the milk solution in the beaker to neutralize the pH. 4. Transfer the casein to a section of nylon hose. Wrap the protein and gently squeeze over the top of the beaker to remove as much of the milk solution as possible. 5. Weigh a plastic boat and record. Add the casein and record the mass on the data sheet. Lactalbumin isolation 6. Heat the mixture to 75 oC for 5 minutes. This precipitates the lactalbumin protein (and any of the lactoglobulin protein in the milk). 7. Use a filter funnel, flask, and filter paper to separate the solid precipitate from the hot solution. 8. Transfer the lactalbumin to a weigh boat and record mass on data sheet. Waste Disposal and Cleanup: Properly dispose of all solutions and solids according to your lab instructor. Thoroughly wash and rinse all glassware and return to their original places. Revision SP11 Page 3 of 7 Austin Peay State University Department of Chemistry CHEM 1021 ISOLATION OF CASEIN FROM MILK THIS PAGE INTENTIONALLY LEFT BLANK Revision SP11 Page 4 of 7 Austin Peay State University Department of Chemistry CHEM 1021 ISOLATION OF CASEIN FROM MILK DATA SHEET Name: ______________________________________ Partner: _____________________________________ 1. Exact mass of dried milk _________________g 2. Mass of isolated casein _________________g 3. Mass of lactalbumin _________________g Using the box of milk to find numbers we can work with to check quality control: 4. Mass of protein listed per serving on milk box _________________g/serving 5. Volume of reconstituted milk per serving (in mL) _________________mL/serving 6. Convert the volume of milk (in #5) to the mass of reconstituted milk. Assume the density of milk is ~1 g/mL. 7. Percent protein in a reconstituted milk sample _________________g # # 100 _________________% Compare your data to the listed nutritional information you determined above: 8. Total mass of protein in your sample (#2 + #3) _________________g 9. Find the percent of total protein in your sample _________________% 100 10. Find the percent of casein in your sample _________________% 11. Find the percent of lactalbumin in your sample _________________% Revision SP11 Page 5 of 7 Austin Peay State University Department of Chemistry CHEM 1021 ISOLATION OF CASEIN FROM MILK POST LAB QUESTIONS Name:__________________________________ Partner: ________________________________ 1. Percent error: Determine the error in this experiment by using the following equation: 100% _________________% 2. Assuming your experiment had some level of error (in other words, you have a non‐zero answer for #1), what do you think could be a reason for your error? ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ 3. What does the acetic acid do the structure of casein that causes it to precipitate? ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ Revision SP11 Page 6 of 7 Austin Peay State University Department of Chemistry CHEM 1021 ISOLATION OF CASEIN FROM MILK PRE‐LAB ASSIGNMENT Name:_________________________________ 1. What is casein? __________________________________________________________________________________ __________________________________________________________________________________ 2. What is the purpose of lactoglobulin? __________________________________________________________________________________ __________________________________________________________________________________ 3. What is an emulsion? __________________________________________________________________________________ __________________________________________________________________________________ 4. What is an example of an emulsifying agent, other than phospholipids? ___________________ 5. If you are lactose intolerant, what enzyme are you lacking? __________________________________________________________________________________ __________________________________________________________________________________ 6. What are the monomers that make up lactose? _______________________________________ 7. Why does milk spoil? Be as specific as possible. __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ Revision SP11 Page 7 of 7