LAB REPORT 1 MIC 254

ISOLATION OF STARTER CULTURES FROM FOOD PRODUCTS

By:

Muhammad Ilham Nur Hakim Bin Ramli (2022775425)

Muhammad Nasren Hisyam Bin Saifuddin (2022744737)
Muhammad Idham Bin Abu Bakar (2022340509)
Nadia Yasmin Binti Mohd Zaki (2022777181)
Farahin Nursyahirah Binti Mahthir (2022553425)

Lecturer: Madam Syazuani Mohd Shariff

Date of Experiment: 29/03/2023

Date of Submission: 04/04/2023

Group: AS1143A1
TITLE
Isolation of Starter Cultures From Food Product.

OBJECTIVES

1) To isolate the starter culture from food products.

2) To observe the morphology of the microorganisms under the microscope.
3) To enumerate the microorganisms in the food product.

INTRODUCTION

Starter culture is the most important component of all commercially produced fermented food
and dairy products such as cheese, yogurt, sour cream and others. It creates various enzymes,
such as lactase, proteases, peptidases, and lipases, to speed up and regulate chemical reactions.
These enzymes are mixed with the substance that is fermenting. Although fermentation products
can be created without starting cultures, their nutritional content, preservation abilities, economic
standing, and sensory qualities can all be improved by adding these cultures. In terms of their
morphology, size, mode of reproduction, and capacity to break down carbohydrates, the starting
microorganisms can differ greatly. The characteristics of a starting culture that produces strong
and reliable lactic acid are those of an ideal starting culture. When provided with the right
organic materials, it grows quickly. In a dynamic setting, it changes quickly.

Yogurt is made from fermentation of lactose (milk sugar) by bacterial enzymes. Several
yogurt varieties contain live bacteria, or probiotics, that were either added after pasteurization or
included in the starting culture. When ingested, these may improve digestive health.
Pasteurization, a heat treatment that destroys the good bacteria in yogurt, is a common practice.
Even some yogurt types that are advertised as having living, active cultures frequently
experience some probiotic loss as a result of different storage temperatures and other variables.
Despite, the label and selecting a probiotic that contains live, active cultures is still a best choice
for finding the most effective probiotics.Yogurt is generated via fermentation with the starter
culture which are Streptococcus thermophilus and Lactobacillus bulgaricus. Lactose metabolism
during yogurt fermentation may result in the production of acetaldehyde due to pyruvate
decarboxylation. The conversion of the amino acid threonine into acetaldehyde and glycine,
however, is the main source of acetaldehyde in these bacteria. Also, it has been demonstrated
that yogurt cultures in the intestinal tract secrete the enzyme lactase, which continues to break
down the lactose in the dairy product. This enables lactose intolerant persons to eat yogurt.

Tempeh is a soybean-based fermented food that is popular worldwide. It is regarded as a

good source of protein and is easily digestible food. Rhizopus oligosporus, a fungus sometimes
known as mould , is grown on soybeans or other plant substrates such as wheat, various grains,
and beans to produce tempeh. It is adored all over the world for its quality, distinct flavour, and
nutritional and therapeutic benefits. Tempeh has a chewy texture due to the white fluffy fungus
mycelium that tightly enwraps the yellow soybeans (or other plant-food utilised) and binds it all
together. The most typical form of sale is in rectangular, inch-thick slabs. As it expands, the
exposed surface takes on a few spores of the fungus, which are black and grey in colour. Tempeh
also provides health benefits. Products made from the Rhizopus oligosporus have some
beneficial impacts on human health. A tempeh diet causes rats' blood cholesterol levels to
decrease. The lecithin, niacin, sitosterol, and unsaturated fatty acids in tempeh are likely to be
responsible for the impact of lowering cholesterol. High antioxidative activity is produced by
components from Rhizopus oligosporus.

Tapai is a fermented product made from starch sources such as glutanious and cassava .
In order to identify the chemical, microbiological, and sensory properties of the probiotic tapai, the
native amylolytic lactic acid bacteria were used in the synthesis of tapai. Amylolytic enzyme is
producing Aspergillus sp. from sweet - flavoured tapai. A variety of starch-based media, including
potato peel, potato dextrose broth, cassava, and maize, were used to test the amylolytic capabilities of
the fungi isolated from sweet tapai. Temperature and pH levels for partially purified amylolytic
enzymes that work best.
MATERIALS

Nutrient agar
PDA agar
Autoclaved distilled water
Gram Stain reagents
Glass slide
Cellophane tape
70% alcohol
Methylene blue
Dropper
Test tubes
Test tube rack
Microscope
Food samples (Yogurt, Tempe, Tapai)

METHODOLOGY

A) Microscopic examination.

Sample: Yogurt
1. A clean glass slide was prepared.
2. A drop of water was placed on the slide.
3. Using a sterile loop, a very small sample was obtained and transferred to the slide.
4. The sample was mixed and spread evenly in circular motion on the slide.
5. The smear was air-dry and fixed by quickly passing over the slide over the Bunsen burner
flame. The fixing was done to prevent the smear from washing off during staining.
6. The slide was flamed a few times only to prevent the alteration of the cell morphology
and induce the stain to decolorize more rapidly.
7. The smear was flooded with a few drops of crystal violet and was left for 1 min. After
that, the stain was then washed gently using tap water.
 8. The smear was flooded again with a few drops of Gram's iodine and was left for another
1 min. The stain was then washed gently with tap water.
9. The slide was tilted and 95% ethyl alcohol was dropped for a few seconds until the
alcohol ran roughly clear. The slide was washed gently with tap water.
10. The smear was counterstained with safranin and was left for 45 seconds. The stain was
gently washed with tap water.
11. The slide was blot dried and the slide was examined under a light microscope.

Sample: Tempe
1. A clean glass slide was prepared.
2. A strip of cellophane tape was cut and gently touched the sticky surface against the
mould growth.
3. The strip was then transferred to a drop of methylene blue stain on a glass slide.
4. The slide was then examined using a light microscope.

Sample: Tapai
1. A clean glass slide was prepared.
2. A drop of water was placed on the slide.
3. A very small sample was obtained using a sterile loop and was transferred to the slide.
4. The sample was then mixed and spread evenly by circular motion on the slide.
5. The smear was air-dry and fixed by quickly passing over the slide over the Bunsen burner
flame. The fixing was done to prevent the smear from washing off during staining.
6. The slide was flamed a few times only to prevent the alteration of the cell morphology
and induce the stain to decolorize more rapidly.
7. The slide was flooded with methylene blue for 1 to 2 min.
8. The stain was washed gently with tap water.
9. The slide was blot dried and was examined using a light microscope.
B) Isolation of starter culture from tapai.

1. The inoculating loop was sterilised in the Bunsen burner by putting the loop into the
flame until it is red hot. The loop was then cooled down.
2. A loopful of tapai samples was streaked over the first quadrant of the PDA plate.
3. The loop was then flamed again and cooled down before going back to the edge of area 1
that just streaked. The streak was extended into the second quarter of the plate (Quadrant
2).
4. The loop was flamed again and cooled down before going back to the area that just
streaked which is area 2. The streak was extended into the third quarter of the plate
(Quadrant 3).
5. The loop was flamed again and cooled down before going back to the area that just
streaked which is area 3. The streak was extended into the fourth quarter of the plate
(Quadrant 4).
6. The loop flamed once more.
7. The plate was labelled at its bottom edge with the required information.
8. The plate was incubated for 24 hours at 37°C in an upright position.
9. The colonies grown on the plate were examined carefully and the observations were
recorded.

C) Enumeration of starter culture from yogurt.

1. 0.5 mL of the sample was weighed and added 4.5 mL autoclaved distilled water. This
preparation produces a sample solution (T1) with a dilution factor 10¹.
2. Four sterile tubes were prepared and added 4.5 mL autoclaved distilled water and labelled
T2, T3, T4 and T5. 0.5 mL of sample solution was inserted from T1 into T2 and was
mixed homogeneously. The tube was labelled as dilution factor 10². The same procedure
for the rest three test tubes was done and labelled as dilution factor 10³, 10⁴ and 10⁵
respectively.
 3. 100 (microliter) of the homogenized mixtures from tubes with dilution factor 10³, 10⁴ and
10⁵ was inoculated on nutrient agar plates and was spread evenly using L shaped
spreader.
4. The agar plate was incubated inverted at 37°C for 48 hours and the observations was
recorded.

RESULTS

A) Microscopic examination

Sample: Yogurt

Microscopic morphology Expected starter culture

Sample: Tempe

Type of spores & hyphae Expected starter culture

Sample: Tapai

Presence of budding cells and acrospore Expected starter culture

 B) Enumeration of starter culture from yogurt

Dilution Number of colonies

10
3 416

10
4 388

10
5 755

Number of bacteria in original sample (use the formula (C×M)/V) =

C = 388
4
M = 10
V = 0.5 ml
Colony forming unit/ml = [(C × M)/ V]
4
388 × 10
=
0.5
4
= 7.76 × 10 CFU/ml

DISCUSSION

In this experiment, three samples which are yogurt, tempe, and tapai were used in this
investigation. Streptococcus thermophilus, also known as yoghurt or lactobacillus bulgaricus, is a
gram positive bacteria. As can be seen under the microscope, it has coccus-shaped cells that are
spherical and non-motile. It uses the facultative anaerobe respiration and does not produce spores
which are non spore forming. Tempe which occurs for spore formation, also known by its
scientific name Rhizopus oligosporus. As can be seen under the microscope, it features hyphae
without septa that resemble umbrellas. For tapai, it has an elongated shape. The end budding
cells and the majority of the budding cells are pseudohyphae. As may be seen under a
microscope, it does not exist for acrospore. For enumeration starter culture from yogurt, the
3 4 5
homogenized mixture from tubes with dilution factor 10 , 10 and 10 were inoculated and
spread evenly on nutrient agar plates. Theoretically, the number of colonies found on the agar
plates would decrease as the dilution factor increases. This is due to the fact that fewer bacteria
would be present in the tubes with a greater dilution factor. However, the result and data that we
obtained was different from the expected.

The obtained data and result that differs from the theoretical values may be caused by
errors and mistakes made by students handling the experiment. For example, a random mistake
when mixing the homogenized mixture with varying dilution factor. Therefore altering the result
data obtained. Another error that may have occurred was the condition of starter culture from
yogurt. The starter culture may develop differently because of certain factors such as the initial
number of healthy bacteria that were spread on nutrient agar plates during enumeration of the
starter culture of yogurt.

CONCLUSION

As a conclusion, only one of the three objectives was achieved which is isolating the starter
culture from food products such as tapai. However, there were some errors that led to the failure
of the experiment. It is important to be careful whenever the experiment is being conducted.
There are a few precautions that could have been taken into account when doing the microscopic
examination. Firstly, the slide should not be flamed too long as it may cause the bacteria to
explode and also alter the cell morphology. Moreover, students should also be careful when
washing the stain from the glass slide and to make sure that the stain is washed gently using tap
water. This is to avoid the smear from being washed away with high pressure from the tap water.
Another precaution that should be taken when doing the enumeration of starter culture from
yogurt is to use the same amount of homogenized mixture to spread on the nutrient agar. This is
to ensure that the amount of bacteria will reduce according to the dilution factor.
QUESTIONS

1) Explain the principle of Gram staining.

The ability of the bacterial cell wall to hold on to the crystal violet dye after solvent
treatment is the central principle of gram staining. Gram-positive bacteria have more
peptidoglycan, whereas gram-negative bacteria contain more lipid.

2) Discuss why microorganism counts are reported as CFU/ml rather than cells/ml.

Because certain bacteria have a propensity to clump or gather and some are nonviable,
not all bacterial cells develop into colonies. Results are therefore expressed in terms of
colony forming units (CFU)/ml of bacterial culture.

3) Discuss the purpose of PDA media.

Yeasts and moulds are grown, isolated, and counted from foods and other materials using
potato dextrose agar. Some dermatophytes are encouraged to produce pigment and mould
spores by the nutrient-rich base (potato infusion).
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