MICRB 202: Introductory Microbiology Lab
Unit VI: Food and Beverage Microbiology
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Unit VI: Food and Beverage Microbiology
Activities:
12.1 Yogurt - Preserving Milk (Ex 18)
12.2 Wine (or Vinegar) (Ex 19)
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Introduction:
It is uncertain when humans began to intentionally preserve foods or make alcoholic beverages
by microbial fermentation reactions. Biochemically, fermentation is the metabolic process of yielding
energy from the catabolism of organic compounds, like glucose, in the absence of oxygen and using an
endogenous carbon source as an electron acceptor. Science only began to understand the role of
microbes in these processes about 150 years ago. However, the utility of fermentation in food
processing very likely pre-dates history.
Ancient historical documents suggest that various fermentation processes were “domesticated”
prior to 2000 BC. Hebrew text tells of Noah taking beer onto the Ark and vinegar used to guard
drinking water from contamination or relieve the physical suffering of crucifixion. Specific fermented
milk and vegetable products have been unique to numerous cultures throughout the world for
centuries. Both yeasts and bacteria are used to produce different products. Today, attention is even
placed at the strain level for some fermented products. Many of the commercial products we consume
today come from these deep cultural roots: beer, wine, vinegar, cheese, yogurt, buttermilk, pickles and
sauerkraut
12.1 Yogurt – Preserving Milk: (Ex 18)
Yogurt is a fermented food which is prepared by growing certain lactic acid bacteria in milk. It
is believed that this fermented form of milk which has been consumed for centuries by peoples of the
Near East (Balkans) was a means of preserving milk for nomadic people. In the United States this
slightly acidic dairy product is prepared commercially from bovine (cow) milk and serves as an
excellent source of protein for many active and health conscious individuals.
Yogurt is produced by inoculating boiled milk with two lactic acid producing bacteria (Figure
1). Streptococcus thermophilis and Lactobacillus bulgaricus are inoculated into boiled milk and the
milk is held at 46º to 48º C for a few hours. At this higher temperature, the thermophilic (heat loving)
bacterium, S. thermophilis converts the milk sugar, lactose, to lactic acid. As this occurs, the lactic
acid causes the milk protein, casein, to coagulate. After the initial incubation at the higher temperature
the product is refrigerated and begins to cool. As the temperature drops L. bulgaricus (a mesophile)
ferments the remaining lactose in the milk to lactic acid as well as minor organic compounds. These
minor fermentation products give yogurt its characteristic flavor and aroma. The lactic acid produced
by these organisms will prevent growth of other types of microorganisms and consequently, prevent
the spoilage of the milk.
MICRB 202: Introductory Microbiology Lab
Unit VI: Food and Beverage Microbiology
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Figure 1: The diversity of fermentation products and intermediates made by various groups if yeasts
and bacteria.
The fermentation of milk is one of the oldest methods of food preservation. Before the days of
refrigerators individuals used fermented milk to increase the shelf life of fresh milk. The acid
produced by fermentation prevented the growth of food spoilage and pathogenic (disease-causing)
microorganisms in milk and also resulted in a product pleasing to many consumers. This exercise
demonstrates a simple method for preparing yogurt, the use of lactic acid bacteria for food
preservation, and the gram stain and morphology of these “yogurt bugs”.
EXERCISE 18:
Making Yogurt:
Materials:
Cultured commercial yogurt without preservatives or other additives.
Commercially prepared whole milk, 250 ml
500 ml beaker sterile
100 mL beaker sterile
Incubator set at 46º to 48º C
pH paper
MICRB 202: Introductory Microbiology Lab
Unit VI: Food and Beverage Microbiology
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Procedure:
1. Preparation of whole milk
a. Scald about 250 ml each of whole and skim milk by heating it just to boiling (do not let it
boil). This procedure kills almost all of the unwanted bacteria (acid production by the
lactic acid bacteria will inhibit the rest). (this may be done for you in advance of class)
b. Cool the milk to 46º to 48º C. Check this with a thermometer. If the temperature is
higher before inoculation the mesophilic L. bulgaricus will die. Check the pH of the
milk before inoculating with the starter culture, and record.
2. Inoculation of milk:
a. Add two teaspoons of commercially prepared yogurt into to a sterile 100 mL beaker. This
commercially prepared yogurt is known as the starter culture and will contain
the organisms necessary to ferment the milk. Add cooled milk to about 50 mL.
b. Thoroughly mix the starter culture and milk in the 100 mL beaker. Thorough mixing is very
important because the starter culture is a gel and is difficult to mix with the milk. When
you have homogenized the mixture add it back into the rest of the milk – inoculation!
3. Incubation and storage:
a. Evenly divide the inoculated milk into two cups or flasks
b. Cover the container with saran wrap and aluminum foil.
c. Incubate this mixture at 46º to 48º C for 3 to 6 hours. Your instructor will check the
progress of the fermentation. This step provides conditions suitable for the
thermophile (S. thermophilis) to ferment the lactose.
d. The container is then removed from the incubator and placed in the refrigerator. It will
take about 2 hours for the temperature to drop from 46º to 10º C. During this time
the mesophile (L. bulgaricus) will begin to grow and ferment the remaining lactose
primarily adding flavor-producing compounds to the yogurt and more lactic acid.
The yogurt will become firmer.
NOTE: Run your own experiment with half of your yogurt batch. Think about how changing
the temperature, or duration of incubation at a particular temperature, may change the outcome
of product. Test this hypothesis and compare it to the flask with the normal protocol.
e. The yogurt will be stored in the refrigerator until the next laboratory period. At this time
no growth of either organism occurs and there should be no further change in the
product.
<<NEXT PERIOD>>
4. Examination of the commercially prepared yogurt:
a. Gram stain the culture. Use a sterilized loop to prepare a heat-fixed smear of the culture.
Gram stain the preparation and examine. You should look for two distinctly
different types of bacteria. Sketch your observations.
b. Determine the pH of the yogurt by sterilizing a loop and using it to transfer a loopful of
yogurt to a piece of pH paper. Examine the opposite side of the paper to determine
the color change of the pH paper.
c. Record the color, aroma, texture and taste along with your pH data.
5. Examination of your yogurt (s):
MICRB 202: Introductory Microbiology Lab
Unit VI: Food and Beverage Microbiology
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a. Gram stain your cultures. Use a sterilized loop to prepare a heat-fixed smear of the culture.
Gram stain the preparation and examine. You should look for two distinctly
different types of bacteria.
b. Determine the pH of the yogurt by sterilizing a loop and using it to transfer a loopful of
yogurt to a piece of pH paper. Examine the opposite side of the paper to determine
the color change of the pH paper.
c. Record the color, aroma, texture and taste along with your pH data.
6. Present all your results in a single well organized table.
7. Was your normal protocol yogurt similar to the commercial product based on the observations?
If not, then offer an explanation. What about your experimental treatment? Did you get the
expected result?
8. Discuss the nutritional value of yogurt as compared to milk and describe the fermentation
process involved in yogurt production.
12.2 Wine (or Vinegar): (Ex 19)
Enology is the “science” of wine production, which involves the fermentation of sugars in fruit
juice, or must, to ethyl alcohol by members of the yeast genera Saccharomyces. The process can be
quite involved (Figure 2), but the only requirements are simple sugar, yeast and anaerobic conditions.
Wines can vary a great deal depending on the grapes, their skins, yeast strain, temperature,
sugar content, storage containers, and aging. The two common forms, red and white wines, differ in
the grape type and processing. White wines can be made from skinless grapes of any color. Red and
other shades of wine get there color from pigments in the grape skin, which is left on during the initial
processing and fermentation. Red wines are fermented at warmer temperatures (24ºC) than white
wines (13ºC). Whether a wine is dry or fruity (sweet) depends on the initial amount of sugar and
completeness of fermentation. As fermentation proceeds, acidity and alcohol content increase, both of
which can limit the growth of yeast due to toxic effects. Therefore, tolerance to acidity and alcohol
content are important characteristics that need consideration in choosing the right yeast strain for a
particular type of wine. Duration of primary and secondary fermentation can influence the production
of minor fermentation byproducts, such as volatile aromatics that can add to a wines bouquet. The
particular material of the storage containers used to age the wine and the duration of aging can further
influence flavor. For example, oak casks will leach wood tannins into the wine, adding an acidic bite
to the wine. The final step in wine production is racking, or the bottling of the wine.
One of the critical safe-guards against spoilage used by most wineries today is pasteurization of
the must prior to fermentation. Pasteurization is basically the heating of the must to about 70ºC for
several minutes to kill all spoilage bacteria. Louis Pasteur (1856) invented this procedure, which is
named after him, in order to save the wine industry of France, which was plagued by spoilage during
long storage on naval and merchant ships. Pasteur identified a yeast, Saccharomyces cerevisia, as
responsible for the desired fermentation produce alcohol and a bacterium as the spoilage agent, which
soured the wine to vinegar. Pasteurization of must was shown to kill the spoilage; if it’s performed
MICRB 202: Introductory Microbiology Lab
Unit VI: Food and Beverage Microbiology
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prior to inoculating the must with a pure yeast culture then the wine would never spoil. This work
also gives credit to Pasteur as the first scientist to realize that microbial metabolism can cause an
alteration in their chemical environment.
Figure 2. The major steps in wine production (Prescott et al, 2001; Fig 41.16).
Saccharomyces cerivaciae is a facultative anaerobic yeast that can ferment sugar to ethanol with the
production of copious amounts of carbon dioxide. Allowing the yeast to ferment sugars after bottling
will result in a carbonated, effervescent wine or Champaign. If conditions are not kept anaerobic the
wine could turn sour, i.e. vinegar.
Aerobic conditions will permit growth of acetic acid bacteria, which further oxidize ethanol to acetic
acid (Figure 1) and are responsible for the tart sour bite vinegar’s taste. Aerobic conditions are
performed intentionally and under controlled conditions during industrial manufacturing of vinegar.
Some type of ethanol containing solution is aerated or exposed to a high surface area substrate that will
promote the growth of the aerobic acetic acids bacteria. Two genera of acetic acid bacteria are
commonly used in commercial vinegar production today. Gluconobacter is well suited for vinegar
production because it does not have a complete Krebs Cycle, so acetic acid accumulates. The other
bacterium, Acetobacter, will further oxidize acetic acid to carbon dioxide and water under aerobic
conditions. We will attempt to make both good and sour wine to demonstrate the fermentation process
and ethanol oxidation.
MICRB 202: Introductory Microbiology Lab
Unit VI: Food and Beverage Microbiology
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EXERCISE 19:
Making Wine or Vinegar:
Materials:
must (grape juice from concentrate with sugar and yeast nutrients added)
Saccharomyces cerevisiae var. ellipsoideus
2 fermentation flasks
2 glass Petri dish lids (as saucers for fermentation flasks)
Balloons
pH test paper
Plastic spoon (for tasting)
Graduated tube
Fill line
Figure 3. Fermentation flask for measuring rates of gaseous products. Note the tube on the left is
graduated to measure the volume of gas produced over time.
MICRB 202: Introductory Microbiology Lab
Unit VI: Food and Beverage Microbiology
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Procedure:
1) Label fermentation flasks at their base as follows:
Y = yeast
Y+O2 = yeast with oxygen exposure
<< Use aseptic technique at all times. >>
2) Fill each fermentation flask with 15 or 30 ml of pasteurized must, depending on your flask size
(Figure 3). You will need to tilt the flask as demonstrated so to fill the gas-tube without spilling.
3) Inoculate must with the instructed volume of yeast culture.
4) To the “Y” flask, carefully attach a balloon to the flask opening to assure anaerobic conditions. The
“Y+O2” flasks will be covered with foil to allow oxygen to pass into the flask. Volunteers can help set
up some non-inoculated flasks with and without balloons to run as a class controls.
5) Use some of the extra pasteurized must to characterize the juice at the start of your experiment.
Record pH, color, aroma and taste (it’s just grape juice!).
6) Every couple days over the next week, measure the fermentation rates. You need to do this at least
five times over the next nine days. I strongly advise the first couple readings be performed on the
second day (early morning and afternoon). Note: once the microbes start to grow exponentially, the gas
production can be extremely rapid. You’ll see bubbles. Here is how to make a rate measurement:
* Tilt the flask to fill the graduation tube (Figure 3).
* Note the time and date.
* After 30 to 60 minutes note the time and record the volume of accumulated gas.
* Divide gas volume by your assay duration (stop minus start time) for a rate in units mL/h.
* Perform this for all treatments.
<< Next Week>>
7) Characterize the product from each flask at the end of your incubation period. Pipette sample
(without disturbing the settled yeast) into a beaker. Record pH, color, and aroma.
8) If Bromocresol Green plates WITH EtOH and those WITHOUT EtOH are available, isolate growth
from each flask of acetic acid bacteria by simply streaking a culture moistened swab once across the
plate. This way you can fit multiple treatments on the same plate. Make sure your plates are well
labeled and that you take not of the arrangement of your inoculum. The pH indicator, bromocresol
green, will be blue-green at pH > 5.4 and yellow at pH < 3.8. Yeast will stop fermenting when pH
drops below about 4.5 and/or when ethanol content reaches > 20%. Incubate at room temperature.
(NOTE: Gluconobacter turns yellow; Acetobacter turns yellow then back to green).
9) Time permitting, view a wet mount of your experimental treatments under the compound
microscope (100x oil objective); sketch and note observations. Can you distinguish yeast from
bacteria? Make a sketch of any yeast and bacteria.
MICRB 202: Introductory Microbiology Lab
Unit VI: Food and Beverage Microbiology
10) Compile class data, and plot rate of gas production versus days of incubation for each treatment.
Add a proper figure legend to the bottom of the plot.
11) Discuss:
When were the fastest rates of gas production, and for which cultures?
Did pH or other characteristics differ between un-inoculated must and other treatments?
Did the un-inoculated control differ from at the start of incubation?
Which flasks became most acidic?
Did any smell like wine or vinegar?
What might need to be done to improve either wine or vinegar production?
How do any of the microscopy or plating results support your conclusions?
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