Download Lab: Cheese Making

Unit I: Introduction to Biotechnology and Laboratory Procedures
Biotechnology I/Zeiher
Lab: Cheese Making
Lab: Modified from Daughtery, E. 2012. Biotechnology Laboratory Manuel. Paradigm
Publishing. pp. 8-11 and Brown, Judy. 2002 “Little Miss Muffet” in Shoestring Biotech. National
Association of Biology Teachers. pp. 139-152.
Introduction:
The cheese-making industry is huge and is a good example of how biotechnology has
improved an industrial process. Have you ever let milk stay too long in the refrigerator or on the
kitchen counter? You will notice that the milk will begin to form clumps. This happens because
the naturally occurring bacteria in the milk is causing the milk to turn sour. This was the original
way that cheese was made
This clumping occurs because milk bacteria have enzymes (protein that speed chemical
reactions) that convert the milk sugar lactose to the product lactic acid. Lactic acid has a low
acidic pH. This acidic pH causes the milk protein casein, to denature or break down and fall out
of solution. Other enzymes, called proteases, may also act on casein. Proteases act as scissors
cutting proteins, such as casein, into smaller fragments that will also fall out of solution. The
lumps of denatured casein are called curds and are the clumps of milk observed when the milk
sours.
Curds, these solid clumps, are pressed together to form cheese. The liquid remaining after
curdling is called whey as was mentioned in the nursery rhyme “Little Miss Muffet”
Little Miss Muffet sat on her tuffett eating her curds and whey”
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.
1.
Aging Method: The milk may simply naturally sour by exposing to air and naturally
occurring bacteria. The curds that form are pressed and used to form cheese.
2.
Bacterial-culture Method: New batches of cheese are started with specific cultures of
Lactobacillus bacteria, bacteria known to make milk curdle found in, for example,
buttermilk and yogurt.
Unit I: Introduction to Biotechnology and Laboratory Procedures
Biotechnology I/Zeiher
3.
Calf Stomach Enzyme Method: One of the earliest methods for making cheese was
accidently found when ancient people used animal stomachs as a canteen to store
milk. They discovered that the milk stored in these stomachs became clumpy. It is now
known that cells that line animal stomachs contain enzymes, specifically proteases. It
was subsequently found that if we add a purified form of a protease, known as rennin,
that it can stimulate the formation of curds by breaking down casein into small
fragments. Rennin is isolated from the cells that line calves’ stomach. To retrieve the
enzyme for commercial use, companies grind up calves’ stomach and separate and
purify the rennin enzyme from other macromolecules (proteins, carbohydrates, nucleic
acids). Because of the animal source of rennin, some vegetarians and vegans do not eat
rennin cheeses (e.g. cheddar cheese, asiago cheese, blue cheese).
4.
Recombinant Enzyme Method: Scientists isolated the gene that encodes for rennin from
calves’ cells and inserted into a plasmid. The recombinant DNA molecule (Calf DNA +
Fungal DNA) was inserted into yeast (a fungus) cells. The yeast cells produce rennin in
larger amounts and faster than normally occurs in the calf’s stomach. Cheese makers
use the genetically engineered enzyme known as chymosin to speed curdling process.
chymosin cheeses include Jack, mozzarella, and most Swiss cheeses.
Biological Question:
Which curdling agent produces cheese at the fastest rate?
Pre-Lab- In your lab notebook
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Write biological question
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Write a hypothesis as an If……. Then statement
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Answer Pre-Lab questions
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Summarize procedures in your own words (IYOW)
Pre-Lab Questions
1. Explain the difference between curds and whey.
2. Why do the solids form in milk when cheese is made? In your answer describe the
science behind the clumping.
3. List the four methods used to make cheese and briefly explain each method
4. What is the difference between rennin and chymosin?
Supplies Needed per lab station
Unit I: Introduction to Biotechnology and Laboratory Procedures
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1 thermometer
1 hot plate
1 100 mL graduated cylinder
3 150 ml flask or beaker
1 clock or timer
1 1-mL pipette
1 bulb or pump
1 stirring rod
1 funnel
3 100-ml beaker
1 balance
1 permanent marker, such as a Sharpie or laundry marker
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150-mL cultured milk
0.2-mL Chy-Max (0.1% w/v)
0.2 mL Rennin (0.1% w/v)
4 15 x 15-cm cheesecloth squares
1 weigh boat
5 highly absorbent paper towels
plastic wrap
Biotechnology I/Zeiher
Materials:
Procedure
1.
2.
3.
4.
5.
6.
Each group will be doing the following treatments: buttermilk, rennin, chymosin, and
scalded whole milk (negative control).
Using a 10-mL pipet and pipet pump, transfer exactly 7 mL of scalded whole milk into a
labeled 15 mL conical tube. (label needs to have the treatment name, your initials, the
date, the period).
Using a pre-set P1000, add 250 µL (0.25 mL) of the curdling agent.
Cap the tube and mix gently by inverting three times. Record the "initial time".
Place the milk-containing portion of the tube in 37oC water for at least 15 minutes.
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, place on the lab bench and let
Unit I: Introduction to Biotechnology and Laboratory Procedures
Biotechnology I/Zeiher
it incubate overnight (at room temperature) and check 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 described below:
Pour the whey and curds through cheese cloth in a funnel into a 10-mL graduated cylinder.
Determine the volume of when collected in the graduated cylinder. By subtraction, determine
the volume of curds as described in calculations section.
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 Curdling Volume of Whey Volume of Curds
(min)
(mL)
(mL)
buttermilk
rennin
chymosin
Distilled water
Calculations
Volume of Curds (mL) = Total Volume of starter milk culture – Volume of Whey (mL)
Data Analysis and Results
1. Draw two graphs:
a. Average time to curdling for each treatment.
b. Average volume of curds produce by each treatment.
2. How well did the single trial your group did support the hypothesis? Explain.
Unit I: Introduction to Biotechnology and Laboratory Procedures
Biotechnology I/Zeiher
3. Averaged data are the best way to answer an experimental question. Explain why.
4. Does it appear that the number of trials for each curdling agent 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.
Extension Activity
Scenario
Assume you are a member of a team of biotechnicians from a cheese making company. The
president of the company offers a significant bonus to the team that designs the most costeffective protocol to increase the yield of cheese. You and the members of your team decide to
accept the challenge. How will your team organize themselves to accept this challenge? Your
task is to develop a protocol that will increase cheese-yield based on what you learned from the
previous experiment and then test that protocol to determine if your ideas are correct.
Review the cheese-making protocol you previously did, then choose a variable that you could
modify that might enhance curd formation.
Brainstorm what variables you could vary
In developing your protocol (Complete this in your Lab Notebook)
1)
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6)
Define your biological question
Based on your biological question, develop a hypothesis
Provide your experimental design – Experimental Group, Control Group, Fixed Variables
List the materials required
Outline the procedure you will use
After this is completed get the instructors initials.
After completion of experiment
1)
2)
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4)
Organize your data in well-constructed table
Show patterns observed in the data graphically
Summarize the patterns observed in the graph but do not interpret the results
Draw conclusions
Unit I: Introduction to Biotechnology and Laboratory Procedures
Biotechnology I/Zeiher
a.
b.
c.
d.
e.
State claims
Use evidence from data to support your conclusions
Reject/Accept hypothesis
Describe possible errors and its effect on results
Make a recommendation to the cheese company supervisor about which
variable should be considered to improve production.
f. State any new questions you might ask based on the conclusions drawn from this
experiment
Technical Standards Lab addresses:
3.0 Demonstrate Critical Thinking and Scientific Problem-Solving Skills
3.1 Identify and use industry-recognized observational methods and skills.
3.2 Identify and structure tractable, easily managed and controlled questions showing evidence of
observation and connection to prior knowledge.
3.3 Develop and test hypotheses utilizing experimental design distinguishing between controls and variables
and use experimental, analytical, and statistical design.
8.0 Demonstrate Basic Lab Skills in the Use of Equipment and Instrumentation
8.1 Use software/hardware for scientific analyses and documentation (e.g. Excel, PowerPoint, Word).
8.2 Identify and demonstrate proper use of laboratory glassware.
8.3 Identify and demonstrate proper use of laboratory balances.
8.4 Identify and demonstrate proper use of micropipettes.
Unit I: Introduction to Biotechnology and Laboratory Procedures
Biotechnology I/Zeiher
Teacher Notes
Chemical Preparation
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 100ml storage bottle. The liquid product contains approximately 0.1% active chymosin. E mporase
comes ready to use. Follow the manufacturer’s recommendation for refrigeration.
Chymosin recombinant rennin can be ordered from Sargent Welch cat# WLB1030
Preparation of scalded milk
Heat whole milk to 82oC and leave at that temperature for 5 min. Store in sterilized bottle.