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Three Fermentation Exercises
Submitted by Dr. Bob Hutkins
I. Background:
Although some microorganisms encountered in nature are responsible for causing disease
of spoilage of foods, there are others that are very useful. In fact, microorganisms are
necessary for the production of many of the foods that consumers enjoy. For example,
yeast are used for the manufacture of bread, wine, and beer; and bacteria are needed to
make yogurt, cheese, pepperoni sausage, and pickles. Even fungi or molds are useful –
they are involved in the manufacture of certain cheeses, soy sauce, and other food
ingredients.
What do these microorganisms actually do in foods? For the most part, their role is rather
simple. They ferment simple sugars (in order to derive energy) and produce metabolic
end products that contribute to the flavor, texture, and overall sensory attributes of that
food. These end-products are usually acids, such as lactic acid, ethanol, or gases such as
carbon dioxide. Acids are made primarily by a group of bacteria known as “lactic acid
bacteria”. Cheese, yogurt, and sausage are examples of fermented foods made by lactic
acid bacteria. Ethanol and carbon dioxide (C02) are made by various means, but are most
often associated with yeast fermentations.
Monitoring the progress of fermentation is relatively easy. When lactose (milk sugar) is
fermented to make yogurt, the acids produced by the culture bacteria cause the milk pH
to decrease from near neutral (about 6.5) to the acid region (4.5). This acidification
causes the milk proteins to coagulate or curdle. In fact, precipitation of milk proteins by
fermentation acids is the principle by which cultured dairy products, such as yogurt and
sour cream, are produced. Acid formation via fermentation can also be measured directly
by use of a pH meter, or indirectly using pH paper. In the case of yeast fermentation, it is
often easier to measure the C02 produced, rather than the ethanol. If a balloon or piece of
plastic tubing is attached to the mouth or opening of the fermentation vessel (i.e., a flask),
the C02 gas can be collected. With the right apparatus, the volume of the evolved gas can
even be measured.
Some fermentations do no involve microorganisms directly. Rather, enzymes produced
by microorganisms are extracted and used to perform specific functions. The best-known
example of such an enzymatic process is the conversion of cornstarch to a product known
as “High Fructose Corn Syrup”. In this process, inexpensive cornstarch, which is a
polymer of glucose and which has essentially no sweetness, is first converted to glucose
syrup by the action of enzymes called amylases. This product (Karo® syrup is an
example) has moderate sweetness. However, the degree of sweetness can be increased
substantially if the glucose is then converted to fructose, a much sweeter sugar. The
conversion of glucose to fructose is achieved by the action of the enzyme, glucose
isomerase. Thus, the glucose concentration decreases, but the sweetness increases, as
fructose is formed. High fructose corn syrup is less expensive than sucrose, which has
led to its widespread use in soda pop, ice cream and many other products.
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II.
Experiment 1……………..Fermentation of sugars by yeasts
A. Purpose: To understand conditions that influence growth and
fermentation of yeast (Saccharomyces cerevisiae).
B. Materials:
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Yeast (Note: “Quick-rise” bakers yeast works better than ordinary
“active dry yeast”)
Sugars, based on availability, but could include glucose (same as
dextrose), fructose (same as fruit sugar), lactose (same as milk
sugar), or sucrose (same as table sugar).
Corn starch
Vinegar
Erlenmeyer flasks, 100 ml or 200 ml volume (or pop bottles)
Balloons (round, 7-10 inch)
Masking tape
C. Procedures:
1. To each flask, add about 1 tablespoon of sugar (about 5 grams) and
80 mL of water.
2. Add about 1 level teaspoon (2.5 grams) of yeast.
3. Mix, then quickly place a balloon on the top and fasten with tape.
4. Incubate the flasks at 40oC (104oF) and visually examine the
expansion of the balloons.
5. Treatments can include:
a. Add 10 ml of vinegar
b. Add 5 grams of cornstarch rather than sugar
c. Incubate at 4oC (use an ice bath) or at 80oC (if an oven is
available).
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III.
Experiment 2…………………How bacteria turn milk into yogurt
A. Purpose: To understand how biological, physical, and chemical activities
are involved in the production of fermented foods.
B. Materials:
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Skim milk (1 gallon will make about
Nonfat Dry Milk (Milk Powder)
One cup of fresh Dannon brand plain yogurt (containing
Streptococcus thermophilus and Lactobacillus bulgaricus).
Plastic cups (8 ounce), with lids
C. Procedures (Note: the first three steps are optional):
1. To 1 gallon of skim milk add about 5 tablespoons (20 grams) of nonfat
dry milk.
2. Mix well and heat or scald this mixture in a large pot to just below a
boil (stirring constantly).
3. Cool to about 45oC (113oC).
4. Add about 4 ounces (100 grams) of fresh Dannon® brand plain yogurt
(about half of an 8 ounce container).
5. Mix well and dispense into clean 8 ounce cups (add about 6 ounces per
cup to yield about 20 containers). Cover each container with a fitted
lid.
6. Incubate in a slightly warm oven (between 40 and 45oC or 105 –
113oF).
7. Remove a container every hour and check the consistency and pH (if a
pH meter is available).
8. When the yogurt has the consistency of thin pudding or when the pH
reaches 4.5 or below, the fermentation is complete.
9. Move the remaining containers to a refrigerator and let cool for about
four hours.
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IV.
Experiment 3…………..Making sweeteners from corn
A. Purpose: To understand how enzymes derived from microorganisms can
be used to convert simple corn sugar into sweeteners used in soda pop.
B. Materials:
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Karo® Syrup
Glucose isomerase enzyme
Magnesium sulfate or magnesium chloride
Glucose test strips
C. Procedures:
1. Transfer 25 grams (or about 25 ml) of Karo syrup into a 100 mL
beaker.
2. Add 25 mL of tap water, and heat to 50-60 C (122 – 140 F), stirring
constantly. Do not overheat.
3. Transfer 1 ml to a 100 mL volumetric flask, and fill with water to 100
ml.
4. Using the glucose test strips, measure the glucose in the diluted
sample.
5. Calculate the actual glucose concentration in the sample by
multiplying the value by 100.
6. To the remaining Karo® Syrup-water mixture, add 5 mg magnesium
sulfate or magnesium chloride, ¼ teaspoon (1 gram) of glucose
isomerase enzyme, and continue stirring.
7. At 20-minute intervals, for up to 60 minutes, repeat steps 3-5.