Prepared by
Nam Sun Wang
Department of Chemical & Biomolecular Engineering
University of Maryland
College Park, MD 20742-2111

Table of Contents


To demonstrate the use of rennet in casein coagulation in different pH conditions.


Cow's milk is rich in a wide range of chemical compounds that can be processed into various dairy products such as cheese, butter, and yogurt. Specifically the milk component involved in cheese production is a soluble protein called casein. The enzyme rennet can be used to catalyze the conversion of casein in milk to para-casein by removing a glycopeptide from the soluble casein. Para-casein further clots, i.e. coagulates, in the presence of calcium ions to form white, creamy lumps called the curd, leaving behind the supernatant called the whey.
         rennet                      Ca++
  casein -------> para-casein (aq.) --------> para-casein (ppt)
The precipitate is soft at this point and can be separated from the whey by the use of cheese cloth. Filtration does not work very well; filter paper clogging is a recurrent problem.

There is no standard method of cheese making; limitless variations exist for all stages of the process: pre-ripening, curdling, addition of artificial ingredients and salt for flavor, and aging. This variation in processing accounts for the wide range of cheeses commercially available, differing in texture and flavor. The curd can also be processed with other techniques to make a variety of desserts. However, all processes have one thing in common: the separation of the curd from the whey.

Three ways of preparing milk for curdling will be introduced in this experiment. Industrially, the lactic acid level in the milk is increased by adding a starter culture of Streptococci or Lactobacilli to the milk and fermenting at 32ºC for 10 to 75 minutes. In addition to biologically converting the lactose present in the milk to lactic acid, these strains of microorganisms also greatly affect the flavor of the final product. Thus, the selection of a suitable strain, the amount of starter culture, and the length of pre-ripening, is of the utmost importance in creating the subtle differences in the final color and aroma that distinguishes an expensive cheese from a cheap one.

If one has not yet acquired a keen palate for cheeses, the second approach should suffice. In this approach, one takes advantage of the existing Lactobacillus culture in buttermilk and uses it as the starting culture. One ml of buttermilk is added per 100 ml of milk, and the mixture is then fermented at room temperature for 4-12 hours. At the end of fermentation, the temperature of the mixture is raised to 32ºC, and artificial coloring is added to the mixture prior to curdling.

The third way to prepare the milk in a short time frame is to add acid (HCl) and to heat to 32ºC. Of course, there leaves much to be desired in this method if you are a cheese connoisseur.

After rennet is added to the pre-cured milk, the coagulation process is started. In cheese making, as coagulation comes to completion, the temperature is gradually raised to about 38ºC. This slightly elevated temperature facilitates the separation of the curd from the whey. A higher temperature also hardens the curd. The curd may be hardened further by cooking it for a longer period of time, either with or without the whey.

After the curd is separated from the whey, salt, seasoning, and other curing and flavoring ingredients are added. The curd is wrapped in cheese cloth and pressed for 12 to 18 hours to remove the additional whey soaked in the curd. The curd hardens and forms a cheese block in the shape of the press as the whey is squeezed out. Finally, the cheese block is dried for 6 hours.

It is now ready for consumption, or it may be left to age in a controlled cool environment (2-13ºC). Although a higher temperature promotes faster curing, there is also a higher chance of spoilage due to undesirable microbial activities at elevated temperatures. Prior to aging, the cheese block is usually wrapped tightly to exclude air and microbial contaminants from entering and spoiling the cheese. One way to accomplish this is to dip the cheese block in a pot of melted wax. During the aging process, many complicated microbial and chemical actions continue to take place in the cheese block. Thousands of techniques exist to develop various distinctive flavors. These reactions are not well characterized; thus, cheese making is still an art rather than a science. Depending on the technique employed, this final aging process takes any where from 2 weeks to 6 months.

Precuring at a Warm Temperature (Acidify the Milk)
Coagulation by Rennet
Separation of the Whey
Flavor addition
Compression of the Curd
Finished Cheese

Figure 1. Steps in cheese making.

List of Reagents and Instruments

A. Equipment

B. Reagents


  1. The T.A. will prepare several batches of cultured milk before the class by mixing 2.5ml of buttermilk (starter culture) to 250ml of normal pasteurized milk in a beaker. The content in the first beaker is to be fermented at room temperature for 12 hours (G); the second for 10 hours (F); the third for 8 hours (E); etc. Do not add buttermilk to the batch labelled A, which is kept free of a starter culture. The number of mixtures with differing pre-curing times depends on the number of students enrolled. Each student will be responsible for analyzing one of these beakers.
           A |                                0 hr
           B |                            2 hr
           C |                        4 hr
           D |                    6 hr
           E |                8 hr
           F |           10 hr
           G |       12 hr
                   NUMBER OF HOURS INCUBATED
  2. Analogous to Step 1., prepare several synthetic "cultured" milk by adding 5-20 ml of 1N HCl to 250 ml of milk in a beaker. Each student will be responsible for one of these beakers. (What happens when lactic acid or citric acid is used in lieu of HCl?)
  3. Record the pH of each batch.
  4. Gently heat the authentically and synthetically cultured milk to 32ºC in a temperature bath.
  5. Stir and add 0.5 ml (approx. 5 drops) of rennet to each beaker.
  6. After the enzyme addition, let the milk stand on the bench top for 30 minutes to allow coagulation to proceed. Do not disturb the coagulation process in the beakers; otherwise, the curds will be broken into pieces too small to filter efficiently with cheese cloth.
  7. After curd formation, break up the curds with a stirring rod and drain the whey through layers of cheese cloth. Filtration through a medium pored filter paper may give better results.
  8. Weigh the amount of curd obtained in its wet state and approximate the yield.
  9. Taste your cheese. (Yum-m-my, I love cheese!) Is there anything missing?


It is generally quite straight forward to locate the sources of enzymes; the natural sources of enzymes are where nature needs them. It comes as no surprise that rennet had traditionally been isolated from the fourth stomach of young calves because digestion by suckling calves is nature's primary way of processing cow milk. However, the ways of nature are not always the most economical from man's perspective when a process is adapted to different uses to benefit mankind in ways not originally intended by the nature. Thus enters the study of biochemical engineering.

Chemical engineering is the study of how to make a large quantity of chemicals in an economical fashion. For example, frequently a chemical engineer must devise a process to mass-produce a polymer that is totally different from the one originally used by an organic chemist working with small test tubes and beakers in a laboratory. Biochemical engineering, being a sub-field of chemical engineering, also deals with the same kinds of problems facing chemical engineering, except that the chemicals are not synthetic (manmade) but biological (naturally existing) in nature. Although rennet is naturally excreted from a calf's stomach lining, extracting it from its natural source is not economical. Other proteases can also convert casein to para-casein, but their action does not stop there. They further degrade the curd to soluble subunits. Fortunately, large quantities of rennet of consistent quality can now be produced easier and cheaper in a well controlled environment by microbial fermentation.

A word of caution is in order here. Enzymes as a class of chemicals are not generally considered as dangerous, toxic, nor poisonous; they do not cause skin irritations or burns as acids or bases. Some exceptions are proteases that catalyze the breakdown of protein molecules to amino acids components. Because meat is mainly protein, protease can digest the soft moist sections of the skin. We can all imagine how that is going to feel. Thus, you should exercise the same caution with enzymes as you do with any other chemicals.


  1. Report your observation of this experiment and discuss the effect of pH on the yield of the curd. Try out other types of processed milk such as nonfat milk and powdered milk. Are they suited for cheese making?
  2. Taste the cheese you have made. How does it taste compared to cottage cheese? (The product should be edible if the glassware is clean, which is exactly what it should be.)
  3. In Step 1, why is buttermilk not added to the batch labelled "A"?
  4. Because its action has long been discovered, rennet does not follow the rules of enzyme nomenclature discussed in class. Its conventional name has already been deep-rooted in the craft of cheese making. Considering the action of rennet, how would you rename the enzyme to conform to the general rules of nomenclature?
  5. What microorganisms are currently used to manufacture rennet? How is rennet isolated?
  6. Why is the coloring added before coagulation instead of afterwards?
  7. Give a list of the chemical components found in milk. In this experiment, you used an enzyme to react selectively with a protein called casein; what other carbohydrates, proteins, and fats are found in milk? List the nutritional information for some popular types of milk and break down each category into individual chemical components. (Example: Nonfat milk has x g/liter of carbohydrates, y% of which is lactose, z% of which is sucrose, etc.)
  8. What are some of the products other than cheese that are derived mainly from milk? Identify the principle component of milk utilized in each product and point out the use of enzymes if applicable. Choose a specific dairy product and give a description of the processes involved in the production.
  9. Milk spoilage can be detected visually by the presence of coagulated curd. Identify the chemical mechanisms responsible for coagulation in this case. Are these chemicals/enzymes produced by contaminant microbial actions? Is the curd resulting from spoilage the same as that you have obtained in class? If they are the same, why would you consume cheese but not spoiled milk?
  10. Comment on ways to improve the experiment.


  1. Prins, J., Microbial rennet, Process Biochem., May, 1970.
  2. Robinson, R.K., Dairy Microbiology, Vol. 2, Applied Science Publishers, New Jersey, 1981.
  3. Richmond, H.D., Dairy Chemistry, A Practical Handbook, Charles Griffin & Co., London, 1930.
  4. Constituents of milk: Jenness, R. and Patton, S., Principles of Dairy Chemistry, John Wiley, 1959, p3.

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Cheese Production from Milk
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Nam Sun Wang
Department of Chemical & Biomolecular Engineering
University of Maryland
College Park, MD 20742-2111
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