LABORATORY REPORT

MIC254
FOOD MICROBIOLOGY

LAB 1 : ISOLATION OF STARTER CULTURES FROM

FOOD PRODUCT
DATE EXPERIMENT : 29 MARCH 2023
DATE SUBMISSION : 4 APRIL 2023

STUDENT NAME STUDENT ID

1. LEEYA UMAIRAH BINTI MOHD SHAFIE 2022791815

2. NUR SABRINA BINTI MUHAMMAD SHAH 2022744707

3. ZUHAIRAH BINTI ISHAK 2022318339

4. MUHAMMAD RAIF HANAFI BIN NASURUDDIN 2022132031

TITLE: ISOLATION OF STARTER CULTURES FROM FOOD PRODUCT.

OBJECTIVE :
1) To isolate the starter culture from food product.
2) To observe the morphology of the microorganisms under the microscope.
3) To enumerate the microorganisms in the food product.

INTRODUCTION:

Starter culture is an essential component of nearly all commercially made fermented or dairy
foods, including cheese, yogurt, sour cream, and others. As a start, bacteria, primarily lactic acid
bacteria and coagulase-negative staphylococci, as well as yeasts and molds, can be used. Starter
cultures are microorganisms that are inoculated directly into food components to produce
predictable and wanted changes in the finished product. These modifications include improved
preservation, increased nutritional content, altered sensory properties, and increased commercial
worth. They can improve the safety of fermented beef products by creating antimicrobial
compounds such as bacteriocins or by quickly acidifying the matrix. Furthermore, starters may
help to standardise product traits and accelerate the ripening process.

Yogurt is produced by bacterial enzymes fermenting lactose (milk sugar). The procedure is
anaerobic, which means it takes place in the absence of oxygen. Lactose, a complicated sugar, is
formed when glucose and galactose, two basic sugars, combine. Lactase, an enzyme produced by
bacteria, converts lactose into these sugars during yogurt manufacture. Lactic acid and
acetaldehyde are byproducts of glucose and galactose metabolism. Lactic acid and acetaldehyde
production reduces the pH of the milk, giving it a sour, tart flavour. The lower the pH, the more
casein coagulates and precipitates, creating the solid curd that is yogurt. Fermentation with the
starting cultures Streptococcus Thermophilus and Lactobacillus bulgaricus produces yogurt. The
physical nature of this food lends itself to further enhancement through the addition of prebiotics
and probiotics, resulting in the development of specific foods with an emphasis on a person's
health.

Tempe is a well-known native Indonesian food made from soybeans. Tempe is a cake made
from cooked, fermented soybeans and created by a solid-state fungal fermentation process that
results in a densely woven cake of mycelia. Rhizopus Oligosporus is the dominant bacterium in
tempe. R. oligosporus-made tempe contains high amounts of free radical scavenging activity,
with two-thirds of this activity coming from the peptides and one-third from the isoflavones
released by fungal-glucosidase from the glycosides. Fresh tempe has a short shelf life due to
microbial enzymatic processes. Fresh tempe eventually turns brown during storage due to the
senescence of the fungal mycelium, the material softens, and an ammoniacal odour develops. If
the tempe is to be stored for later use, it is typically blanched, sun-dried, or refrigerated.

Tapai is a fermented product derived from starches like glutinous rice and cassava. The
indigenous amylolytic lactic acid bacteria were used in the manufacture of tapai, and the
chemical, microbiological, and sensory properties of the probiotic tapai were discovered. The
starter culture for tapai fermentation is a mixed culture of microbes. Ragi is traditionally
prepared under comparatively poor aseptic circumstances by people untrained in microbiology.

PROCEDURE/METHODS
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 by circular motion on the slide.
5. The smear was allowed to air-dry and was fixed by quickly passing the slide over the
Bunsen burner flame. The fixing will prevent the smear from washing off during
staining.
6. Do not flame the slide too much as it could alter the cell morphology and the stain was
induced to decolorize more rapidly.
7. The smears were flooded with a few drops of crystal violet and leave it for 1 minute. The
stain was washed gently with tap water.
8. Then, the smears were flooded with a few drops of Gram’s iodine and left it for 1 minute.
The stain was washed gently with tap water.
9. The slide was tilted and the 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 left it for 45 seconds. The stain was
washed gently with tap water.
11. The slide was blotted dry before examining it using 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 mold
growth.
3. The strip was transferred to a drop of methylene blue stain on a glass slide.
4. Examined it using a light microscope.

Sample: Tapai

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 by circular motion on the slide.
5. The smear was allowed to air-dry and fixed by quickly passed the slide over the Bunsen
burner flame. The fixing will prevent the smear from washing off during staining.
6. Do not flame the slide too much as it could alter the cell morphology and induced the
stain to decolorize more rapidly.
7. The slide was flooded with methylene blue for 1 to 2 minutes.
8. The stain was washed gently with tap water.
9. The slide was blotted dry before examining it using a light microscope.

B) Isolation of starter culture from tapai

1. The inoculating loop was sterilized using the Bunsen burner by putting the loop into the
flame until it was red hot. Allow it to cool.
2. A loopful of tapai samples was streaked over the first quadrant of the PDA plate
(approximately ¼ of the plate).
3. The loop was flamed again and let it cool down. Going back to the edge of area 1 that had
just streaked, extend the streaks into the second quarter of the plate (Quadrant 2).
4. The loop was flamed again and let it cool down. Going back to the area that you just
streaked (area 2), extend the streaks into the third quarter of the plate (Quadrant 3).
5. The loop was flamed again and let it cool down. Going back to the area that you just
streaked (area 3), extend the streaks into the center a fourth of the plate (Quadrant 4).
6. The loop was flamed once more.
7. The plate was labeled at its bottom edge with the required information.
8. The plate was incubated at 37°C for 48 hours in an upright position.
9. The colonies grown on the plate were carefully and observations were recorded.

C) Enumeration of starter culture from yogurt.

1. 1 g of the sample was weighed and 9 ml of autoclaved distilled water was added. This
preparation produced a sample solution (T1) with a dilution factor 101.
2. Four sterile tubes added with 9 ml autoclaved distilled water were prepared and T2, T3,
T4 and T5 were labelled. 1 ml of sample solution from T1 was added into T2 and mixed
homogeneously. The tube was labeled as dilution factor 102. The same procedure was
done for the three test tubes were labelled 103, 104 and 105 respectively.
3. 100 μl of the homogenized mixture ( from tubes with dilution factors 103, 104 and 105) on
nutrient agar plates were inoculated and spread evenly using L shaped spreader.
4. The agar plates were incubated inverted at 37°C for 48 hours.
RESULTS :

A) Microscopic examination.

Sample: Yogurt (yakult)

Microscopic morphology Expected starter culture

Sample : Tempe

Microscopic morphology Expected starter culture

Sample: Tapai

Presence of budding cells and ascospore Expected starter culture

B) Isolation of starter culture from tapai

C) Enumeration of starter culture from yakult.

Dilution Number of colonies Results

10
3 151

10
4 169

10
5 187

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

3
Colony forming unit/ml = (151× 10 )/0.1
6
= 1.51× 10 CFU/ml
DISCUSSION :

A) Microscopic examination.

Sample: Yogurt ( yakult)

In this experiment, the yakult result showed that there were only 2 to 3 bacteria are visible
under the microscope. This happened because the smear was washed off during the staining. To
avoid the problem, fix the smear by quickly passing the slide over the Bunsen burner flame.

The expected starter culture should have Streptococcus Thermophilus and Lactobacillus
bulgaricus. Streptococcus Thermophilus is Gram-positive bacteria, has coccus-shaped
(spherical), non-motile, facultative anaerobe and a non-spore forming. While Lactobacillus
bulgaricus is gram-positive bacteria, has rod-shaped, non-motile, facultative anaerobe, and also a
non-spore forming.

Sample: Tempe

In this experiment, the tempe result showed that there were microbes that was observed but
it was not from the slide but from the lens of the light microscope. The microbes that was
observed were probably because the objective lens was not clean. The microbes from the slide
itself was not observed because student that handle the experiment does not follow the lab
manual. Therefore , student should follow lab manual properly in order to avoid human error.

Sample: Tapai

In the experiment, the tapai result showed that there were no microbes that could be seen
under a microscope as the objective lens was kind of dirty. The effect is we were not able to see
any bacteria inside tapai and your imagery will not be as sharp or high quality if you do not clean
it. To avoid the problem, use special lens paper to clean the lens.

The expected culture starter is it must has rhodotorula glutinis inside tapai. Aerobic yeast R.
glutinis has pink, smooth colonies that appear moist. The form of the cells can be spherical,
ellipsoidal, or elongated. Some varieties of rhodotorula glutinis produce remaining
pseudomycelium and reproduce asexually through multilateral or polar budding. A pigmented
yeast related to the Basidiomycota phylum named Rhodotorula glutinis is particularly significant
for the food industry due to its biotechnological potential and safety implications. The
fermentation process in tapai involves the fermentation of glutinous rice starch which is sugar
into alcohol by yeast, moulds, and other bacteria. During fermentation, acids form in tapai giving
the product a little acidic flavour so it helps enhance the aroma of food and extend the shelf life
of foods.

B) Isolation starter culture from tapai

In the experiment, the figure shows the result of isolating starter culture for 48 hours from
tapai. There is a visible culture in the media as seen in the mentioned figure. We can observe that
every quadrant has a culture. Using the PDA agar, we discovered that there are bacteria called
amylolytic acid in the tapai. The typical amylolytic acid is positive bacteria and non-motile.
The error might happen because the inoculating loop is too hot and kills the bacteria so that’s
why quadrant 4 only has a few bacteria. To avoid this problem, the inoculating loop must be
cooled because the excessive heat could kill the bacteria once they contact our loop before
collecting a sample of bacteria.
C) Enumeration of starter culture from yakult.

In this experiment we used yakult for enumeration of starter culture. Yakult is fermented milk
drinks containing Lacticaseibacillus paracasei Shirota. We have used nutrient agar to support the
growth of a wide range of microorganisms. We took 0.5 ml of yakult and mixed it
homogeneously with 4.5 ml of distilled water that has been labeled as T1. After that the sample
solution from T1 was mixed with 4.5 ml of distilled water which is labeled as T2. The same
procedure was repeated for T3, T4 and T5. We observed that the color of yakult from T1 is
cloudiness to T5 colorless. Then, we inoculate the homogeneous mixture from tubes T3, T4 and
T5 on the nutrient plate agar plate by using L-shaped spreaders. The L-shape allows for
application across the full area of the agar in the plate or petri dish. A smooth spreading surface
with a small upward return minimizes the risk of damaging the agar sample. The agar plate was
kept in an inverted position during incubation for 48 hours. It is to prevent condensation droplets
that might fall on the agar surface as it can lead to contamination.

Based on the result, T3 has less number of colonies that grow on the agar plate and the
morphology of the bacteria is in a punctiform shape. While T4, it shows the irregular shape of
bacteria colonies. The last agar plate T5 contains more colonies that were cultured on the agar
plate. Based on the theoretical it should be T3 has a lot of colonies because of the higher
concentration of yakult. There might be some error during spreading the solutions by using the
same dropper each of the test tubes.

CONCLUSION:

In conclusion, we have successfully isolated the starter culture from food products such as tapai.
Tapai is one of the fermented products that is made from starch such as glutinous rice and
cassava. Besides, we have observed the morphology of the microorganisms under a microscope.
For example, Lactobacillus casei is a bacteria that is contained in yakult. It is non- motile and
rod-shaped. Lastly, we have enumerated the microorganisms in the food product by using a
colony counter. It is to specify the number of microbial colonies present on agar plates to make
working in the lab more efficient .
DISCUSSION QUESTIONS:

1. Explain the principle of Gram staining.

- the ability of the bacterial cell wall to retain the crystal violet dye during
solvent treatment. Gram-positive microorganisms have higher
peptidoglycan content, whereas gram-negative organisms have higher
lipid content.

2. Discuss why microorganism counts are reported as CFU/ml rather than cells/ml.
- Because not all bacterial cells produce colonies, as some bacteria tend to
clump or aggregate, and some are nonviable. For this reason results are
reported as colony forming units (CFU)/ml of bacterial culture.

3. Discuss the purpose of PDA media.

- Potato Dextrose Agar is used for the cultivation, isolation and enumeration
of yeasts and molds from foodstuffs and other materials.
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