Name_________________________________________Date____________________Period_

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Laboratory 1 Cheese Production: The Evolution of Cheese-Making Technology

(this activity is from your Biotechnology Lab Manual pgs. 6-11) Please pay attention to the
vocabulary terms in bold.

Background
The cheese-making industry is huge and has a great number and variety of products
(see figure 1.6). Cheese-making is a good example of how biotechnology has improved an
industrial process. In the past, people made cheese simply by letting the naturally occurring
bacteria in milk turn the milk sour. In that process, the bacteria use milk sugar (lactose) as
an energy source and product lactic acid, a waste product. Lactic acid also causes the
mixture of have a mild to slightly bitter taste. Along with other flavorful compounds, the lactic
acid gives the cheese a characteristic flavor.

The milk bacteria produce special enzymes (protein that speed reactions) that convert the
lactose to lactic acid. Lactic acid has a low pH (the hydrogen ion concentration or a measure of
the acidity) and causes the milk protein casein, to denature (unwind) and fall out of solution.
Other enzymes, called proteases, may also act on casein. Proteases cleave (cut) proteins,
such as casein, into smaller fragments that will also fall out of solution. The lumps of
denatured casein are called curds. Curds are pressed together to form cheese. The liquid
remaining after curdling is called whey. (You may have learned the nursery rhyme: "Little
Miss Muffett sat on a tuffet eating her curds and whey. Along came a spider and sat down
beside her and frightened Miss Muffett away.")

Using early methods, cheese makers started new batches with a small amount of cheese
(containing the enzyme-producing bacteria) they had saved from a batch of curdled milk that
produced a good cheese. Although some cheese is still produced in this fashion, today, most
commercially made cheese is produced in one of the four ways listed below. In each method,
sterilized milk is used as a starting reagent.

The Leave-it-to-chance Method: The milk may simply be left to age, exposed to air and
naturally occurring bacteria (see Figure 1.7).

The Bacterial-culture Method: New batches of cheese are started with specific cultures of
selected bacteria. These "known" bacteria also make the enzymes that curdle milk. Buttermilk
has a good culture of Lactobacillus bacteria and can be used as a "starter" (see Figure 1.8).

The We-need-a-cow-stomach-for-this Method: New cultures may be started by the addition of

purified enzymes, such as rennin, which is retrieved from the cells lining the stomachs of
calves (see Figure 1.9). Rennin is a type of protease, and like other proteases, it cleaves the
casein into small fragments that settle out as curds. When calves nurse (yes, baby cows drink
their mother's milk), their stomach cells produce rennin to digest the milk protein. To retrieve
the calves' enzyme for commercial use, companies grind up the calf stomachs and purify the
rennin enzyme from all the other compounds made by the cells (why is this process
disadvantageous?). For this reason, some vegetarians and vegans do not eat rennin cheeses.
There are many rennin cheeses, including Asiago, most brie, most cheddar, and Roquefort.

The Genetically-trick-fungus-into-making-cow-enzyme Method: New cultures may be started

by adding purified enzymes produced by genetic engineering (see Figure 1.10). Scientists
found the DNA code for the cheese-making enzymes produced by calves in regular cow cells.
They cut out the cow's rennin cheese-making code (gene) and inserted it into fungus cells.
Fungus cells then read the cow DNA and synthesized (made) the rennin enzyme, which
scientists called "chymosin". Then, cheese makers used the genetically engineered enzymes to
speed curdling. Now, fungal cultures (growing fungus cells) produce the curdling enzymes in a
faster, cheaper manner, and in larger amounts than can be produced inside multicellular
organisms, such as cows. Chymosin cheeses include Jack, mozzarella, most Swiss cheeses,
plus many others.

Scientists work to create new and improved versions of cheese-curdling enzymes, as well as to
improve the yields and qualities of cheeses. Modern-day cheese makers want to produce large
amounts of high-quality cheese in the most economical way.

Objectives
To determine which curdling agent produces cheese the fastest.
To determine which curdling agent produces the most cheese.
To examine numerical data to support predictions.
To examine variables that can lead to invalid experiments.

Hypothesis
In the space below, predict which curdling agent will produce the largest volume of cheese in
the shortest amount of time (you can choose rennin--from cows or buttermilk--fermented and
contains active cultures / bacteria).
__________________________ will produce the largest volume of cheese in the shortest
amount of time because _____________________________
___________________________________________________________________________
__________________________________________________.

Materials
15 mL sterile tubes, 3 per group
10 mL pipet
Pipet pump
Whole milk
P1000 micropipet
P1000 micropipet tips
Buttermilk
Rennin, bovine (from cows)
Test tube rack
25 mL graduated cylinder
Plastic funnel
Filter paper, 4
Permanent marker

Procedure
1. Each group will be doing the following treatments: buttermilk, rennin, whole milk (negative
control)...one per person.

2. Using a 10-mL pipet and pipet pump, transfer exactly 7 mL of whole milk into a labeled 15
mL conical tube. (label needs to have the treatment name, your name, the date, the period).

3. Using a pre-set P1000, add 250 L (0.25 mL) of the curdling agent assigned to you.
Remember, you need to have each person do one treatment.

4. Cap the tube and mix gently by inverting three times. Record the "initial time".
5. Place the milk-containing portion of the tube deep in your armpit, like a thermometer, and
incubate it there for at least 15 minutes.

6. Check for curdling every 5 minutes, recording the time to curdle in minutes. To check for
curdling, gently tilt the tube, being careful not to break up any curds. Curds are large lumps of
solidified milk. After 15 minutes, place the tube upright at room temperature and check for
curdling every 15 minutes for the next 2 hours. If curdling has not occurred within 2 hours,
continue to check once every 4 hours. With the greatest accuracy possible, record the time, in
minutes, until the milk curdles.

7. If curdling has not occurred by the end of the lab period, bring the tube home (at room
temperature) and back to class the next day. Keep the tube upright so any curds will fall to
the bottom.

8. When you return to lab the next day, measure the volume of curds (solids) and whey
(liquid) in the tube. You may be able to read the volume of each directly from the tube. If not,
filter the curds as shown in Figure 1.11 and described below:
Pour the whey and curds through a paper filter / funnel into a 10-mL graduated cylinder.
Determine the volume of when collected in the graduated cylinder. By subtraction, determine
the volume of curds.

9. In your lab notebook, make a data table similar to Table 1.2 and record the data for your
sample plus one each of the other variable groups. This will give you data for one
experimental trial of each curdling agent. Record the name of the person from whom you
obtained the data. As a class, we will average the data for each experimental group.

Table 1.2 The Effects of Cheese-Curdling Agents on Curdling Time and Volume

Curdling Agent Time to AVERAGE Time Volume of Volume of

Curdling (min) to Curdling Whey (mL) Curds (mL)
(min)

buttermilk

rennin

milk (control)
Data Analysis
1. Using Microsoft Excel, produce two graphs:

a. Average time to curdling for each enzyme treatment.

b. Average volume of curds produce by each enzyme treatment.

2. How well did the single trial your group did of the experiment support the hypothesis?
Explain.

3. Averaged data are the best way to answer an experimental question. Explain why.

4. Does it appear that the number of replications for each curdling agent was sufficient? Yes or
no? Explain your answer.

5. Do you think the whey-o-meter was adequate for an accurate determination of whey
volume and, indirectly, curd volume? Why or why not? If yes, explain why. If no, suggest a
better system to determine the volume of curds or the volume of whey.

Conclusion
Address the following in each paragraph of the conclusion:

Paragraph 1:
Describe the results of your experiment. Include evidence (data) and explanations.
Discuss how well the results support the hypothesis.

Paragraph 2:
Identify the sources of error AND explain what might happen as a result of this error.
ex: "Too much rennin was added. This may have caused the milk to curdle faster than
expected because more the ratio of curdling agent to milk was higher than in any other
experimental group."

Paragraph 3:
Imagine you are an employee at a cheese company. Make a recommendation to your
company supervisor about which curdling agent should be used for production of cheese or the
target of continued testing.
Be sure to sign and date at the bottom of your conclusion.