Microbiology Laboratory Activity

Cheese Making

Group 3
Rioja, Andrea Lou BSChem – 3
Sinco, Ariane Rose BSBio – 2
Tamondez, Rejie BSBio – 2
Torrento, Ruzzel BSBio – 2
Villafuerte, Caren Grace BSChem – 3
Villoso, Kiarrah Nicolle BSChem – 3
Date: June 23, 2021

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 casein 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 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.
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.
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

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.
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.

Figure 1. Non-Rennet Cheese (White Cheese)

The non-rennet cheese shown on Figure 1 was easily and quickly made with very simple
ingredients. It has similarities to cottage cheese, ricotta and mozzarella. The home-made cheese tastes
quite the same to mozzarella cheese.
1. Describe the role of microorganisms in cheese making process.

The primary purpose or role of microorganisms in cheese making is induce the fermentation
process, to convert lactose in milk into lactic acid. Starter cultures are used early in the cheese making
process to assist with coagulation by lowering the pH. The metabolism of the starter cultures
contributes desirable flavor compounds, and help prevent the growth of spoilage organisms and
pathogens. The starter culture changes the cheese microenvironment, affecting a variety of factors,
including pH, redox potential, levels of organic acids such as lactate and acetate, and other nutrients.
As some of the lactic acid bacteria (LAB) within the starter culture begin to die, the cells release enzymes
that further break down milk proteins, mainly casein, to small peptides and amino acids. Adjunct
cultures are used to provide or enhance the characteristic flavors, textures, and often giving cheeses a
pleasant sweet flavor and promoting the growth of tyrosine crystals. Yeasts and molds are used in some
cheeses to provide the characteristic colors and flavors of some cheese varieties.

2. What conditions affect fermentation?

The growth of yeast is supported by the existence of basic compounds such as fermentable sugars,
amino acids, vitamins, minerals and also oxygen. Most yeasts require an abundance of oxygen for
growth, and a basic substrate such as sugar. Some yeasts can ferment sugars to alcohol and carbon
dioxide in the absence of air but require oxygen for growth. They produce ethyl alcohol and carbon
dioxide from simple sugars such as glucose and fructose.
Yeasts are active in a broad temperature range from 0 to 50° C, with an optimum temperature
range of 20° to 30° C. The optimum pH for most microorganisms is near the neutral point (pH 7.0). Molds
and yeasts are usually acid tolerant and are therefore associated with the spoilage of acidic foods. Yeasts
can grow in a pH range of 4 to 4.5 and molds can grow from pH 2 to 8.5, but favor an acid pH (Mountney
and Gould, 1988).
In terms of water requirements, yeasts are intermediate between bacteria and molds. Normal
yeasts require a minimum water activity of 0.85 or a relative humidity of 88%.
Yeasts are fairly tolerant of high concentrations of sugar and grow well in solutions containing
40% sugar. At concentrations higher than this, only a certain group of yeasts (osmophilic type) can
survive. There are only a few yeasts that can tolerate sugar concentrations of 65-70% and these grow
very slowly in these conditions (Board, 1983). Another group which can tolerate high salt concentrations
and low water activity is Zygosaccharomyces rouxii, which is associated with fermentations in which
salting is an integral part of the process.

3. Does yeast die during fermentation?

In natural fermentations, there is a progressive pattern of yeast growth. Several species of yeast
are active for the first two to three days of fermentation. Yeast will become dormant and eventually die
after a few weeks to months, after any food sources, like priming sugar, have been consumed.
REFERENCES

https://www.ncbi.nlm.nih.gov/books/NBK562892/
http://www.milkfacts.info/Milk%20Processing/Cheese%20Production.htm
http://www.fao.org/3/x0560e/x0560e08.htm