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
Extended Essay - Biology
Shin-Ae Lee
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 4째C 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 37.5째C. 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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Extended Essay - Biology
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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 Page 3 of 51
Extended Essay - Biology
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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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Extended Essay - Biology
Shin-Ae Lee
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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Extended Essay - Biology
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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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Extended Essay - Biology
Shin-Ae Lee
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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Extended Essay - Biology
Shin-Ae Lee
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,â€&#x; which in this context mean that sodium chloride was dissolved in 100 ml of distilled water.
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Extended Essay - Biology
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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
10% NaCl Solution (w/v)
MRS Agar Solution
Beakers
Distilled Water
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Extended Essay - Biology
Shin-Ae Lee
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 ÎźL of pure aqueous doenjang extract using the micropipette. 4. To maintain equilibrium, add the same amount of pH buffer (500 ÎźL) to the extract of the rightly labeled microcentrifuge using the micropipette. 5. Thoroughly mix each microcentrifuge using the electronic vortex mixer.
6 7 8
See appendix 4. This process is repeated until sufficient amount of extract is obtained for the investigation. See Appendix 5.
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6. Incubate these microcentrifuges in the refrigerator at 4째C 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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Extended Essay - Biology
Shin-Ae Lee
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
Volume of Sterilized
Total Volume
Extract at Different pH
Distilled Water / ml
/ ml
Levels / 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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Extended Essay - Biology
Shin-Ae Lee
Table 3.5.3: Dilution table of doenjang extract at different NaCl concentrations Dilution
Volume of Doenjang
Volume of Sterilized
Total Volume
Extract at Different pH
Distilled Water / ml
/ ml
Levels / 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 ÎźL 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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Extended Essay - Biology
Shin-Ae Lee
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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Extended Essay - Biology
Shin-Ae Lee
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
26
15
10-3 Dilution Plates
A
B
6
1
2
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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Extended Essay - Biology
Shin-Ae Lee
Table 4.1.2: Dilution Plates of doenjang extract at pH 3
pH 3 -2
10 Dilution Plates
A 52
B 38
42
30
10-3 Dilution Plates
A 10
B 2
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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Extended Essay - Biology
Shin-Ae Lee
Table 4.1.3: Dilution Plates of doenjang extract at pH 5
pH 5 -2
10 Dilution Plates
A 61
B 29
63
26
10-3 Dilution Plates
A 15
B 8
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 -2
10 Dilution Plates
A 79
B 50
71
52
10-3 Dilution Plates
A 10
B 1
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 -2
10 Dilution Plates
A 60
B 24
50
30
10-3 Dilution Plates
A 10
B 3
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 -2
10 Dilution Plates
A 0
B 0
0
0
10-3 Dilution Plates
A 0
B 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
Molds / CFU per 0.05ml
0.05ml pH
Diluti
Plate 1
Plate 2
Average
Plate 1
Plate 2
Average
10-2
26
15
20.5
6
5
5.5
10-3
1
1
1
2
0
1
10-2
52
42
47
38
30
34
10-3
10
14
12
2
6
4
10-2
61
63
62
29
26
27.5
10-3
15
16
15.5
8
4
6
10-2
79
71
75
50
52
51
10-3
10
19
14.5
1
4
2.5
10-2
60
50
55
24
30
27
10-3
10
8
9
3
0
1.5
10-2
-
-
-
-
-
-
10-3
-
-
-
-
-
-
on
pH 1 pH 3 pH 5 a
pH 7
pH 11 pH 14 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 -3
10 Dilution Plates
A 20
B 6
15
7
10-4 Dilution Plates
A 23
B 0
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 -3
10 Dilution Plates
A 26
B 12
27
13
10-4 Dilution Plates
A 5
B 1
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 -3
10 Dilution Plates
A 14
B 10
29
13
10-4 Dilution Plates
A 1
B 6
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 -3
10 Dilution Plates
A 32
B 15
30
23
10-4 Dilution Plates
A 0
B 1
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 -3
10 Dilution Plates
A 4
B 11
22
12
10-4 Dilution Plates
A 1
B 0
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
Molds / CFU per 0.05 ml
0.05 ml NaCl a
0%
10% 20%
Dilution
Plate 1
Plate 2
Average
Plate 1
Plate 2
Average
10-3
20
15
17.5
6
7
6.5
10-4
23
0
11.5
0
2
1
10-3
26
27
26.6
12
13
12.5
10-4
5
35
20
1
2
1.5
10-3
14
29
21.5
10
13
11.5
10
1
0
0.5
6
0
3
10-3
32
30
31
15
23
19
10-4
-
-
-
1
0
0.5
10-3
4
22
13
11
12
11.5
10-4
1
3
2
0
1
0.5
-4
30% 40% b
NaCl 10%
-
(-) = no activity (b) = Negative Control (a) = Positive Control
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4.3 Data Processing for Calculating CFU Calculations for CFU/ml of doenjang solution -
Number of CFU at 10 đ?›‚ dilution plate (per 50 ÎźL = 0.05 ml) =
ď ą
Number of CFU at 100 dilution plate (per 50 ÎźL = 0.05 ml) =
ď ą ď‚´10ď Ą
Number of CFU at 100 dilution plate (per 5 ml) =
ď ą ď‚´10ď Ą ď‚´100
Number of CFU at 100 dilution plate (per 1 ml)
ď ą ď‚´10ď Ą ď‚´100 =
5 Calculations for CFU/ml of original doenjang
Number of CFU at 100 dilution plate (per 1 ml) of original doenjang concentration
ď ą ď‚´10ď Ą ď‚´100 =
5
ď‚´2
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 ď€ď Ą dilution plate (per 0.05 ml) ď‚´ 100 ď‚´ 10ď Ą ď‚´2 5
=
20.5 ď‚´100 ď‚´10 2 ď‚´2 5
=
205,000 ď‚´2 5
= 82,000 Page 29 of 51
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= 8.2 104 CFU/ml Table 4.3.1: Number of CFU/ml in original concentration of doenjang affected by different pH levels Doenjang at different pH
B (a)
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
buffer
(a) = Calculation refers to
Number of CFU at 10 dilution plate (per 0.05 ml) 100 10 2 5
(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
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
concentration / %
(a) = Calculation refers to
Number of CFU at 10 dilution plate (per 0.05 ml) 100 10 2 5
(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 55
LAB Mold Total (LAB+Mold)
50.4
Number of CFU/ml Ă— 104
50 45 40
35.8 32.4
35 30
24.8
25 13.6
15 5
20.4
18.8
20 10
30.8
30
10.4
8.2
11
20 10.8
2.2
0 pH 1
pH 3
pH 5
pH 7 (Control)*
pH 11
pH 14
pH Buffer Level * = 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 (%) LAB 25
Mold Total (LAB+Mold)
Number of CFU/ml Ă— 105
22.5
20
20 17.5
15.6
15
13.2
12.5 9.6
10 7.5 5
12.4
10.6
9.8
8.6
7.6
7 5
4.6
10
20
5.2 4.6
2.6
2.5 0 0 (Control) *
30
40
Salt Concentration / %
* = 0% NaCl is interpreted as a controlled value as it was substituted with distilled water (neutral medium).
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Extended Essay - Biology
Shin-Ae Lee
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, Page 34 of 51
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Shin-Ae Lee
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 Page 35 of 51
Extended Essay - Biology
Shin-Ae Lee
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/mlĂ—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 canâ€&#x;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 Page 36 of 51
Extended Essay - Biology
Shin-Ae Lee
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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Extended Essay - Biology
Shin-Ae Lee
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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Shin-Ae Lee
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 Page 39 of 51
Extended Essay - Biology
Shin-Ae Lee
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 Page 40 of 51
Extended Essay - Biology
Shin-Ae Lee
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. Page 41 of 51
Extended Essay - Biology
Shin-Ae Lee
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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Extended Essay - Biology
Shin-Ae Lee
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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Extended Essay - Biology
Shin-Ae Lee
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." Page 44 of 51
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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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Extended Essay - Biology
Shin-Ae Lee
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 isnâ&#x20AC;&#x;t as extreme. This peculiar taste is produced by a bacterium called Bacillus, which is inferred to have antibiotic characteristics.
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Extended Essay - Biology
Shin-Ae Lee
2. Journal: Analysis of microbial communities in doenjang, a Korean fermented soybean paste, using nested PCR-denaturing gradient gel electrophoresis
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Extended Essay - Biology
Shin-Ae Lee
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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Extended Essay - Biology
Shin-Ae Lee
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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Shin-Ae Lee
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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Extended Essay - Biology
Shin-Ae Lee
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
pH level
compared to distilled water 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
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