Cheese making Demonstration: Engineered Enzymes
Rational: The cellular energy unit focuses on enzymes, photosynthesis, and cellular respiration. The cheese
making demonstration is a very visual way for students to first be introduced to enzymes and see them in
action. The primary function of the demonstration is to initiate the questioning process of what before white
boarding in groups.
Background: The process of making cheese has existed for centuries; its origin can be traced back as far as
1,000 B.C. Like many scientific discoveries; the process of cheese making was most likely discovered
purely by accident.
Primitive tribes were primarily hunters and gatherers, occupying an area only as long as it suited their
needs, exploiting its natural resources, and then moving their herds to a newer, more fertile location. Because
resources were scarce, they utilized every source of food and supplies to its fullest extent, including those
provided by their herd animals. The tribesmen stored their milk in bladders made from the stomachs of
animals killed for food where, they discovered, the milk formed soft lumps that could be eaten; this type of
cheese was similar to today's cottage cheese. Though the tribesmen did not know why this process was
occurring, they began to intentionally produce this new source of food, becoming the first to engage in the
new enterprise of cheesemaking.
Although cheesemakers knew that a certain combination of ingredients, placed under certain
conditions, would produce cheese, they did not know the scientific reason why or how this happened. As
technology advanced, however, scientists unraveled the mystery of how efficient cellular reactions occurred
when they discovered enzymes.
Enzymes are protein molecules with the ability to catalyze, or change the rate of, chemical reactions,
while the enzymes themselves remain unchanged after the reaction has occurred. As scientists investigated
the role of enzymes in biochemical processes, they soon discovered that chemical reactions that usually took
hours to complete occurred in seconds when enzymes were added. They also discovered that enzymes are
very specific molecules; a specific enzyme can only catalyze a specific reaction, such as putting together
amino acids to form a larger, more complex protein molecule. A different enzyme may in turn break a larger
protein molecule into small amino acids, making them much more useful at the cellular level.
When the early nomads placed milk in their primitive "canteens", the stomach bladders, enzymes
began to work immediately. The stomach lining of some animals contains an enzyme called rennin, which
reacts with casein (milk protein) to cause the protein to coagulate, or clot, forming the basis for the simplest
type of cheese.
In the modern cheesemaking industry, certain microorganisms that contain an enzyme called
rennilase, which reacts much the same way as rennin, is added to cause the reaction. Different
microorganisms are used to produce the distinct flavors of different types of cheese. The solid portion of the
milk, called the curd, is separated from the remaining liquid, called whey; treating the curd after it is
removed produces soft, creamy cheese like cottage cheese. When the moisture is removed from the curd, and
the curd is salted and allowed to age, or "ripen", harder, sharper-flavored cheese such as cheddar or Colby is
produced.
Warning: All procedures must be done in a sterile setting if you wish taste the cheese you make. Your
table should be sterilized, hands must be washed, and all lab instruments must be new or
thoroughly cleaned.
Materials:
Whole Milk (D)
Rennin enzyme solution (1 tablet dissolved in 60ml DI), approximately 0.5g per 60 ml or follow
directions on bottle.
Sterile 50ml tubes filled with 45ml of Whole D Milk.
*Larger quantities of milk can be used in a flask for a more dramatic demonstration.
Procedure:
1. Warm the whole milk to approximately 45 - 55°C in a microwave, hotplate, or hot water bath.
2. Fill each plastic cup or sterile tube approximately half full with the warmed milk.
3. Immediately add 1 ml of the rennin solution (mixed 1 tablet to 60ml DI). Mix well with a stirring stick.
4. Allow the mixture to sit undisturbed for at least 30 minutes for best results, however visual results can be
seen within a few minutes for classroom discussion.
For faster reaction times, Emporase (Carolina Biological) may be used.
5. Separate the curds (solid white masses) from the whey (remaining liquid) by filtering the mixture
through two pieces of cheesecloth over a sink or container.
6. Allow the whey to drain from the curd for a few minutes. Gently squeeze the curd to remove any excess
moisture.
Taste at your own risk, although probably safe because we were operating with sterile materials there is still
some risk to consuming warm dairy products. You can add salt, rewrap the cheese, and set it aside to harden.
Potential White Boarding Questions:
How do the two tubes compare?
What happened to the milk when rennin was added to the milk? Describe what you observed over the
thirty minute period.
Milk is an emulsion of fat in water, with a typical composition for cow's milk of 87% water, 4% fat, 4%
protein, and 4% carbohydrate. Of these constituents, it is a family of proteins called casein that holds the
emulsion together. Chymosin is a protease, a class of enzymes that react with and degrade other proteins.
Many proteases will degrade casein. When this happens, the emulsion is broken, and calcium caseinate
precipitates as the curd. Acid also will cause the formation of curd. However, chymosin in its pure form is
unique in its ability to cleave casein in a selective way that causes coagulation without producing other
breakdown products that may contribute off-flavors to the curd, and ultimately to the cheese.
What effect do you think changing the amount of milk fat would have on the formation of the cheese?
What are the benefits to producing chymosin through Biotechnology compared to extracting it from
calf stomachs?
Why are microorganisms important to cheese manufacturing?
How Cheeses Are Made
The great variety of cheeses are produced by changing conditions, such as temperature, strain of lac
to bacteria used, amount of salt added, length of time cheese is ripened, and strain and type of ripening
organisms added.
Cheddaring is a term used in the ripening process of some cheeses. Proteolytic or lipolytic
bacteria or fungi are fermented in large vats, and the cheddaring enzymes are extracted. These enzymes are
added to the curds with salt. The term comes from Cheddar, England,- where the process developed in the
seventeenth century.
Swiss cheese and other cheeses with holes are made from the action of proPionibacterium,
which produces carbon dioxide bubbles that are trapped in the cheese. Propionic and succinic acid give the
cheese its distinctive flavor.
Blue cheeses are formed when fungal spores germinate in the cheese. English Stilton cheese
tastes different from Danish Blue because different fungal strains are found, and thus used, in England and
Denmark. These fungal spores need oxygen to germinate, so cheesemakers puncture the cheese with needles
to allow air to enter the interior of the cheese.
Cheeses with rinds, such as Brie, are formed when the outside of the cheese is wiped with
organisms that secrete enzymes, which penetrate the cheese and produce flavor chemicals. These increase
the density of the cheese near the outside.
Cheese is often aged to ripen the flavor. In order to protect the cheese from drying out and
microbial contamination, it is wrapped tightly or dipped into melted wax.