BIOPROCESSING PRACTICALS:

PRODUCTION OF CHEESE LAB

REPORT
MRS. AE KANEME
GROUP: B5
DUE DATE: 17th OCTOBER 2022
 Group names Student numbers
e. zekala 219164568
N.MASHIANE 220000387

T.SHOSHA 219156921

PROJECT 3: CHEESE
PRODUCTION USING A
KIT.
Tittle: Cheese production using a kit.
INTRODUCTION
Cheese is a diary product, derived from milk and produced in wide ranges of flavours,
textures and forms by coagulation of the milk protein casein. It compromises proteins and
fat from milk, usually the milk of cows, goats or sheep. Over a thousand types of cheese
exists and are currently produced in various countries. The type of cheese which will be
produced in this experiment is the Colwick Cheese which is a fresh cheese invented around
the 17th Centuries in the village of Colwick, south of Nottingham on the River Trent. The
cheese has recently been revived using traditional methods and milk from rare Red Poll
Cattle. Colwick Cheese is a soft, curdy cheese, because of the way it is made it has a unique
shape, forming a bowl. Colwick cheese is normally made using moulds similar to cake tins,
many households which traditionally make their own cheese would tip the curds into a
cloth, rather than a mould (David, 2007).

Cheese is a very popular food that can be found on the shelves of any grocery store, through
cheese found in the dairy section is often a highly processed food containing additives and
preservatives. In contrast, homemade cheese can be free of all the colouring and chemical
stabilizers used to make cheese more attractive and stable for store shelves. Homemade
cheese will be better-tasting, more alive, and more versatile than any commercially
processed cheese (Banks et al., 1989).

Figure1: shows the home-made cheese (Visser, 2020).

Milk is an emulsion of milk fat globules in an aqueous portion of milk contains a variety of
substances including lactose, protein, minerals, and certain vitamins. At its heart cheese
making is the process by which we remove water from milk, concentrating the fat and
protein. Water makes up the majority of milk. Fat globules and casein are what gives the
milk its colour. Light is scattered when it hits the globules and casein micelles and gives the
opaque white colour milk is known for. The name of the sugar found in milk is Lactose,
which is a disaccharide, in cheese the bacteria cultures added during the production process
consume lactic acid. This process happens more and more over extended periods of time.
With enough age, all the lactose has been converted into lactic acid. For many reasons
cheese can be safely consumed by those who suffer from lactose intolerance. The fat
portion of milk exists in globular structures. These milk fat globules are complex structures
with multiple layers and membranes. The homogenization process breaks up the larger fat
globules into smaller ones. These smaller globules stay distributed in the watery phase of
milk better and are less likely to rise to the top and form a “cream line”. Fat soluble vitamins
(A, D, E, and K) and their precursors (e.g., beta-carotene) are found in this portion of milk
(Niewwoudt, 2019).

Milk is heated, whether it be raw or pasteurized, as the first step of the cheese making
process to create a desired temperature for lactic acid bacteria to thrive. Lactic acid bacteria
consume lactose present in milk and produce lactic acid, which acidifies the milk and inhibit
the growth of potentially harmful bacteria (Johnson, 2017).
Like any other living organism, bacteria require a certain temperature range to live and
flourish. The specific temperature range depends on the type of bacteria, thus for this
experiment heating of milk under the temperature of 32 degrees Celsius was necessary
hence the Lactic acid bacteria are tolerant of the temperature used. Therefore, heating was
needed to reach desired range for the Lactic acid bacteria (Sapkota, 2020).
In microbiology, streaking is a technique used to isolate a pure strain from a single species
of microorganisms, often bacteria. Samples can be taken from the resulting colonies and
microbial culture can be grown on a new plate so that the organism can be identified,
studied or tested. The dilution or isolation by streaking was first developed in Koch
laboratory, which involves the dilution of bacteria by systematically streaking them over the
exterior of the agar in a petri dish to obtain isolated colonies. The technique is done by
diluting a comparatively large concentration of bacteria to a smaller concentration. The
decrease of bacteria should show that colonies are sufficiently spread apart to affect the
separation of the different types of microbes. Streaking is done using a sterile tool, such as a
cotton swab or inoculating loop (Brucker, 2012).

Gram staining is a common technique used to differentiate two large groups of bacteria
based on their different cell wall constituents. The gram stain procedure distinguishes
between gram positive and gram-negative groups by colouring these cells red or violet.
Gram positive stains violet due to the presence of a thick layer of peptidoglycan in their cell
walls, which retains the crystal violet these cells are stained with. Alternatively, Gram
negative bacteria stain red, which is attributed to a thinner peptidoglycan wall, which does
not retain the crystal violet during the decolouring process (Colco, 2005).
A microscope is an instrument that produces enlarged images of small objects, allowing the
observer an exceedingly close view of minute structures at a scale convenient for
examination and analysis. It is basically used to see objects that are too small to be seen by
the naked eye. The microscope may provide a dynamic image or one that is static (Ford &
Brian, 1992).
A catalyse test is a test used to demonstrate the presence of catalase, an enzyme that
catalyses the release of oxygen from hydrogen peroxide. It is used to differentiate those
bacteria that produce an enzyme catalase, such as staphylococci, from non-catalase
producing bacteria such as streptococci. Normally 3% hydrogen peroxide is used for the
routine culture while 15% hydrogen peroxide is used for detection of catalyse in anaerobes.
The enzyme catalyse mediates the breakdown of hydrogen peroxide into oxygen and water.
The presence of enzymes in a bacterial isolate is evident when a small inoculum is
introduced into hydrogen peroxide, and the rapid elaboration of oxygen is evident by lack of
bubble production. The culture should not be more than 24 hours old. Bacteria thereby
protect themselves from the lethal effect of hydrogen peroxide which is accumulated as an
end product of aerobic carbohydrate metabolism (Reiner, 2016).

AIM:
To make production of cheese using a kit.

OBJECTIVES
1. To make production of cheese
2. To develop the basic structure of cheese.
3. To isolate and identify organisms in the cheese.
4. To perform streak plate
5. To perform gram stain
6. To perform catalase test

PART ONE: PRODUCTION

REQUIREMENTS:
• 2 litres full cream milk (not long-life milk)
• One capsule cheese culture
• One capsule rennet
• Water
• Beaker
• Cheese former
• Cheese cloth

PROCEDURE:
All the apparatus were rinsed with sterilizing liquid. The milk was then poured into the
beaker. Then the milk was heated by putting it in the water bath. A broke opened capsule’s
powder of the cheese culture was poured into the milk. The full contents of one rennet was
dissolved in a little amount of water in a separate beaker then put in the milk. The milk was
stirred constantly until the temperature reached 32°C. It was then removed from the water
bath, covered with foil and was let to stand for one hour to allow it to coagulate. The two
halves of the cheese former were lined, with the strainers in place, with the cheese cloth,
and the curd was transferred to each half. A big spoon was used. After about an hour, the
cloth was pulled upwards and inwards to curl the outside edges of the curd. This was done
another 3 or 4 times during the day. The curd was then let to drain for another 24-36 hours.
At that point, the cheese had been firm enough to handle. Then the cheese former was
removed and the cheese cloth was peeled off. The cheese was then ready to be eaten or
kept covered in the fridge for up to seven days.

PART TWO: ISOLATION AND IDENTIFICATION OF ORGANISMS IN THE CHEESE

REQUIREMENTS:
• Finished cheese product
• Nutrient agar
• MacConkey agar

PROCEDURE:
The cheese was streaked out onto the Nutrient agar and MacConkey agar. Then incubated
at 37°C overnight. Then colonies were described. The most common colony was selected on
the two plates and Gram stains were performed, Gram staining.
A slide with a heat fixed smear was used. Gently flooded with crystal violet for 1 minute and
then washed with distilled water. Then was gently flooded with grams iodine for 1 minute
and washed with distilled water. Acetone was applied drop by drop for 5-10 seconds and
immediately rinsed with water. The smear was flooded with safranin to counterstain for 45
seconds. Rinsed with distilled water then blot dried. The slides were then examined under
the microscope.
Catalase test
A tube with hydrogen peroxide was taken. Then a wire loop with culture was dipped in the
tube and results were observed... Then a catalase test was done on the same colonies.
Based on the results, a tentative identification was done.
RESULTS
Ingredients used to produce cheese, these ingredients used ripen the milk, form curd,
plain yoghurt and add flavour to the finished cheese.

Figure 1(a): full cream milk, it is coagulated and form curds which later becomes the cheese

Figure 2: A consistently stirred milk solution with all the ingredients until reached 32
degrees Celsius, the solution is consistently stirred to firmly mix all the ingredients, while
reading the thermometer.

Figure 3: Milk solution covered with foil to allow coagulation.

Figure 4: a final product of cheese

Figure 5: MacConkey agar plate of organisms from the colwick cheese, colonies appearing
red due to the acidic product they released when they use lactose as a carbon and energy
source.

Figure 6: Growth on a streaked out MacConkey agar

Figure 7: Gram stain from MacConkey plate without salt

Figure 8: Gram stain from MacConkey with salt

Figure 9: Gram stain from Nutrient Agar plate

Figure 10: Catalase positive result from Nutrient Agar sample
Observable bubbles on top, meaning the organism produces the enzyme catalase and
respire using oxygen hence producing bubbles.

Figure 11: Catalase positive result from MacConkey agar without salt
Less bubbles, because the organism is either anaerobic or facultative anaerobic and do not
use oxygen as a terminal electron acceptor hence test positive.
Figure 12: MacConkey agar with salt
Less bubbles, because the organism is either anaerobic or facultative anaerobic and do not
use oxygen as a terminal electron acceptor hence test positive.

Table 1: The results of gram staining and catalase test of a cheese microorganisms on both
Nutrient agar and MacConkey.

Features Nutrient agar MacConkey agar

Colour Purple Purple
Shape Spherical shape Rod shape
arrangement In chains Irregular grapes like clusters
Size average bacillus is 0.5-1.0 average coccus is about 0.5-
wide by 1.0-4.0 micrometre 1.0 micrometre in diameter,
long
organisms Streptobacillus Staphylococci bacterium
Gram positive/negative Positive Positive
Catalase test Positive Positive

DISCUSSION:

The milk is shown in figure 1, its role in this experiment is to coagulate the milk and form
curd which later becomes the cheese.
It is generally quite straight forward to locate the sources of enzymes; the natural sources of
enzymes are where nature needs them. It comes as no surprise that rennet had traditionally
been isolated from the fourth stomach of young calves because digestion by suckling calves
is nature's primary way of processing cow milk. However, the ways of nature are not always
the most economical from man's perspective when a process is adapted to different uses to
benefit mankind in ways not originally intended by the nature. Thus, enters the study of
biochemical engineering.

Chemical engineering is the study of how to make a large quantity of chemicals in an

economical fashion. For example, frequently a chemical engineer must devise a process to
mass-produce a polymer that is totally different from the one originally used by an organic
chemist working with small test tubes and beakers in a laboratory. Biochemical engineering,
being a sub-field of chemical engineering, also deals with the same kinds of problems facing
chemical engineering, except that the chemicals are not synthetic (manmade) but biological
(naturally existing) in nature. Although rennet is naturally excreted from a calf's stomach
lining, extracting it from its natural source is not economical. Other proteases can also
convert casein to para-casein, but their action does not stop there. They further degrade the
curd to soluble subunits. Fortunately, large quantities of rennet of consistent quality can
now be produced easier and cheaper in a well-controlled environment by microbial
fermentation.

Streaking method was performed using both Nutrient agar and MacConkey agar plates as
shown in figure 5 and 6 above. Gram staining was also performed as shown in figure 7, 8
and 9 above. In the gram staining experiment (figure 9), a slide of pure culture was observed
under microscope under 1000x magnification using oil immersion. The culture observed
appeared as purple rod shaped bacterium (Streptobacillus) arranged in chains, an average
bacillus is 0.5-1.0 wide by 1.0-4.0 micrometre long, resulting in the culture being Gram
positive. In the gram staining experiment (figure 7 and 8), a slide of pure culture was
observed under microscope under 1000x magnification using oil immersion. The culture
observed appeared as purple spherical bacteria (staphylococci bacterium) arranged in
irregular, often grape like clusters, an average coccus is about 0.5-1.0 micrometre in
diameter, resulting in the culture being Gram positive.
The positive catalase test (nutrient agar), shown in figure 10, produces bubbles on top
meaning the organism produces the enzyme catalase and respire using oxygen hence
producing bubbles. In figure 11 and 12, less bubbles are observed in the positive catalase
test (MacConkey agar) because the organism is either anaerobic or facultative anaerobe and
do not use oxygen as a terminal electron acceptor hence test positive.
Conclusion
The production of cheese using a kit was successfully done. The ingredients that were used
all worked as expected. The content of one capsule of cheese culture helped to ripen,
allowed fermentation to begin and pH to decline as acidic fermentation product
accumulate. The gram staining was performed and the organisms were gram positive
bacterium. The catalase test performed with organisms isolated in MacConkey and Nutrient
agar plates were positive. We conclude by saying the organisms found are Staphylococcus
and Bacillus because their morphologies matches with the ones we found after performing
the Gram staining.
References
1. Catalase test [online]. Virtual Interactive Bacteriology Laboratory. Available from:
https://learn.chm.msu.edu/vibl/content/catalase.html. [Accessed: 13 October 2022].
2. Martin, J., 2020. Vegetarian Rennet Tablets - Makecheese.Ca - Homemade Cheese.
[online] Make Cheese Inc. | Cheese Kits, Ingredients & Supplies. Available at:
<https://www.makecheese.ca/products/rennet-tablets> [Accessed 13 October
2022].
3. Classification of Cheese". Www.egr.msu.edu. Archived from the original on
November 24, 2011. Retrieved March 23, 2011.
4. Reiner, K., 2016, Catalase test protocol. American society for food microbiology. 11
November 20.
5. Hayaloglu, A.A., 2016, Microbiology of cheese, Inonu University, Malatya, Turkey.

6. Brucker, M.Z., 2012, viability and qualifications stains. Montana State University,
Bozeman.

7.