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Transcript of IB EE on the effect of pH and salt on the growth of Lactobacillus in daejang
Extended Essay - Biology Shin-Ae Lee
International Baccalaureate
Extended Essay
Biology
In Vitro Study of the Effect of pH and Salt Concentration on the
growth of Lactic Acid Bacteria and Mold in Doenjang (Korean
Fermented Soybean Paste)
Word Count: 3993
Name: Shin-Ae Lee
Candidate Number: 002213-062
School: Taejon Christian International School
Exam Session: May 2010
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Abstract
Doenjang (Korean fermented soybean paste) is a traditional fermented soybean food
in Korea. It not only has been consumed as food, but has also been used as folk medicine for
emergency treatments. This is due to lactic acid bacteria (LAB) and probiotic mold in
doenjang.
This research investigated the in vitro effect of pH and salt on the microbial growth
of LAB/mold in doenjang using the standard viable plate count method. This research was
divided into two parts: incubating doenjang extract at different pH and at various salt
concentrations. Doenjang extracts were incubated separately at pH 1, 3, 5, 7, 11, 14, and in
0%, 10%, 20%, 30% and 40% salt concentrations in 4°C for 2 days. After incubation the pH
and doenjang solutions were diluted to 10-3
, and salt concentrations and doenjang to 10-4
.
These were plated out on MRS agar and were further incubated for 15 hours in 37.5°C. The
bacteria concentrations were determined by counting the colony-forming unit (CFU).
Results revealed that there was viable growth of LAB/mold in pH 1, 3, 5, 7 and 11,
but not in pH 14. The CFU of LAB/mold in pH 1 decreased by 79% in comparison to pH 7,
the optimum level. The CFU of LAB/mold increased from pH 1 to 7, but decreased from pH
7 to 14. Whereas, there were viable cell counts in doenjang at all salt concentrations from 0%
to 40%. The CFU of LAB/mold increased from 0% to 30% salt concentration and decreased
drastically from 30% to 40%. The optimum level was shown at 30% salt concentration.
In conclusion, LAB/mold were viable in all salt concentrations and all pH levels
except for pH 14.
(Word Count: 279)
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Table of Contents
Abstract ................................................................................................................................... 2
Table of Contents ................................................................................................................... 3
1. Introduction .......................................................................................................................... 5
1.1 About Doenjang.......................................................................................................... 5
1.2 Rationale of Study ...................................................................................................... 5
1.3 About Bacteria in Doenjang ....................................................................................... 6
1.4 Aim ............................................................................................................................. 7
2. Variables ............................................................................................................................... 8
2.1 Independent Variable.................................................................................................. 8
2.2 Controlled Variable .................................................................................................... 8
2.3 Dependent Variable .................................................................................................... 8
3. Procedures ............................................................................................................................ 9
3.1 Preparation of MRS Agar Plate .................................................................................. 9
3.2 Preparation of Sodium Chloride Solution .................................................................. 9
3.3 Method for Sterilizing ................................................................................................ 9
3.4 Preparation of Aqueous Doenjang Extract at Different pH Levels and Salt
Concentration ............................................................................................................. 9
3.5 Dilution of Aqueous Doenjang Extract .................................................................... 10
3.6 Method for Plating on MRS Agar Plate ................................................................... 13
3.7 Incubation ................................................................................................................. 14
3.8 Bacteria Count .......................................................................................................... 15
4. Data Collection ................................................................................................................... 16
4.1 Raw Data for Doenjang at pH Levels....................................................................... 16
4.2 Raw Data for Doenjang at Salt Concentrations ........................................................ 23
4.3 Data Processing for Calculating CFU ...................................................................... 29
4.4 Data Presentation ...................................................................................................... 32
5. Data Analysis ...................................................................................................................... 34
5.1 Observation of the Effect of pH on Doenjang .......................................................... 34
5.2 Observation of the Effect of Salt Concentration on Doenjang ................................. 35
6. Discussion............................................................................................................................ 38
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6.1 Effect of pH on Doenjang ......................................................................................... 38
6.2 Effect of Salt on Doenjang ....................................................................................... 40
6.3 Limitations and Improvements ................................................................................. 41
6.4 Further Investigation ................................................................................................ 41
7. Conclusion .......................................................................................................................... 43
8. References ........................................................................................................................... 44
9. Appendix ............................................................................................................................. 46
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1. Introduction
1.1 About Doenjang
Doenjang (Korean fermented soybean paste) is a traditional fermented soybean food
that was developed in Korea along with other processed soybean foods (1). Because of its rich
protein source as well as its taste for enhancing foods, doenjang has been an essential part of
Korean food from history. Doenjang has also been used as a folk medicine for emergency
treatments, believed to remove toxins from insect or snake bites, or simply for stopping
bleeding, etc. Its medicinal functions were first described in Dongeuibogam (1613 A.D.),
which was a popular traditional Korean medical text (1).
There are mainly two different kinds of doenjang: one made by the conventional type,
and one by the improved type. These two doenjangs differ in methods of making1, as well as
their tastes: the traditional type gives a strong, stinging smell with a salty taste, while the
improved type isn‟t as extreme. This peculiar taste is produced by a bacterium called Bacillus,
which is inferred to have antibiotic characteristics.
1.2 Rationale of Study
Since I was young, I‟ve heard many series of the medical efficacy of the Korean
fermented soybean paste, doenjang, from my grandparents. In the early 1900‟s when medical
wasn‟t well developed yet in Korea, doenjang took place as medicines in many ways. When
my grandfather got a bruise from bumping his head on the edge of the desk, his mother
pasted a spoonful of doenjang on the bruise to arrest bleeding. My grandmother used to paste
doenjang on her leg when it got swollen from being bitten by bugs or from getting scalded.
As such, doenjang in Korea has been used as a folk remedy from the old times until this day.
1 See Appendix 1.
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I remember my mother fixing me doenjang soup when I had stomach troubles. This sparked
my curiosity, wondering, „how can a food have such medical efficacy.‟ As a biology student, I
soon became interested in the chemical elements of doenjang and whether it really has some
sort of medical function. Then after few days, I found a thought-provoking journal2 written
by few Korean researchers about the microbial communities in doenjang. Interestingly
enough, it was written that “several types of lactic acid bacteria [LAB] including L.
mesenteroides, T. halophilus, and E. faecium were observed as the predominant bacterial
species” in doenjang (3). Knowing that LAB is pro-biotic, I found it worthy to further
investigate to what extent this characteristic is preserved in affect to different pH levels and
salt concentration.
1.3 About Bacteria in Doenjang
According to previous researches, several microorganisms were identified in
doenjang. These include molds such as Aspergillus Mucor and Rhizopus species that were
detected in meju3. Recently in 2007, Korean researchers have investigated the microbial
communities in doenjang and announced an unexpected observation that Staphylococcus
equorum and some lactic acid bacteria are the dominant species in doenjang, instead of
previous founding that Bacillus. subtilis is the primary bacteria in doenjang (3). In addition to
these microorganisms, several fungi and yeast species were also found to be present in
doenjang (3).
2 See Appendix 2.
3 Meju is a dried, fermented soybean block, which is further fermented with salt water to become doenjang.
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1.4 Aim
The aim of this investigation is to explore the effect of different pH levels and salt
concentration in doenjang, and whether LAB/mold will be viable in extreme pH and salt
conditions.
Therefore, my precise research question is:
In Vitro Study of the Effect of pH and Salt Concentration on the growth of
Lactic Acid Bacteria in Doenjang (Korean Fermented Soybean Paste)
This investigation is divided into two parts: investigating in different pH levels and
in different salt concentrations. These investigations are made possible by plating out on
MRS4 agar, which cultivates LAB/mold.
4 See Appendix 3.
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2. Variables
2.1 Independent Variable
2.1.1 pH
Doenjang is incubated at different pH level in the refrigerator for 2 days and further
incubated after plating in a MRS agar. The different pH levels are pH 1, 3, 5, 7, 11, and 14.
2.1.2 Salt
Doenjang is incubated at different salt concentration, controlled by the amount of
sodium chloride dissolved in 100 ml of distilled water. Salt concentration will be varied by
0%, 10%, 20%, 30% and 40% NaCl (w/v)5.
2.2 Controlled Variable
The fixed variables are the temperature of the room, refrigerator, and incubator; the
time of incubation in the refrigerator and in the incubator; the pH and volume of MRS agar
plate; the amount of solution inoculated on the MRS agar.
2.3 Dependent Variable
Number of bacterial colonies forming on the surface of the MRS agar plate is
counted in colony forming unit (CFU). To avoid plentiful bacteria covering the petri dish,
serial dilution technique is used to dilute the samples. After incubation, the bacteria counted
will go through further calculation to get the amount of bacterial population before dilution.
5 (w/v) indicate „with volume,‟ which in this context mean that sodium chloride was dissolved in 100 ml of
distilled water.
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3. Procedures
3.1 Preparation of MRS Agar Plate
1. Add 70 g of MRS agar powder to 1,000 cm3 of distilled water using a volumetric
flask.
2. Heat the mixture while stirring to dissolve the powder completely.
3. After the mixture has boiled for 1 minute, remove from heat and pour it into a glass
bottle.
4. Leave the glass bottle cap loosened to allow steam to escape and prevent explosion in
the autoclave. Sterilize the agar solution in the pressure autoclave.
5. Pour approximately 30 cm3 into each petri dish and cover the lids after it has been
hardened.
3.2 Preparation of Sodium Chloride Solution
1. Add 10g of sodium chloride to 100 cm3 of distilled water using a volumetric flask.
2. Stir until completely dissolved.
3. Repeat step 1 and 2 by substituting 10g with 20g, 30g, and 40g for 10%, 20%, 30%
and 40% NaCl Solution (w/v).
3.3 Method for Sterilizing
3.3.1 Essential Apparatus to sterilize:
Cheese cloth
MRS Agar Solution
10% NaCl Solution (w/v)
Beakers
Distilled Water
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3.3.2 Method of sterilizing
Sterilizing all solutions and instruments to be used in the experiment, including the
essential apparatus listed above, is a significant step in this investigation, as it deals with
bacteria. This will be done by using a pressure autoclave.6
3.4 Preparation of Aqueous Doenjang Extract at Different pH Levels and
Salt Concentration
3.4.1 Preparation of aqueous doenjang extract7
1. Spray alcohol (70% ethanol) on the lab table and leave it until completely dried.
2. Place the sterilized cheese cloth on the lab table and place approximately 30 g of
doenjang8.
3. Squeeze out doenjang extract in a sterilized beaker.
3.4.2 Preparation of aqueous doenjang extract at different pH Levels
1. Purchase the following pH buffers from Carolina: pH 1, 3, 5, 7, 11 and 14. (pH 7 can
be substituted with autoclaved distilled water.)
2. Label 6 microcentrifuges as the different pH levels.
3. Fill in 500 μL of pure aqueous doenjang extract using the micropipette.
4. To maintain equilibrium, add the same amount of pH buffer (500 μL) to the extract of
the rightly labeled microcentrifuge using the micropipette.
5. Thoroughly mix each microcentrifuge using the electronic vortex mixer.
6 See appendix 4.
7 This process is repeated until sufficient amount of extract is obtained for the investigation.
8 See Appendix 5.
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6. Incubate these microcentrifuges in the refrigerator at 4°C for 2 days.9
3.4.3 Preparation of aqueous doenjang extract at different salt concentration
1. Prepare the following sterilized salt concentration solution: (0%, 10%, 20% and 30%)
NaCl (w/v). (0% NaCl can be substituted with sterilized distilled water.)
2. Repeat from step 2 to 6 of 3.4.2, but using salt concentration instead of pH buffers.
3.5 Dilution of aqueous doenjang extract
Serial dilution technique is used to avoid too much bacteria from covering the petri
dish, which gives difficulty in counting the bacterial population. After 2 days of incubation in
the refrigerator, leave the microcentrifuges of aqueous doenjang extract at different pH levels
and salt concentration at room temperature for 10 minutes.
9 This is to ensure that the bacteria in doenjang are completely affected by the specific medium.
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Diagram 3.5.1: Procedure for serial dilution of doenjang at different pH and salt
concentration
Table 3.5.2: Dilution table of doenjang extract at different pH levels
Dilution Volume of Doenjang
Extract at Different pH
Levels / ml
Volume of Sterilized
Distilled Water / ml
Total Volume
/ ml
10-1
0.1 0.9 1.00
10-2
0.1 (10-1
)* 0.9 1.00
10-3
0.1 (10-2
)* 0.9 1.00
* Extract taken from the previous dilution.
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Table 3.5.3: Dilution table of doenjang extract at different NaCl concentrations
Dilution Volume of Doenjang
Extract at Different pH
Levels / ml
Volume of Sterilized
Distilled Water / ml
Total Volume
/ ml
10-1
0.1 0.9 1.00
10-2
0.1 (10-1
)* 0.9 1.00
10-3
0.1 (10-2
)* 0.9 1.00
10-4
0.1 (10-3
)* 0.9 1.00
* Extract taken from the previous dilution.
3.6 Method for Plating on MRS Agar Plate
3.6.1 Plating of aqueous doenjang extract at different pH level
Plate out doenjang extract with pH 1, 3, 5, 7, 11, and 14 incubated for 2 days in the
refrigerator on MRS agar plate with dilution 10-2
and 10-3
.
3.6.2 Plating of aqueous doenjang extract at different salt concentration
Plate out doenjang extract with (0%, 10%, 20% and 30%) NaCl incubated for 2 days
in the refrigerator on MRS agar plate with dilution 10-3
and 10-4
.
1. Label the bottom of the petri dishes as labeled on the microcentrifuge.
2. Use a micropipette to drop 50 μL of solution in the middle of the rightly labeled MRS
agar plate.
3. Use a sterile cotton swab to swab the surface of the nutrient agar in the direction as
showed in diagram 3.6.3.1.
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Diagram 3.6.2.1: Direction of swabbing on the MRS agar plate
4. Close the lid of the petri dish.
5. Replication is necessary for a more accurate data. Therefore, repeat step 1 to 4 using
the same solution.
6. Repeat step 1 to 5 for different diluted solutions of pH and salt.
3.6.3 Plating of negative controls
Plate out negative controls in duplicate to check if there are any kinds of
contamination. Plate out 50 μL of sterilized distilled water and 50 μL of 10% NaCl using the
same method of plating on MRS agar.
3.7 Incubation
A total of 44 petri dishes are placed in an electronic incubator upside down.10
Adjust the
temperature of the incubator to 37.5°C. Keep them in the incubator for 15 hours.
10
This is to prevent water vapors from dropping on the surface of the MRS agar.
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3.8 Bacteria Count
According to previous researches, it is expected to see bacteria colonies and molds
on the MRS agar plate after incubation.
1. After incubation for 15 hours, take out the MRS agar plates and leave it in room
temperature to cool down.
2. With the agar plate upside down, count the CFU of LAB by marking dots on the petri
dish when a round bacteria (LAB) is found.
3. Count the CFU of mold by marking dots with a different color on the petri dish when
a blurred colony (mold) is found.
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4. Data Collection
4.1 Raw Data for pH and Doenjang
Table 4.1.1: Dilution Plates of doenjang extract at pH 1
pH 1
10-2
Dilution Plates A B 10-3
Dilution Plates A B
26 6
1 2
15 5
1 0
(*) = Replicate 1
(**) = Replicate 2
A = Number of CFU of LAB (per 50 μL dilution plated)
B = Number of CFU of molds (per 50 μL dilution plated)
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Table 4.1.2: Dilution Plates of doenjang extract at pH 3
pH 3 10
-2 Dilution Plates A B 10
-3 Dilution Plates A B
52 38
10 2
42 30
14 6
(*) = Replicate 1
(**) = Replicate 2
A = Number of CFU of LAB (per 50 μL dilution plated)
B = Number of CFU of molds (per 50 μL dilution plated)
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Table 4.1.3: Dilution Plates of doenjang extract at pH 5
pH 5 10
-2 Dilution Plates A B 10
-3 Dilution Plates A B
61 29
15 8
63 26
16 4
(*) = Replicate 1
(**) = Replicate 2
A = Number of CFU of LAB (per 50 μL dilution plated)
B = Number of CFU of molds (per 50 μL dilution plated)
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Table 4.1.4: Dilution Plates of doenjang extract at pH 7
pH 7 10
-2 Dilution Plates A B 10
-3 Dilution Plates A B
79 50
10 1
71 52
19 4
(*) = Replicate 1
(**) = Replicate 2
A = Number of CFU of LAB (per 50 μL dilution plated)
B = Number of CFU of molds (per 50 μL dilution plated)
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Table 4.1.5: Dilution Plates of doenjang extract at pH 11
pH 11 10
-2 Dilution Plates A B 10
-3 Dilution Plates A B
60 24
10 3
50 30
8 0
(*) = Replicate 1
(**) = Replicate 2
A = Number of CFU of LAB (per 50 μL dilution plated)
B = Number of CFU of molds (per 50 μL dilution plated)
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Table 4.1.6: Dilution Plates of doenjang extract at pH 14
pH 14 10
-2 Dilution Plates A B 10
-3 Dilution Plates A B
0 0
0 0
0 0
0 0
(*) = Replicate 1
(**) = Replicate 2
A = Number of CFU of LAB (per 50 μL dilution plated)
B = Number of CFU of molds (per 50 μL dilution plated)
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Table 4.1.7: Data collection from doenjang extract at different pH level
Lactic Acid Bacteria / CFU per
0.05ml
Molds / CFU per 0.05ml
pH Diluti
on
Plate 1 Plate 2 Average Plate 1 Plate 2 Average
pH 1 10-2
26 15 20.5 6 5 5.5
10-3
1 1 1 2 0 1
pH 3 10-2
52 42 47 38 30 34
10-3
10 14 12 2 6 4
pH 5 10-2
61 63 62 29 26 27.5
10-3
15 16 15.5 8 4 6
a pH 7
10-2
79 71 75 50 52 51
10-3
10 19 14.5 1 4 2.5
pH 11 10-2
60 50 55 24 30 27
10-3
10 8 9 3 0 1.5
pH 14 10-2
- - - - - -
10-3
- - - - - -
b Distilled Water -
(-) = no activity
(b) = Negative Control
(a) = Positive Control
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4.2 Raw Data for Salt concentration and Doenjang
Table 4.2.1: Dilution plates of doenjang extract at 0% NaCl
0% NaCl 10
-3 Dilution Plates A B 10
-4 Dilution Plates A B
20 6
23 0
15 7
0 2
(*) = Replicate 1
(**) = Replicate 2
A = Number of CFU of LAB (per 50 μL dilution plated)
B = Number of CFU of molds (per 50 μL dilution plated)
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Table 4.2.2: Dilution Plates of doenjang extract at 10% NaCl
10% NaCl 10
-3 Dilution Plates A B 10
-4 Dilution Plates A B
26 12
5 1
27 13
35 2
(*) = Replicate 1
(**) = Replicate 2
A = Number of CFU of LAB (per 50 μL dilution plated)
B = Number of CFU of molds (per 50 μL dilution plated)
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Table 4.2.3: Dilution plates of doenjang extract at 20% NaCl
20% NaCl 10
-3 Dilution Plates A B 10
-4 Dilution Plates A B
14 10
1 6
29 13
0 0
(*) = Replicate 1
(**) = Replicate 2
A = Number of CFU of LAB (per 50 μL dilution plated)
B = Number of CFU of molds (per 50 μL dilution plated)
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Table 4.2.4: Dilution Plates of doenjang extract at 30% NaCl
30% NaCl 10
-3 Dilution Plates A B 10
-4 Dilution Plates A B
32 15
0 1
30 23
0 0
(*) = Replicate 1
(**) = Replicate 2
A = Number of CFU of LAB (per 50 μL dilution plated)
B = Number of CFU of molds (per 50 μL dilution plated)
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Table 4.2.5: Dilution Plates of doenjang extract at 40% NaCl
40% NaCl 10
-3 Dilution Plates A B 10
-4 Dilution Plates A B
4 11
1 0
22 12
3 1
(*) = Replicate 1
(**) = Replicate 2
A = Number of CFU of LAB (per 50 μL dilution plated)
B = Number of CFU of molds (per 50 μL dilution plated)
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Table 4.2.6: Data collection from doenjang extract at different salt concentration
Lactic Acid Bacteria / CFU per
0.05 ml
Molds / CFU per 0.05 ml
NaCl Dilution Plate 1 Plate 2 Average Plate 1 Plate 2 Average
a 0% 10-3
20 15 17.5 6 7 6.5
10-4
23 0 11.5 0 2 1
10% 10-3
26 27 26.6 12 13 12.5
10-4
5 35 20 1 2 1.5
20% 10-3
14 29 21.5 10 13 11.5
10-4
1 0 0.5 6 0 3
30% 10-3
32 30 31 15 23 19
10-4
- - - 1 0 0.5
40% 10-3
4 22 13 11 12 11.5
10-4
1 3 2 0 1 0.5
b NaCl 10% -
(-) = no activity
(b) = Negative Control
(a) = Positive Control
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4.3 Data Processing for Calculating CFU
For example, in doenjang at pH 1, the average number of CFU at 10-2
dilution plate per 0.05
ml is 20.5.
Number of CFU/ml = Number of CFU at 10 dilution plate (per 0.05 ml) 100 10
25
= 220.5 100 10
25
= 205,000
25
= 82,000
Calculations for CFU/ml of doenjang solution
Number of CFU at 10-𝛂 dilution plate (per 50 μL = 0.05 ml)
=
Number of CFU at 100 dilution plate (per 50 μL = 0.05 ml)
= 10
Number of CFU at 100 dilution plate (per 5 ml)
= 10 100
Number of CFU at 100 dilution plate (per 1 ml)
= 10 100
5
Calculations for CFU/ml of original doenjang
Number of CFU at 100 dilution plate (per 1 ml) of original doenjang concentration
=
10 1002
5
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= 48.2 10 CFU/ml
Table 4.3.1: Number of CFU/ml in original concentration of doenjang affected by
different pH levels
Doenjang at
different pH
buffer
A B (a)
C
LAB Mold LAB Mold
pH 1 20.5 5.5 8.2 2.2 10.4
pH 3 47 34 18.8 13.6 32.4
pH 5 62 27.5 24.8 11 35.8
pH 7 (b)
75 51 30 20.4 50.4
pH 11 50 27 20 10.8 30.8
pH 14 0 0 0 0 0
(a) = Calculation refers to Number of CFU at 10 dilution plate (per 0.05 ml) 100 10
25
(b) = Controlled value
A = Average number of CFU at 10-2
dilution plates per 0.05 ml for duplicate samples
B = Number of CFU/ml in pure doenjang × 104
C = Total number of CFU/ml of LAB and molds at in pure doenjang × 104
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Table 4.3.2: Number of CFU/ml in original concentration of doenjang affected by
different salt concentrations
Doenjang at
different salt
concentration / %
A B * C
LAB Mold LAB Mold
0 (b)
17.5 6.5 7.0 2.6 9.6
10 26.5 12.5 10.6 5.0 15.6
20 21.5 11.5 8.6 4.6 13.2
30 31.0 19 12.4 7.6 20
40 13.0 11.5 5.2 4.6 9.8
(a) = Calculation refers to Number of CFU at 10 dilution plate (per 0.05 ml) 100 10
25
(b) = Controlled value
A = Average number of CFU at 10-3
dilution plates per 0.05 ml for duplicate samples
B = Number of CFU/ml in pure doenjang × 105
C = Total number of CFU/ml of LAB and molds at in pure doenjang × 105
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4.4 Data Presentation
Graph 4.4.1: Number of CFU/ml in Doenjang at different pH Buffer
Levels
8.2
18.8
24.8
30
20
2.2
13.611
20.4
10.810.4
32.435.8
50.4
30.8
0
5
10
15
20
25
30
35
40
45
50
55
pH 1 pH 3 pH 5 pH 7 (Control)* pH 11 pH 14
pH Buffer Level
Nu
mb
er o
f C
FU/m
l × 1
04
LAB
Mold
Total (LAB+Mold)
* = pH 7 is interpreted as a controlled value as it was substituted with distilled water (neutral medium).
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Graph 4.4.2: Number of CFU/ml in Doenjang at different Salt
Concentrations (%)
7
10.6
8.6
12.4
5.2
2.6
5 4.6
7.6
4.6
9.6
15.6
13.2
20
9.8
0
2.5
5
7.5
10
12.5
15
17.5
20
22.5
25
0 (Control) * 10 20 30 40
Salt Concentration / %
Nu
mb
er o
f C
FU/m
l × 1
05
LAB
Mold
Total (LAB+Mold)
* = 0% NaCl is interpreted as a controlled value as it was substituted with distilled water (neutral medium).
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5. Data Analysis
5.1 Observation of the Effect of pH on Doenjang
5.1.1 LAB
Generally, the growth of LAB was dominant over the growth of mold in doenjang at
all pH levels, excluding pH 14. According to graph 4.3.1, the number of LAB started to
develop consistently from pH 1 to pH 7, reaching up to 30×104 CFU/ml. However, after pH 7,
it accompanied by a 33% sudden decrease in pH 11, and eventually showed no viable cell
growth in pH 14, an extreme alkaline solution.
In comparison to the controlled value—pH 7— viable cell counts decreased from (30
to 8.2)×104 CFU/ml with a 73% decrease in the number of LAB in doenjang at pH 1 (11). In
pH 3, the number decreased by approximately 37%, and in pH 5, by 17%. This general trend
suggests that as the acidity decreased, the number of LAB in doenjang increased.
In pH 14 of an extreme level of alkalinity, no growth of LAB was observed. This
indicates that, in opposition to the growth of LAB in acidic condition, as alkalinity increased,
the number of LAB in doenjang decreased. Overall, the observation of no visible growth at
extreme pH 14 but in pH 1 proposes that LAB grows better in acidic condition than alkaline.
This also suggests that LAB in doenjang has a remarkable ability to remain viable under a
broad range of pH conditions (10).
5.1.2 Mold
Generally, according to graph 4.3.1, the viable cell counts of mold at different pH
levels were always less than the number of LAB in doenjang. Moreover, the growth of mold
in doenjang at different pH levels showed no consistency. From this, we can hypothesize that
the growth of mold in doenjang is not affected by the acidity nor the alkalinity of the solution,
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except for the fact that there was no growth in extreme alkaline solution, pH 14.
Mold was most viable in pH 7, the controlled value, reaching up to 20.4×104 CFU/ml.
However, it showed the least survivability in pH 1, which decreased to 2.2×104 CFU/ml with
an 89% decrease from the controlled value. Furthermore, there was no viable growth of mold
in pH 14. This suggests that acidity and alkalinity somehow affects the growth of mold in
doenjang.
5.1.3 Total (LAB/Mold)
The general trend of the growth of LAB/mold is following the trend of the growth of
LAB in doenjang at different pH levels. There is an increase growth of LAB/mold as the
acidity decreases. When pH 1 was compared to pH 7, there was a 79% decrease in the viable
cell count. Though there was a 9% small increase of LAB/mold from pH 3 to pH 5,
consistent increase can be seen from pH 1 to pH 7 as the there is an increment of 29% from
pH 5 to pH 7. Then after, as the alkalinity increased from pH 7, the total number of
LAB/mold decreased from (50.4 to 30.8)×104
CFU/ml. In pH 14 LAB/mold were both unable
to survive in extreme alkaline concentration. Overall, this general trend supports that
LAB/mold are tolerant in extreme acidic level, but not towards extreme alkaline level. In
other words, the survivability of LAB/mold proves to be absent in extreme alkaline solution.
5.2 Observation of the Effect of Salt Concentration on Doenjang
5.2.1 LAB
According to graph 4.3.2, LAB in doenjang has the ability to survive under a broad
range of salt concentration from 0% to 40%. This graph also suggests that the optimum viable
cell count of LAB is found in 30% salt concentration, with approximately 56% increase in
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comparison to the controlled value—0% salt concentration. The viable cell count of LAB in
0% to 30% salt concentration all showed development. However, in an extreme 40% salt
concentration, a rapid decrease was shown with approximately 26% decrease and the least
viable cell count of LAB. This is the only decrease in comparison to the controlled value.
Overall, the growth of LAB is not consistent in effect to different salt concentration, though it
was survivable in all concentrations from 0% to 40%.
5.2.2 Mold
Overall, according to graph 4.3.2, there were growth of mold at all different salt
concentrations, but were less than the growth of LAB at different salt concentration. The
controlled value showed the least viable mold with 2.6 CFU/ml×105. Growth of mold was
still shown in the extreme, 40% salt concentration, but decreased about 39% from the growth
of mold in 30% salt concentration. It also showed no difference in the number of growth from
20% salt concentration. The maximum growth was observed in 30% salt concentration with a
66% increase compared to the controlled value. Predominantly, it is displayed in graph 4.3.2
that mold is survivable at all salt concentrations, but can‟t conclusively state which is the
optimum level for mold at different salt concentration.
5.2.3 Total (LAB/Mold)
The general trend suggests that the total of LAB/mold showed the most growth in
30% salt concentration, with a 52% increase in comparison to the controlled value. Though
there was a decrease of viable cell counts from 10% to 20% salt concentration, 15% decrease
seems insignificant in this trend as the number increased by 34% from 20% to 30%, and
decreased 51% from 30% to 40% salt concentration. Overall, the graph shows that LAB is
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dominant over the growth of mold, and that LAB/mold at 30% salt concentration showed the
most growth. It is significant to notice that at 40% salt concentration, the growth of
LAB/mold decreased in a high percentage, but were viable to survive in all salt
concentrations, including the extreme 40% concentration.
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6. Discussion
6.1 Effect of pH on doenjang
In cellular respiration, which is the breakdown of organic compounds, a chain of
reaction—glycolysis—takes place that converts glucose into a substance called pyruvate.
Fermentation is the process of this pyruvate being broken down anaerobically, producing
either lactate (lactic acid) or ethanol (alcohol) and CO2 (7).
Figure 6.1.1 Process of glycolysis and anaerobic fermentation
Image taken from: Prentice Hall Biology Textbook
In figure 6.1.1, it is shown that 2 NADH adds with 2 hydrogen molecules to convert
to 2 NAD+. This process in anaerobic fermentation is crucial as 2 NAD+ is being converted
to 2 NADH in the glycolysis. Thus, hydrogen ion concentration is very significant in the
overall process of cellular respiration.
Changing the pH level has the potential to disturb the whole process of fermentation.
This is because when the pH of a growth medium is changed, it also means that the hydrogen
ion concentration is being changed11
. Hydrogen ions have the potential to disrupt the bonds
that maintain the tertiary shape of the enzymes. These bonds are primarily hydrogen bonds
11
See appendix 6.
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and ionic interaction beween oppositely charged amino acids. Due to the broken bonds,
enzymes gets denatured and affects the efficiency of the catalysis as the active site no longer
maintains its original shape. Thus, since this catalysis is what causes the metabolic reactions
to occur, pH has the potential to affect the metabolic pathway in fermentation.
LAB/mold is an essential factor in the fermentation of doenjang. It is evidently
shown in graph 4.3.1 that the number of CFU/ml of LAB/mold changes at different pH levels.
The inability of LAB/mold to survive in pH buffer 14 can be due to the extreme
concentration of alkalinity in the solution. pH 14 consists of 1/10,000,000 hydrogen ion
concentration in comparison to distilled water. Thus, this concentration could affect the LAB
metabolism pathway that disrupts the survivability of LAB/mold due to the enzyme being
denatured. Though pH 1, another extreme pH buffer level, has the least viable LAB/mold, the
growth of LAB/mold itself in such an acidic level is showing that doenjang is high acid
tolerant. This may suggest that there are acid tolerant mechanisms in LAB/mold in doenjang
that prevent the enzyme from being denatured. This leads to a hypothesis that these
mechanisms may be the one removing the hydrogen ions out—an ionic pump (proton pump)
that pumps out hydrogen ion to prevent the buildup of hydrogen ion. Another suggestion is
that the enzymes involved in metabolic or fermentation pathway are resistant towards acidic
condition.
In pH 14, LAB/mold in doenjang wasn‟t able to survive through the extreme alkaline
concentration. This might be due to enzymes which are very susceptible towards high
hydroxide ion concentration. At extreme alkaline environment, the enzymes which are
involved in fermentation or metabolic pathway can be easily denatured due to the high
hydroxide ion concentration, by disrupting the tertiary structure of the enzyme. Another
possible reason is that LAB/mold doesn‟t possess any alkaline tolerant mechanism to pump
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out hydroxide ions that get into the cell. Thus, it can be deduced that enzyme and membrane
bound protein are highly sensitive to a change of pH, but less if it is acidic.
6.2 Effect of Salt on Doenjang
According to a microbiology research journal, “During the fermentation process,
addition of NaCl effectively inhibited the growth of aerobic bacteria and clostridia, but not
yeasts (8).” Since fermentation is a metabolic process that happens in anaerobic condition,
this suggests to us that LAB inhibited the growth of other pathogenic bacteria which are
susceptible to pH solution. This founding also directs us to the fact that NaCl improved the
quality of fermentation. This premise reflects on graph 4.3.2 as the viable mold in doenjang at
different salt concentrations is generally greater than the controlled value.
The ability to survive in all salt concentrations show that LAB in doenjang is
halophilic—survivable in environments with high salt concentration. It also shows that
salinity promoted the growth of the useful bacteria while inhibiting the growth of the
unfavorable bacteria. This might suggest that LAB/mold have mechanism which can
maintain the osmolarity of the cell and prevent them from dehydration. Possible mechanisms
like membrane bound protein pump, which pumps in water from its surrounding might help
to maintain its osmolarity. Another possible mechanism might be due to the presence of
Na+/K
+, which are embedded on the plasma membrane and help to regulate the movement of
Na+ ions into the cell. This pump will remove any accessible Na+ that diffuse in, and thus
maintain its osmolarity, enabling LAB/mold to survive under all different salt concentrations.
There might be osmotic regulation performed by enzymes which are osmotolerant
and are able to function at high salt concentration (12). Outer membrane bound protein for
LAB might have transport mechanisms which function as osmoregulants that help LAB to
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survive in high osmotic stress. Their outer membrane structure and phospholipid bilayer
composition might be different so as to allow them to survive in these conditions (13).
6.3 Limitations and Improvements
There is a high percentage of uncertainty to the viable cell count as it was difficult to
count the exact number of LAB/mold due to its similar appearance after a certain stage of
growth. Another uncertainty is that the number of CFU for LAB is difficult to determine as
the growth of mold will tend to cover up LAB. Thus, this bacteria cell count is biased and
uncertain. Period of incubation should be shortened, so that the mold will not overgrow on
the MRS agar, covering the LAB.
Due to time constrain, duplicate trial was done for this investigation. Thus, the result
is only limited to the data collected from the two trials.
Isolating and identifying the dominant LAB strain could not be done due to lack of
expertise and resources. Thus, all different kinds of LAB strains were counted together.
Moreover, MRS agar supports the growth of LAB as well as normal bacteria. Thus, the
number of CFU/ml of LAB/mold cannot be conclusively stated that they are actually
LAB/mold. Selective agar medium like Nitrite Actidione Polymyxin (NAP) or Raka Ray
Agar should be adopted, which only supports the growth of LAB.
6.4 Further Investigation
The dominant strain of the bacteria grown on the MRS agar can be isolated for
further investigation. Identifying this strain could lead to producing favorable characteristics,
and thus giving commercial values of the necessary production. Growing together the
probiotics of other food can provide a solution for people who are in need of certain nutrients.
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With this concept of synergism, culturing these probiotics can give rise to a production with
favorable characteristics in it.
The exact optimum condition of pH and salt concentration for the growth of
probiotic in doenjang can be further investigated. Finding out the optimum condition could
maximize the growth of the beneficial bacteria in doenjang. This could both support the
identified probiotic, as well as maximize their viability for their growth in a beneficial
environment.
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7. Conclusion
The initial aim of this research was to investigate the effect of pH levels and salt
concentration on the growth of LAB/mold in doenjang, and whether LAB/mold are still
viable in those extreme conditions. Results showed that LAB/mold survived in all tested pH
levels (pH 1, 3, 5, 7, 11, and 14), except in pH 14. The optimum growth occurred in pH 7, the
controlled value of the experiment. This possibly suggests that LAB/mold in doenjang consist
some sorts of acid tolerant mechanisms that support the growth even in extreme acidic
conditions.
The data also displayed that LAB/mold survived in all salt concentrations, from 0%
to 40% salt concentration with an optimum growth occurring in 30% salt concentration.
Again, it can be hypothesized that there are salt tolerant mechanisms in LAB/mold in
doenjang that helps maintain the osmolarity of the cell.
Thus, this research shows that LAB/mold are viable in acidic medium and high salt
concentrations.
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8. References
1. Park, Kun-Young, Jung, Keun-Ok. Fermented Soybean Products as Functional Foods:
Functional Properties of Doenjang (Fermented Soybean Paste). CRC Press, Print.
2. Unknown Author, Doenjang. 2009. Absolute Astronomy. 19 May 2009
<http://www.absoluteastronomy.com/topics/Doenjang>.
3. Kim, Hae-Yeong. "Analysis of microbial communities in doenjang, a Korean
fermented soybean paste, using nested PCR-denaturing gradient gel electrophoresis."
International Journal of Food Microbiology 265 no. 271 (2009):
4. deMan, Rogosa and Sharpe. 1960. J. Appl. Bacteriol. 23:130.
5. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical
microbiology, 9th ed. American Society for Microbiology, Washington, D.C.
6. BOOKRAGS STAFF. "Lactic Acid Bacteria". 2005. January 19 2010.
<http://www.bookrags.com/research/lactic-acid-bacteria-wmi/>.
7. Allott, Andrew. Mindorff, David. Biology Course Companion. New York: Oxford
University Press, 2007.
8. Y., Cai, S. Ohomomo, M. Ogawa, S. Kumai. "Effect of NaCl-tolerant lactic acid
bacteria and NaCl on the fermentation characteristics and aerobic stability of silage."
Journal of Applied Microbiology 83 no. 3 (1997): 307-317.
9. Baker, Ron. "pH and Fermentation." Ask A Scientist. Available from
http://www.newton.dep.anl.gov/askasci/mole00/mole00902.htm. Internet; accessed
24 January 2010.
10. Warnecke, Tanya, Gill Ryan T. "Organic acid toxicity, tolerance, and production in
Escherichia coli biorefining applications." Microbial Cell Factories (2005): 3.
11. Lee, S.K., Ji G.E., Park Y.H.. "The viability of bifodobacteria introduced into kimchi."
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The Society for Applied Microbiology (1998): 2.
12. Measures. J.C. (1975). The role of amino acids in osmoregulation of non-halophilic
bacteria. Nature 257:398-400.
13. Tsui, P., Helu, V. and Freundlich, M. (1998). Altered osmoregulation of ompF in
integration host factor mutants of Escherichia coli. J. Bacteriol. 170:4950-4954.
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9. Appendix
1. Method of making Doenjang
The traditional doenjang first starts with the preparation of meju, which is a naturally
fermented soybean block, as well as the main ingredient in making doenjang. Meju is a dried,
soybean block of solely crushed soybeans that were soaked in water for 12 hours and cooked
for 4 hours at 100°C. The enzymes in the fermentation of soybeans are mainly from the
microorganisms of meju. These soybean blocks are dried for 3 days in the air, tied up with
rice straw, and then traditionally hung at the edge of an eave for 1 to 2 months to initiate
natural fermentation, which involves Bacillus sp., molds, and yeasts on the outside of the
meju (1). It is in this process of fermentation that Bacillus subtilis, a type of bacteria in
doenjang, reproduce, consuming soybean protein and water in the meju. When the process of
fermentation is finished, these bacteria are transformed into spores and endospores, which is
the cause of the unpleasant ammonia smell produced during the fermentation. After the whole
process of fermentation, the meju are put into large opaque pottery jars with brine and left to
further ferment. It is at this stage of fermentation that various beneficial bacteria transform
the mixture into a further vitamin-enriched substance (2). Once the fermentation process is
done, the liquids and solids are separated. This solid part is the Korean fermented soybean
paste, doenjang. There are mainly two different kinds of doenjang: one made by the
conventional type, and one by the improved type. These two doenjangs differ in taste: the
traditional type gives a strong, stinging smell with a salty taste, while the improved type isn‟t
as extreme. This peculiar taste is produced by a bacterium called Bacillus, which is inferred
to have antibiotic characteristics.
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2. Journal: Analysis of microbial communities in doenjang, a Korean
fermented soybean paste, using nested PCR-denaturing gradient gel
electrophoresis
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3. MRS
Lactobacilli MRS Agar are used to isolate, enumerate, and cultivate Lactobacillus species.
They are based on the formulations of deMan, Rogosa, and Sharpe. (4) The expected
appearances of Lactobacilli are large, white colonies on the surface of the MRS Agar. (5)
DifcoTM
Lactocabilli MRS Agar from Becton Dickinson was used for this investigation.
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4. Method of Sterilizing using the Autoclave
1. Sterilize all solutions and instruments to be used in the experiment, including the
essential apparatus listed above.
2. Bottle caps of the liquid solutions should not be tightly screwed to avoid pressure
accumulation within the bottles.
3. Cheese cloth is put into a dry beaker and enclosed with aluminum foil to avoid from
getting wet from water vapor.
4. Pour water on the bottom of the steel plate in the autoclave*.
5. Enclose the pressure valve and all the other caps.
6. Once it reaches to pressure 15psi, control the heat to maintain this stage for 10 more
minutes.
7. Open the pressure valve and release the steam.
8. Take out the containers and leave it in room temperature to cool down.
* Pressure Steam Sterilizer Electric Model No.25X from All American
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5. Type of Doenjang Used
“Traditional, commercial, ripen fermented doenjang without preservatives. Didn‟t apply any
sort of heat to preserve enzymes in doenjang.”
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6. pH Scale
pH is in a logarithmic scale, and thus a change of one pH unit becomes a factor of 10 in
hydrogen ion concentration (10).
Concentration of hydrogen ions
compared to distilled water
pH level
10,000,000 pH 0
1,000,000 pH 1
100,000 pH 2
10,000 pH 3
1,000 pH 4
100 pH 5
10 pH 6
1 pH 7
1/10 pH 8
1/100 pH 9
1/1,000 pH 10
1/10,000 pH 11
1/100,000 pH 12
1/1,000,000 pH 13
1/10,000,000 pH 14