Department of Biological Science
Faculty of Science
Bachelor of Science (Hons) Biotechnology/ Bachelor of Science (Hons) Food Science
Year/Semester: 2023/June Trimester
UDBB3234/UDAF2234: FERMENTATION TECHNOLOGY
Submission Date: 17th July 2023
Lecturer: Dr. Lee Kok Chang
Practical Title: Isolation and Screening of Extracellular Enzyme-Producing
Microorganisms
Practical Date: 3rd July 2023
No Student Name ID Pre-lab – 15% Total Marks
. (100%)
1 Chong Zhe Dian 2104642
Marking Scheme:
Marking Criteria Marks
Abstract – 20%
Results – 20%
Discussion – 25%
Conclusion – 5%
References – 5%
Format – 10%
Total Mark – 85%
Abstract
Nutrients such as proteins, carbohydrates, lipids, etc. are needed by the human body in
order to maintain growth and health. The main sources of such nutrients are through the
consumption of foods in our daily life. However, it is crucial to note that many of these
substances in foods exist in a state of high molecular weight molecules which is practically
impossible for our body to absorb. The solution is to initiate the breakdown of these molecules
into much smaller and simpler substances which can then be transported through the cell
membrane and the action of the breakdown can be done through relatable exoenzyme. For
example, amylase for breaking down starch into glucose, lipase for lipid into glycerol and fatty
acid and protease for protein into amino acid. The main objective of this experiment is to isolate
and screen the amylase-, protease-, lipase-producing microorganisms from various local sources
through the usage of three different agar tests. In this experiment, rotten lettuce stem was used as
sample throughout the experiment. Firstly, both specific positive and negative controls were used
for each type of agar. Next, 10 gram of sample was mixed with 90 milliliter of distilled water in
stomacher bag. The sample solution was then separate into heat treatment and non-heat
treatment, where heat treated is heated at 80°C for 10 minutes. Both types of solution were then
diluted up to dilution factor of 10-4 and all of the dilutions were plated once on all types of agar.
Note that a single-line inoculation was made for dilution of 10 -1. The plates were inoculated at
37°C and checked for results. This experiment aims to figure out presence specific
microorganisms through Gram’s iodine solution for starch agar and formation of clear zone for
milk agar and tributyrin agar. Both starch and tributyrin agars have shown positive results,
whereas, solely milk agar presented all negative results.
Results
Table 1: Observations and results observed for all sources of cultures incubated on three types of
agars.
Starch hydrolysis Tributyrin hydrolysis Casein hydrolysis
Sources of
cultures Appearance of Appearance of Appearance of
Result Result Result
medium medium medium
Control Absence of (+) for Presence of (+) for Absence of (-) for
blue-black clear zone
colour around around
positive positive positive positive clear zone positive
control and control, control and control, around both control,
presence of (-) for absence of (-) for positive and (-) for
blue-black negative clear zone negative negative negative
colour around control around control controls control
negative negative
control control
10-1 Absence of Absence of Absence of
(L) isolate isolate isolate
Absence of Absence of
10-1 Absence of
(-) clear zone (-) clear zone (-)
(S) isolate
around isolate around isolate
Presence of Absence of
Heated
Absence of
10-2 (-) clear zone (+) clear zone (-)
isolate
around isolate around isolate
Absence of Absence of Absence of
10-3 (-) (-) (-)
isolate isolate isolate
Absence of Absence of Absence of
10-4 (-) (-) (-)
isolate isolate isolate
Small white
Small white
opaque, big
Small opaque and
milky opaque
10-1 yellowish small yellow
and small
(L) opaque isolate opaque
yellow opaque
was observed isolates were
isolates were
observed
observed
Non-heated
Absence of
Absence of
clear zone Absence of
10-1 blue-black
(+) around big (-) clear zone (-)
(S) colour on
area growth of around isolate
whole agar
isolates
10-2 Presence of (-) Absence of (-) Absence of (-)
blue-black clear zone clear zone
colour around around big around isolate
isolate area growth of
isolates
Presence of
Presence of Absence of
blue-black
10 -3
(-) clear zone (+) clear zone (-)
colour around
around isolate around isolate
isolate
Presence of
Presence of Absence of
blue-black
10-4 (-) clear zone (+) clear zone (-)
colour around
around isolate around isolate
isolate
*(L) represent single-line streaking inoculation and (S) represent spread plate streaking
inoculation
Discussion
Figure 1: Starch agar plate Figure 2: Starch agar plate Figure 3: Milk agar plate
of positive control (right) of positive control (left) of positive control (left)
and negative control (left) and negative control and negative control
after flooding of Gram’s (right). (right).
iodine solution.
Referring to Table 1, it is shown that positive and negative controls of all three agars
displayed expected results with the exception of the positive control on milk agar (Figure 3).
The positive control used during single-line streaking inoculation in milk agar was Bacillus
cereus which theoretically should be able to hydrolyse the protein casein as stated by Yang et al.
(2021). For the above statement, the area around Bacillus cereus isolate should exhibit the
presence of a clear zone that indicates the action of protease on casein. Thus, the negative result
shown might be the accidental inoculation of the negative control, Escherichia coli, due to
confusion during the experiment. However, through the observation of fully negative results
from milk agar in Table 1, there is a low possibility of unintentional exclusion of casein, the
crucial indicator component, during the preparation of milk agar.
Figure 4: Single-line Figure 5: Spread plating Figure 6: Spread plating
streaking inoculation of inoculation of non-heated inoculation of non-heated
non-heated sample with sample with dilution of 10- sample with dilution of 10-
dilution of 10-1 on starch 1
on starch agar plate prior 1
on starch agar plate after
agar plate. to flooding of Gram’s flooding of Gram’s iodine
iodine solution. solution.
First of all, the observation of absence of isolate growth can be studied for all heated
samples inoculum in starch agar plate in Table 1. The particular reason might be the inexistence
or minute amount of thermophiles present in sample solution. According to Sun et al. (2022), the
possible microorganisms that subsist in lettuce are Bacillus cereus, Cronobacter sakazakii,
Pseudomonas spp., Clostridium botulinum, etc. The soft rot of lettuce, which is more relatable to
the sample used, was said to be contributed by Pseudomonas spp., Bacillus spp. and Clostridium
spp. (Lunt, 2013). The above statement provides an understanding of the possible higher ratio of
stated microorganisms to be presented in the sample used. However, Pseudomonas spp. are
mostly not heat resistant, especially at high temperatures such as 80°C performed in the
experiment. On the other hand, Bacillus cereus and Clostridium spp. were able to resist heat
through the formation of spores, but the absence of isolate growth might because of lack of spore
activation throughout the experimentation period.
Small yellowish opaque isolates can be observed in Figure 4 for the single-line streaking
inoculation for the non-heated sample. Therefore, it can be concluded that the isolate is non-heat
tolerable as lack of isolates observed in heated sample. The identity of the isolate was
unidentifiable due to the lack of information and the possible large amount of microorganisms
that might be involved in the spoilage, storage, contamination, etc. of samples. In Figure 5, an
observation was made that white fuzzy growth covered the whole starch agar plate which
indicates possible fungal growth. The possible microorganism is Rhizopus spp. as it is both
common in vegetables and has the ability to degrade starch (Figure 6) mentioned by Litchfield
(2009). Starch agar mainly consists of nutrients for supporting microorganism growth and starch
as carbohydrate simultaneously an indicator. The principle of starch agar test is the ability of
Gram’s iodine solution to react with starch to form blue-black colour; whereas, the lack of blue-
black colour indicated that starch was being hydrolysed by amylase (Aryal, 2019).
Figure 7: Spread plating Figure 8: Single-line Figure 9: Spread plating
inoculation of heated streaking inoculation of inoculation of non-heated
sample with dilution of 10- non-heated sample with sample with dilution of 10-
2
on tributyrin agar plate. dilution of 10-1 on 2
on tributyrin agar plate.
tributyrin agar plate.
Figure 10: Spread plating inoculation of
non-heated sample with dilution of 10-3 on
tributyrin agar plate.
Figure 11: Spread plating inoculation of
non-heated sample with dilution of 10-4 on
tributyrin agar plate.
The principle of tributyrin agar test is the presence of lipase and its ability to break down
lipids to form a clear zone as stated in the article by Sourav (2022). It is also stated that the
opaque agar was formed due to the emulsion of tributyrin oil and the breakdown of lipids will
unable emulsion, thus, forming clear zone shown in Figure 7, Figure 10 and Figure 11.
Similarly to the results of starch agar above, most heated sample plates do not exhibit microbial
growth with two exceptions of dilution 10 -1 and 10-2 (Figure 7) in which sole one showed
positive result. On the other hand, for the non-heated results, at least two types of isolates were
studied being, small white opaque and small yellow opaque isolates referring to Figure 8.
According to Gupta, Gupta and Rathi (2004), some of the possible lipase-producing
microorganisms relating to the sample used are Bacillus spp., Pseudomonas spp., Staphylococcus
aureus, etc. Referring to Table 1, the dilution of 10-1 and 10-2 (Figure 9) exhibit microbial
growth but not positive results. This phenomenon might be due to the suppression of lipase-
producing microorganisms within other dominant species at such low dilution factor.
sample with dilution of 10- streaking inoculation of
1
on milk agar plate. non-heated sample with
dilution of 10-1 on milk
agar plate.
Figure 12: Spread plating
inoculation of heated
Figure 13: Single-line
inoculation of non-heated
sample with dilution of 10-
3
on milk agar plate.
Figure 14: Spread plating
Referring to Table 1, milk agar plates all showed negative results which indicates the
nonexistence of protease-producing microorganisms. Milk agar consists of casein and glucose as
carbon sources for microbial growth, yeast extract as source of vitamins and agar as solidifying
agent. According to Aryal (2019), the presence of proteinase, caseinase, will hydrolysed casein
to form soluble nitrogen compounds which are responsible for the clear zone in positive results.
In the heat treated samples, little amount of isolates in even low dilution factor (Figure 12) made
it clear that most of the microorganisms in sample are not heat tolerant as seen in prior agar as
well. As for non-heat treated sample, there are at least three types of microorganisms present as
seen in Figure 13 and Table 1. However, the isolates were deemed not proteinase-producing due
to lack of clear zone in all 10 -1 to 10-4 dilutions (Figure 14). Do note that there is a possibility of
accidentally excluding casein during the preparation of milk agar as negative result was observed
in positive control as well as stated above.
Conclusion
In conclusion, this experiment is deemed successful overall due to the presence of
amylase-producing and lipase-producing microorganisms detected with the exception of
protease-producing microorganisms. This undetected microorganism might be due to lack of
such microflora in sample used or problem that arose during the preparation of milk agar, such as
the unexpected exclusion of casein during production. Moreover, all control has shown expected
results as well only for positive control, Bacillus cereus, in milk agar being a problem. After the
search for informative articles and researches online, the microorganisms that are possibly
related to sample used, rotten lettuce stem, might be Bacillus cereus, Pseudomonas spp.,
Clostridium spp., Rhizopus spp. and Staphylococcus aureus. A more detailed experiment should
be carried out with the aim of more correctly ensuring the identity of microorganisms detected.
References
Aryal, S., 2019. Casein Hydrolysis Test - Principle, Procedure, Uses and Interpretation. [online]
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test/ [Accessed 15 July 2023].
Aryal, S., 2019. Starch Hydrolysis Test - Principle, Procedure, Uses and Interpretation. [online]
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Sourav, 2022. Lipid Hydrolysis Test Principle, Procedure, Result. [online] Microbiologynote.
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Investigation on the Microbial Diversity of Fresh-Cut Lettuce during Processing and
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Turnitin Checking
