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Engineered Enzymes for Cheese Production: Little Miss Muffet
(Adapted from Shoestring Biotechnology pp.139-152)
Glencoe Biology “The Dynamics of Life”
Chapter. 6 Life Sciences, Sections 6.3
This is also aligned to PSAE 12.11.10
Guiding questions
How do enzymes facilitate chemical reactions? What experimental design increases the yield of cheese
from a sample of milk?
Context of Lesson
A fundamental difference between living and nonliving things is the central role of enzymes in living
things. Enzymes are proteins that facilitate chemical reactions. In this lesson, students are provided with
a protocol to determine the effects of manipulating variables such as pH, temperature and milk fat
content on cheese production with the bioengineered chymosin. Chymosin is a protease- a class of
enzymes that react with and degrade other proteins.
Main Goals/ Objectives:
By the end of this lesson, students will be able to:
 Compare the effectiveness of bioengineered chymosim with natural form of chymosin.
 Describe cheese-making from modern and historic perspectives.
 Describe the role of biotechnology in cheese-making.
Nature of Science: Integrated Theme
 Explain that scientific knowledge is tentative due to new evidence or new
interpretation.
 Distinguish observations from inferences, explain that inferences should be based on
observations, and explain that the development of scientific knowledge involves both
observations and inferences so scientific knowledge is partially inferential.
 Explain that scientists’ creativity influences their doing inquiry so they may have
different observations and interpretations of the same phenomena.
 Explain that scientific knowledge should be based on empirical data.
Scientific Inquiry: Integrated Theme
 Explain that scientific investigations all begin with a question, but do not necessarily test
a hypothesis.
 Explain that there is no single scientific method and provide at least two different
methods.
 Explain that inquiry procedures are guided by the question asked.
 Explain that all scientists performing the same procedures may not get the same results.
 Explain that inquiry procedures can influence the results of an investigation.
 Explain that research conclusions must be consistent with the data collected.
 Explain that scientific data are not the same as scientific evidence.
 Explain that explanations are developed from a combination of collected data and what
is already known.
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General Alignment to Standards
11A. Know and apply the concepts, principles and processes of scientific inquiry.
ILS 11.A.4b. Conduct controlled experiments or simulations to test hypotheses.
ILS 11.A.4c. Collect, organize and analyze data accurately and precisely.
ILS 11.A.4e. Formulate alternative hypothesis to explain unexpected results.
12A.
Know and apply concepts that explain how living things function, adapt and change.
ILS 12A.4b. Describe the structure and organization of cells and tissues that underlie
basic life functions including nutrition, respiration, cellular transport, biosynthesis and
reproduction.
13B.
Know and apply concepts that describe the interaction between science, technology
and society.
ILS 13B.4b. Analyze a particular occupation to identify decisions that may be influenced
by knowledge of science.
PSAE STANDARD 11A – SCIENTIFIC INQUIRY
 11.11.01 Understand and follow procedures relating to scientific investigations,
including understanding the design and procedures used to test a hypothesis, organizing
and analyzing data accurately and precisely, producing and interpreting data tables and
graphs, performing appropriate calculations, applying basic statistical methods to the
data, identifying appropriate conclusions, making predictions, and evaluating competing
models.
 11.11.02 Distinguish among the following: observing, drawing a conclusion based on
observation, forming a hypothesis, conducting an experiment, organizing data,
comparing data.
PSAE STANDARD 12A –LIVING THINGS
12.11.10 Understand that enzymes are proteins that catalyze biochemical reactions and
that the activity of enzymes depends on the temperature, ionic conditions, and the pH
of the surroundings.
PSAE STANDARD 13B –SCIENCE, TECHNOLOGY, AND SOCIETY
 13.11.06 Analyze scientific breakthroughs in terms of societal and technological effects.
 13.11.08 Analyze careers and occupations that are affected by knowledge of science.
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Facilitation:
Day 1 (45 minutes)
Introduce the lab concept, protocol and develop a flow chart for the protocol. Refer to Shoestring
Biotechnology for teacher background information pages 125-136.
Days 2 (45 minutes)
Conduct experiment.
Day 3 (45 minutes)
Analyze and discuss data.
Day 4 (45 minutes)
Perform Student designed experiments
TEACHER TIPS
 You might find rennin, now called chymosin, in the grocery store. Compare the effectiveness of
these forms of rennin with the bioengineered form of chymosin.
 Identify the source of the enzyme you are using. Determine whether it is from calves’ stomach or a
microbial source.
 Buttermilk is used for the stock culture because of its acid content, which speeds up the reaction.
Active plain yogurt*(Lactobacillus) may also be used as a starter.
 Field testers found that skim milk curds were filterable within 3 minutes.
Equipment and Materials Need
For instructor preparation of the protocol, supply the following:
Equipment:
 1 balance
 1 100-mL storage bottle with cap
 1 3-L storage bottle with cap or 1-gallon plastic milk jug
Materials:
 1 bottle Chy-Max (CHR-Hansen #72879, # 72873) or Emporase (Carolina Biological #BA20-2375)
 1 bottle chymosin
 600-mL buttermilk or active culture plain yogurt
 3 L whole milk
For instructor- led preliminary instruction, supply the following for a class of 24 students:
Equipment: 1 VCR and monitor (optional)
Materials: 1 video Biotechnology: Careers for the 21st Century (optional)
Teacher directions for setting up the lab
For instructor- led protocol, supply the following for each group of 3-4 students in a class of 24 students:
Equipment:
 1 thermometer (°C)
 1 500-mL beaker or 1 quart saucepan
 1 hot plate
 1 50-mL graduated cylinder
 1 150-mL flask or beaker
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 1 clock or timer
 1 1-mL pipette
 1 bulb or pump
 1 stirring rod
 1 funnel
 1 100-ml beaker
 1 balance
 1 permanent marker, such as a Sharpie or laundry marker
Materials:
 100-mL cultured milk
 0.2-mL Chy-Max or 0.1 ml Emporase
 4 15 x 15-cm cheesecloth squares
 1 0.95-L resealable, plastic sandwhich bag
 1 weigh boat
 5 highly absorbent paper towels
 1 10 x 10-cm square of plastic wrap
DIRECTIONS FOR SETTING UP THE LAB
One day ahead of the lab
Chymosin Bioengineered Enzyme Solution
Prepare stock solution of selected enzyme at manufacturer’s suggested concentration. For example,
Chy-Max is prepared at a 1.0g per 60-ml deionized water. Store in tightly capped 100-ml storage bottle.
The liquid product contains approximately 0.1% active chymosin. Emporase comes ready to use. Follow
the manufacturer’s recommendation for refrigeration.
Stock Milk Culture
 Prepare Stock Milk Culture in the ratio of 1 part buttermilk to 5 parts milk. If prepared the same day,
the ratio is 1 part buttermilk to 4 parts milk. For a class of 24 students to run the protocol once, add
90-mL buttermilk and 450-ml milk to a 1-L container. Cap and store in the refrigerator.
 Students should be organized into multi-tasked lab teams of 3 to 4. Each student team should
assume a different role during the lab experience. Roles should be defined after watching video or
as you do in your class.
 Each student teams can investigate different types of milk using the Protocol for Positive Control.
Modifications/Accommodations:
 This lesson could be implemented as a four day lab, as written in the Shoestring Biotechnology book.
Day One - pre-lab and develop a flow chart for the protocol, Day Two-conduct the experiment, Day
Three- analyze and discuss data and Day Four perform student designed experiments.
 For students who require more guidance in the lab (for safety or other reasons), have he/she paired
with a student who needs less guidance.
 Reduce the number of variables tested.
 Use a turkey baster instead of 10-ml pipette if students are having difficulty with fine motor control.
 This is a good lab for collaborative groups for students with varying English language abilities and
academic abilities because the lab is data-oriented and hands-on.
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

This is a visual lab. There is an observable substance collected that may be difficult for some learners
with visual impairments. Have them work with students with no visual impairment.
Use graphic organizers and Venn diagrams to reinforce concepts taught in this lab.
Pedagogical Information (Note: These can be integrated)
The following is a chart of some concepts covered in this lab and some student misconceptions of those
concepts:
Correct Conception
Misconception
Many people do science. Especially important Only scientists do science.
are the biotechnicians. Their many skills make
science happen every day.
Cheese flavors are the product of microbial
Cheese has been acted on only by microbes
action with aging.
when you see it covered with fur-like growth.
DAY 1 (45minutes) Student Introdcution to Protocol and Show Video
Introduction to the Lesson:
Bell Ringer:
Begin the lesson by showing your students, a 10 x 10 cm piece of cheesecloth. Ask the students, “Have
they ever seen cheesecloth before? How can it be used?” Possible responses might be: It can be used
as a filter or strainer. It can be used for making tofu. It can be used for bundling herbs, as in sachets.
Discussion:
Tell students that ancient people used animal stomachs and bladders as water canteens. At some point,
someone must have filled an animal canteen with milk instead of water. Many hours later, the milk
clotted into soft, creamy lumps with a sharp, satisfying taste. These curds were the first cheese. Have
students recall the nursery rhyme, “Little Miss Muffet”, ask students, “What did Little Miss Muffet eat?”
They should recall, ‘curds and whey’.
Little Miss Muffet sat on a tuffet,
eating her curds and whey.
Along came a spider, who sat down beside her,
and frightened Miss Muffet away!
Explain that the term curds, describes coagulated milk proteins that forms when milk clots. Whey is the
watery part that remains. The early peoples had no understanding of the fact the transformation of
their milk was a result of the action of an enzyme that was released from the lining of the animal’s
stomach. We know now that the substance which caused the curdling was the enzyme rennin, now
called chymosin.
Today, we often see whey floating at the top of a yogurt container. Also we eat curds and whey when
we eat cottage cheese and other dairy products.
Milk is an emulsion of fat in water. The typical composition for cow’s milk is 87% water, 4% fat, 4%
protein and 4% carbohydrates. Briefly have students, pair-share, with the student next to him/her, on
the various types of milk that he/she has seen or tasted. Ask, “What other types of milk are there
besides whole milk?” Possible responses: 2% milk, 1% milk, skim milk, Soy milk, Buttermilk , camel
milk, Goats milk, etc…
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Tell students that in this lab they will be investigating how to increase the yield of cheese from samples
of milk.
At this time show the video, Biotechnology: Careers for the 21st Century, if available. If not, begin the
investigation by reviewing the safety rules for this lab.
Safety
 Wear goggles, gloves, and protective aprons or lab coats.
 Wash hands before and after the lab activity.
 Exercise caution when using chymosin. Proteases such as chymosin can digest soft, moist
sections of the body. Enzymes as a class of chemicals are not considered to be toxic,
poisonous, or dangerous. An exception is proteases that catalyze the breakdown of proteins.
 Do not eat any material produced during this lab.
DAY 2 ( 45 minutes) Conduct Lab
For each team of 3-4 students, supply the following:
Equipment:
 6 150-mL Erlenmeyer flasks
 1 37°C water bath
 1 5-mL graduated pipette
 1 funnel
 1 permanent marker or wax pencil
 6 weigh boats
 1 balance
Materials
 600 mL skim milk, 2% milk, Soy milk, Goat’s milk
 0.6 mL chymosin enzyme
 1 vial of pH paper
 6 15 x 15-cm cheesecloth pieces
Protocol for Positive Control
1. Prepare a 37°C water bath by filling a 500-mL beaker or 1 quart saucepan half full of water. Place it
on a hot plate.
2. Place 50-mL Stock Milk Culture into a 125-mL flask or beaker.
3. Warm the cultured milk product to 37°C on the hot plate.
4. Remove milk from heat and note the time.
5. Add o.20-mL Chy-Max or 0.1-mL Emporase enzyme. Swirl to mix.
6. Place in the 37°C hot-water bath for 5 minutes.
7. Record how long it takes for the milk to curdle.
8. Use a balance to determine the mass of 4 cheesecloth layers.
9. Line a funnel or a plastic filter holder with the cheesecloth.
10. Pour the warmed milk with enzyme through the cheesecloth into a 100-mL beaker to separate the
curds from whey. (See Figures 2 and 3, p 144 in Shoestring document attached).
11. Gently squeeze the cheesecloth to remove as much of the whey as possible.
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12. Place the curd on the cheesecloth on 5 layers of heavy paper towel to stand overnight. Label and
cover lightly with plastic wrap. (This should remove much of the moisture in the cheesecloth that
can be attributed to the whey.)
13. Take the pH of your stock solution ad record in your notebook.
14. The next day, mass the curd and cheesecloth. Subtract the mass of the 4 layers of cheesecloth to
determine that mass of the curds.
15. Remove whey from cheesecloth and place in resealable plastic bag.
16. Label bag.
17. Save curd in the refrigerator to age into cheese. Check on the curd periodically over a 3-month time
frame.
DAY 3 ANALYZE AND DISCUSS DATA
SAMPLE DATA ANALYSIS AND DATA INTERPRETATION
Students should measure their milk and cheese product carefully. In order to quantify their data,
students should have a least 3 trials for each experiment.
Milk sample
Starter culture
pH
Curdle time
Curd mass
(skim) mL
(buttermilk) mL
(minutes)
(grams)
125.0
0.0
7.0
>60
0.0
120.0
100.0
6.5
>30
5.0
115.0
110.0
6.0
15
16.3
110.0
115.0
5.7
11
11.9
100.0
120.0
5.5
1
12.5
0.0
125.0
4.0
Did not curdle
0.0
Table 1. Data for effect of pH change of the culture solution on curd formation time.
( Shoestring Biotechnology- page 147 for data table and graph)
Homework Questions
1. Explain the difference between calf chymosin and bacterial recombinant chymosin.
2. Why is buttermilk used as a starter?
3. How does this lab relate to your study of enzymes and substrates?
4. Research a cheese and explain how it is made. (cheddar, swiss, blue, Brie, Gouda etc)
Analysis/Assessment:
1. How does the lab differ from the real production of cheese?
2. What could have caused errors in this experiment?
3. How could your experimental results be used by the biotechnology industry?
Conclusion and Discussion
1. Which variables are important to control?
2. What are your positive and negative controls?
3. What role does biotechnology play in our everyday lives?
4. Have students write a reflection in their lab journals on two things that they liked about the lab and
two things that they learned from the lab.
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DAY 4 STUDENT DESIGNED EXPERIMENTATION
Repeat Protocol
Conclusion and Discussion
1. Which variables are important to control?
2. What are your positive and negative controls?
3. What role does biotechnology play in our everyday lives?
4. Have students write a reflection in their lab journals on two things that they liked about the lab and
two things that they learned from the lab.
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