Materials Included In Kit

Additional Materials Required

Safety Precautions

This laboratory examines the chemistry involved in the first steps in cheese-making. Most conditions in the laboratory are not ideal nor are they safe for food production, and, therefore the products produced must not be tasted or eaten. Remind students to wash hands thoroughly with soap and water prior to leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. The materials used in this lab may be disposed of according to Flinn Suggested Disposal Method #26a.

Teacher Tips

• The protease chymosin is an excellent enzyme for all basic studies relating to enzyme chemistry. It is safe, can be observed with the naked eye, and experiments can be done quickly with simple equipment.

• Because of the way chymosin is produced, it is a perfect opportunity for classroom discussions of biotechnology advances in sequencing genes, gene therapy, cloning and the production of genetically improved foods.
• You will want to ensure a coagulation time of about 5–10 minutes for normal classroom timing. Try your milk with your chymosin solution before doing the laboratory. Experiment with enzyme concentration and pH if necessary until you are within the time frame you desire. Aging milk in advance can be helpful in speeding up coagulation. Let the milk set out at room temperature for about 6–8 hours.
• Enzymes, such as chymosin, decrease in effectiveness with age. Therefore, in Part I, step 6 of the student procedure could require anywhere from 2–15 drops to coagulate milk. Again, test before class so students can achieve optimal results in the given amount of time.
• To speed up coagulation, you can add 3–4 drops instead of 2 drops of enzyme solution.
• The enzyme manufacturer suggests that 2–3 g of chymosin powder will curd 100 L of milk under the right conditions. Since you will want to see coagulation in a very short time, we recommend a recipe as follows for your initial experimentation. Dissolve 0.3 g of chymosin in 100 mL of bottled water (pH tends to be 7.0). Use two drops for each 10 mL sample of milk. This kit contains 2 grams of chymosin which should allow you to coagulate more than 100 L of milk!
• You can make a small concentrated stock solution of chymosin for students who want to test the effects of varying enzyme concentration.
• Students who perform experiments on temperature will quickly realize that the reaction rate is greatly affected by slight temperature changes.
• Vinegar is a safe material to use to create acidic conditions in pH variation experiments. Sodium bicarbonate can be used to create basic conditions.
• The curding times for different types of milk (e.g., skim, fat free, 2%, half and half, whole milk, powdered milk) will force careful readings of milk labels and hypotheses about milk contents.
• The open-ended suggestions in the cheese research section can be extremely beneficial in achieving higher level reasoning skills suggested in the National Science Standards. Chymosin affords a great opportunity to do higher level science with an inexpensive set of materials.
• Interdisciplinary spin-offs might include: History of cheese-making, cheese variety and cultural preferences, economics of cheese-making, microbial cultures and cheese varieties, security of brand names and the ownership of culture strains.
• Keep the enzyme and its solutions refrigerated when not in use.

References

Special thanks to John Fedors for the development of this activity.

Harewood, K.; Hinman, R., The American Biology Teacher, 1999, 61, 4, 288–293.

Introduction

When Little Miss Muffet sat on her tuffet, she was eating coagulated milk! Coagulating milk into curds and whey is the first step in cheese-making.

Concepts

• Proteases

• Bioengineering
• Enzyme activity
• Cheese-making

Background

Milk is often considered to be one of nature’s most “complete” foods with its mixture of fats, proteins, and other nutrients. Milk is an emulsion, a mixture of two normally immiscible liquids, fat and water. The fat and water are held together by a natural emulsifying agent, called casein, the name for a group of proteins. If the protein (casein) is destroyed, the emulsion breaks down into its component parts and the insoluble calcium caseinate and the fat precipitate out of the suspension. This “solid” portion that precipitates out of milk is called the curd. The “liquid” portion of the milk that remains after the curding process is called the whey.

The process of separating milk into curds and whey has been used for centuries. The process of forming curds has several advantages for preserving and using milk as a food source. Milk has a very short shelf-life and spoils very easily. (Bacteria like it too!) Turning milk into curds does several things to help keep milk protein unspoiled. In solid form, the milk (curds) can be dried and treated in a variety of ways to prevent bacterial growth and spoilage. In addition, the curding process helps concentrate the food value into a small, solid material that is easier to store. Further treating the curds and turning it into cheese gives a whole new set of food textures and tastes. Different curing techniques, subtle variations in additives, varietal cultures of bacteria/fungi, and processing stages produce the differences in taste, texture, color and aroma of cheeses. However, most cheeses begin using the same coagulation procedure used in this laboratory activity.

The consistency and amount of curd produced from a batch of milk depends upon the availability and concentration of chymosin, the enzyme that converts liquid milk to curds and whey. Historically, the enzyme source used by cheesemakers has been from extracts taken from calves’ stomachs while they are still nursing their mothers’ milk. The stomach extract, called rennet, provides a high concentration of enzyme and works very quickly to curd milk. The chore of maintaining calves on a milk diet and extracting the stomach enzymes has prompted the continual exploration of alternative enzyme sources for making cheese.

Protein-splitting enzymes are called proteases. Proteases break proteins into small chains of amino acids. Chymosin is a protease that reacts with casein in a very specific way, coagulating the milk protein and producing a curd with little or no taste. Other proteases break casein proteins into a variety of smaller chains of amino acids which often result in “off-flavors” in cheese.

With the relatively recent advances in DNA biotechnology, an entirely new source of curding enzyme has been developed. The first step in the development process was the identification of the DNA chromosomal fragment that encoded for the enzyme, chymosin. Once the gene sequence was isolated, it was then recombined with a cleaved bacterial plasmid. This plasmid, with the encoded chymosin DNA, was then introduced into a bacterial cell. The bacterial strain, which can be grown in large quantities, is now used to produce the identical enzyme that was previously produced by calves’ stomachs. The enzyme produced by the bacteria can be harvested, dried and stored until needed. The production and use of this bacteria-produced enzyme, identical to natural calf chymosin, was one of the first biotechnology products that was approved by the FDA (Food & Drug Administration) for humans. The resulting enzyme product, chymosin, is a biotechnology product that has the benefits of nearly unlimited supply, 100% activity versus 60–90% for calf extract, identical to the natural product, much cheaper, and it eliminates the need to maintain milk-fed calves.

Materials

Safety Precautions

Once food items are brought into the lab, they should be treated as chemicals. Never eat anything in the lab or any food that has been in the lab. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Part I. Enzyme Action

1. Read the following observation tips: Milk curd formation will be observed in a test tube. Since curd is a white precipitate in a white liquid some practice may be required. Best results can be seen by tilting the tube on its side and rotating the tube. The first sign of coagulation is the formation of a creamy film on the side of the test tube. Viewing the tube in bright light is a good idea.
2. Fill a 250-mL beaker with 150 mL of 40 °C tap water. This will serve as the water bath.
3. Label two test tubes: “M” (milk only) and “ME” (milk plus enzyme).
4. Place 10 mL of milk in each of the two labeled test tubes.
5. Place the two test tubes in the water bath and allow the tubes to warm for about two minutes.
6. Place 10–15 drops of chymosin solution into the test tube labeled “ME.”
7. Check both test tubes every minute following the observation tips from step 1.
8. Record your data in a chart like the following and then answer the following questions:

1. How much time was there between the start and completion of coagulation for each tube?
2. Why was test tube “M” used? Was it necessary?

Part II. Curds and Whey

1. Place 2–3 layers of cheesecloth inside a funnel placed over an empty beaker (see Figure 1).
2. Pour the contents of test tube “ME” into the funnel containing the cheesecloth strainer.
3. Allow the liquid to drain through the cheesecloth and into the beaker for several minutes.
4. When the curd has stopped draining, take the cheesecloth containing the curd out of the funnel. Spread out the cheesecloth on top of dry paper towels. Pat the curds dry with additional paper towels.
5. Observe the curds and whey. Examine the liquid in the beaker and the solid on the cheesecloth. Record your observations. Estimate how much of the original 10 mL is curd and how much is whey.
6. The curds can be carefully removed from the cheesecloth, dried further and pressed into a more solid mass. Store the curds for 24 hours, then examine again.

Reminder: Do not eat or taste the curd product.

1. Form a research group to investigate one variable that might affect the chymosin/milk curding reaction. Possible variables to consider include: temperature, pH, fat content of milk, brands of milk, enzyme concentration and substrate concentration.
2. Design your experiment and write your plan in detail. Be sure to include controls.
3. Discuss your selection of materials and hypotheses with your teacher.
4. Work with your teacher to secure all necessary materials.
5. Conduct your experiment. Record your results.
6. Write a complete laboratory report for your experiment. Include: (a) statement of your hypotheses; (b) description of your procedures and experimental design (including any safety precautions followed); (c) record of your data and observations; (d) interpretation of your results; and (e) further activities for investigation.
7. Further topics to discuss/research:

1. What different experiments performed by different teams might be combined to make cheese even faster?
2. How do cheesemakers make all the various kinds of cheeses? What happens after the initial curding process?
3. What precautions must be taken in the making of cheese?
4. What is spoiled cheese? How do you know if it is spoiled?
5. What can be added to cheese to make it more marketable?
6. Are there any potential medical problems associated with cheese consumption?