Microbiology Laboratory Activity: Cheese Making

Name:Minguez, Matthew Dominic Date: June 19, 2021

Group No: 2

Dairy Fermentation

Milk is an excellent food source for humans and bacteria alike. It is full of vitamins, fats,
minerals, nutrients and carbohydrates. It is rich in the protein case in which gives milk its
characteristic white color. The most abundant carbohydrate is the disaccharide lactose, “milk
sugar.” At room temperature, milk undergoes natural souring caused by lactic acid produced
from fermentation of lactose by fermentative lactic acid bacteria. This accumulation of acid (H+
ions) decreases the pH of the milk and cause the casein to coagulate and curdle into curds and
whey. Curds are large, white clumps of casein and other proteins. Whey is the yellow liquid that
is left behind after the casein has formed curds. Thus, bacteria obtain nutrients from the milk,
inadvertently curdle it and humans use it as the first step in making many dairy products.

The microbes important for dairy product manufacturing can be divided into two groups,
primary and secondary microflora. Products undergoing fermentation by only primary
microflora are called unripened and those processed by both primary and secondary microflora
are called ripened. Primary microflora are fermentative lactic acid bacteria which cause the milk
to curdle. During dairy product production, milk is first pasteurized to kill bacteria that cause
unwanted spoilage of the milk and of the downstream milk products. Primary microflora
consists of certain kinds of Lactococcus, Lactobacillus and Streptococcus that are intentionally
added to pasteurized milk and grown at 30°C or 37°C (temperature depends on the bacteria
added). Secondary microflora includes several different types of bacteria (Leuconstoc,
Lactobacillus, and Propionibacterium), yeasts and molds; they are only used for some types of
surface ripened and mold ripened cheeses. The various combinations of microflora determine
what milk product you will end up with.

Different unripened milk products are created by using various starting products and bacteria.
For buttermilk production, Lactobacillus bulgaris (named for its country of discovery, Bulgaria)
is added to skim milk to curdle it. Leuconostoc is then added to thicken it. Sour cream is made
the same way except cream is used instead of skim milk. During yogurt production, dry milk
protein is added to milk to concentrate the milk before addition of actively
growing Streptococci and Lactobacilli. Butter is produced by curdling and slight souring
from Streptococci growing in sweet cream. Leuconostoc is then added so it can synthesize
diacetyl, a compound that gives butter its characteristic aroma and taste. The milk is then
churned to aggregate the fat globules into solid butter.
Thus milk type and bacteria will determine the dairy product produced.

Cheese is an important product of fermentative lactic acid bacteria. Particularly in the past,
cheese was valued for its long shelf life. Due to its reduced water content, and acidic pH,
bacterial growth is severely inhibited. This causes cheese to spoil much more slowly than other

1
milk products. Consequently, the art of cheese production has spread throughout Europe, each
country manufacturing many different types of cheeses. Cheese production has three steps:
curd formation, curd treatment and curd ripening.

1. Curd formation can use mare, ewe, cow or goat milk to produce “sour” or “sweet” curd. Sour
curd is produced by fermentative lactic acid bacteria as mentioned above. Sweet curd is
produced by adding an enzyme called renin instead of bacteria to curdle the milk. The curd is
separated from the whey by draining. The curd can be used directly to make unripened cheeses
such as ricotta or cottage cheese or can undergo further processing to make a ripened cheese.

2. Curd treatment consists of condensing and squeezing to form dense, hard curd. It is then
molded into the desired shape, salted and mixed with different types of secondary microflora.

3. Secondary microflora ripen the cheese and will determine the final texture and aroma of
each type of cheese. For hard ripened cheeses such as Cheddar, curds are further compressed
and the bacteria particular for the cheese is added. The Cheddar is wrapped in wax or plastic to
prevent contamination and then incubated to allow the bacteria to do its work. For soft ripened
cheeses such as Camembert and Limburger, a microbe, usually mold, is added to the surface of
the cheese that produces a protein-digesting enzyme. This enzyme breaks apart the curds and
causes the cheese to become creamy and spreadable.

Many cities have long held traditions and nuances for producing a particular cheese i.e. the
limestone caves in Roquefort, France which have constant heat and humidity that create
unique and delightful cheeses.

Industrial processes using fermentation

Fermentation by bacteria, yeast and mold is key to the production of fermented foods.
Fermenting yeast produces the alcohol in beer and wine. In fact, the smell of fresh baked bread
and rising dough can be attributed to alcohol produced from yeast. Fermentation is used to
make many ethnic foods such as sauerkraut and miso. Soy sauce is produced by fermenting
Aspergillus ortzae, a fungus, growing on soy beans. Erwinia dissolvens, another type of bacteria,
is essential for coffee bean production; it is used to soften and remove the outer husk of beans.
Finally, fermentation of milk produces most dairy products. Without microbes, we would not be
able to eat many types of different food that we enjoy today. Table 1 shows example of several
foods that are produced through fermentation with specific organisms.

2
The Biochemical Process

All organisms need energy to grow. This energy comes from the reduction of adenosine
triphosphate (ATP) into adenosine diphosphate (ADP) and results in the release of energy and a
phosphate group. In this way ATP serves as a storage molecule of energy which can be used by
the cell. But where does the ATP come from? Cells get their ATP from the controlled chemical
breakdown of glucose to form two molecules of pyruvate. This process requires two molecules
of ATP but results in the release of four molecules or a net gain of two molecules of ATP. This
process is referred to as glycolysis and is illustrated in Figure 1. Once pyruvate is formed, it can
be processed in several different ways. Mammalian cells usually process pyruvate by putting it
into the tricarboxylic or Kreb’s cycle. In the presence of oxygen, oxidative phosphorylation
produces more ATP from the byproducts of the Kreb’s cycle reactions. This is referred to as
aerobic respiration. However, when oxygen is limiting, other processes must be used in order to
deal with pyruvate. This is done through anaerobic respiration or fermentation and involves the
breakdown of pyruvate into simpler compounds. Two of the most important fermentation
processes which are used on an industrial scale are ethanol or lactic acid fermentation. This is
illustrated in Figure 1.

3
Non-Rennet Cheese (White Cheese)

Recipe adapted from Hill, A.R. (2006)

Heat-acid or no-rennet Queso Blanco is a white, semi-hard cheese made without culture or
rennet. It is eaten fresh and may be flavored with peppers, herbs and spices. It is considered a
"frying cheese" meaning it does not melt and may be deep fried or grilled. Queso Blanco can
also be made by renneting whole milk with little or no bacterial culture.

Ingredients:

 4.6 fl. oz. (137 ml) vinegar (5% acetic acid)

 9.2 fl oz. (274 ml) distilled water
 1.0 gallon (3.76 L; 8 lb; 3.63 kg) heat-treated or pasteurized milk
 1 teaspoon (4.6 g) salt
 Spices to taste

4
Procedure:

1. Heat milk to 176°F (80°C) for 20 minutes.

2. Add vinegar (5% acetic acid) to water and then add slowly to the hot milk until the whey
is semi-clear and the curd particles begin to mat together and become slightly stretchy.
You should be able to stretch a piece of curd about 0.39 inches (1 cm) before it breaks.
It may not be necessary to add all of the vinegar.
3. Separate the curd by filtering through a cloth bag until free whey is removed.
4. Work in salt and spices to taste.
5. Press the curd (high pressure is not required).
6. Package curd in boilable bags (vacuum package if possible) and place in boiling water for
5 minutes to sterilize the surface and prevent mould growth.
7. Queso Blanco may keep for several weeks if properly packed and stored in a refrigerator,
but should be eaten in as fresh a state as possible.

Cheese Made with Rennet (Neufchatel)

Recipe adapted from C. McDaniel and P. Kendall (2004)

Neufchatel (New-sha-TEL) is a milky-white cheese with a soft, smooth texture and is low in fat if
made with reduced-fat or fat-free milk.

Ingredients:

 1 gallon (3.79 L; 8.6 lb; 3.9 kg) pasteurized milk (any level of fat)
 One tablet rennet or 1 tsp liquid rennet dissolved in 1/2 cup tap water
 1/2 cup (4 fl oz; 114 g) fresh cultured unsalted buttermilk or 1/4 cup (2 fl oz; 57 g) fresh
plain yogurt for culture
 3 teaspoons (14 g) salt (optional)

Procedure:

1. Put 1 gallon of milk into the upper part of a double boiler. Add enough water in the
bottom of double boiler to prevent milk from scorching. Stir in buttermilk or yogurt and
warm slowly to 92 to 94°F (33.33 to 34.4°C). Maintain this temperature range through
Steps 2 and 3.
2. Add rennet mixture and stir into milk for 2 to 3 minutes. Allow milk to set undisturbed
for about 30 minutes or until a firm gel forms. To test for curd formation, cut a slit in the
curd with a metal spatula, slip under the curd and lift slightly. If the cut in the curd
breaks clean, it is ready for Step 3.
3. Cut the curd into approximately 1-inch cubes. Stir gently and continuously for 20 to 30
minutes to help firm curds. Keep the temperature range 90 to 94°F (32.22 to 34.4°C).
4. Pour off whey (yellow liquid). Allow the curds to settle, and dip out the remaining whey.
5. Add 1 teaspoon salt, mix gently. Wait 5 minutes and mix in the second teaspoon salt.
Wait 5 more minutes and mix in the last teaspoon salt.

5
6. Divide the curds into two batches and proceed as follows for each batch.
7. Line two coffee cans with clean cheesecloth or muslin. Place half of the cheese curd
inside each lined can. Fold the cloth over the top and add the follower.
8. Apply pressure by pressing with a weight, such as a number 303 can of food, until the
surface is smooth (2 to 4 hours). Do this in the sink to allow the whey to drain out of the
cans.
9. Remove the formed cheese and the cloth. Wrap the cheese tightly in plastic or in waxed
paper and store in refrigerator. It will keep for seven to ten days under refrigeration. It
can be frozen for four to six months. However, freezing lowers the quality of the cheese.

6
Non-Rennet Cheese (White Cheese) Production

7
Answer the following questions:

1. Describe the role of microorganisms in cheese making process.

- Cheese contains a large amount of microorganism such as bacteria, yeast,

and filamentous fungi (molds), which are one of the few essential ingredients
in cheese making. The cheese making process makes condition to overcome
milk protein to repulse which is responsible for keeping the milk in liquid
form, this conversion process makes the milk more acidic, thus needing a
lowering of pH by means of fermentation, the proteins and sugars within the
milk is broken down to make it into simpler product which can be stored for
longer period than the original substance, this is called microbial
fermentation, changing substance while relying on the microbes metabolism.
Microbial fermentation preserves the cheese thus making it last longer than
of fresh cheese which should be consumed immediately after it is
manufactured, whereas aged cheese lasts for 12 months or more.

Source: (American Society for Microbiology, 2015)

2. What conditions affect fermentation?

1) Temperature
 Temperature plays a critical role in fermentation. Yeast needs to be
warm enough to be healthy, but too warm will stress the yeast. Too
cool and the yeast will be sluggish and sleepy. As temperature
increases, fermentation rate accelerates.
2) Pressure
 Pressure also helps suppress unwanted esters caused by higher
fermentation temperatures. By fermenting under pressure at high
temperature, you can make very clean lagers in a fraction of the time.
it reduces esters and the formation of certain undesirable compounds.
3) pH
 Most of the biological components of a fermenting substance are
microorganisms, mainly yeasts and molds, which are in turn made up
of many smaller structural and metabolic proteins (the latter falling
into the more specific protein category of the enzymes). These
organisms evolved in acidic conditions; therefore, their proteins have
evolved to conform to their optimal shapes and functions in slightly
acidic conditions. Moreover, a slightly acidic pH deters the formation
of competing bacterial colonies, helping fermentation to proceed

8
 unobstructed. However, as environmental pH increases into
moderate and high acidity (generally in the range of pH 6.0 and
higher), proteins start to denature. There are a few exceptions to this
pattern, usually where a low (basic) pH generates essential
ingredients for a specific fermentation process.

Source: (AUEssays, 2018) & (Wright, 2005)

3. Does yeast die during fermentation?

- Yes, yeast die during fermentation. It is well known that during fermentation
the end product of this process is Alcohol. Alcohol in the liquid is great for
making a beverage but not for the yeast cells. Yeast will typically die once the
tolerance level for alcohol is reached. In addition, Brewer’s yeast for example
has 5% greater tolerance rate for alcohol when relatively compared to other
kinds of yeast. Once the alcohol level is beyond of what can be tolerated the
yeast cannot continue fermentation. To further elaborate, the enzymes
involve in the conversion of sugar to CO2 and Alcohol is killed by alcohol at
high concentrations of alcohol. The death or denaturation of the enzyme
leads to the death of the yeast.

Source: (FAO, n.d.) & (YoBrew, 2006)

9
References
American Society for Microbiology. (2015). FAQ: Microbes Make the Cheese: Report on an
American Academy of Microbiology Colloquium held in Washington, DC, in June 2014.
Washington (DC). NCBI. https://doi.org/doi: 10.1128/AAMCol.June.2014
AUEssays. (2018). Rate of Fermentation. AUEssays.
https://www.auessays.com/essays/biology/the-rate-and-process-of-fermentation-biology-
essay.php#citethis
FAO. (n.d.). Fermented and vegetables. Food and Agriculture Organization.
http://www.fao.org/3/x0560e/x0560e08.htm
Wright, R. (2005). MASS SPECTROMETRY | Gas Analysis (P. Worsfold, A. Townshend, & C. B. T.-E.
of A. S. (Second E. Poole (eds.); pp. 493–501). Elsevier.
https://doi.org/https://doi.org/10.1016/B0-12-369397-7/00363-0
YoBrew. (2006). Alcoholic fermentation, of sugar into CO2 and alcohol (p. 1).
https://www.yobrew.co.uk/fermentation.php

10
Appendices
images are in no particular order

11