Effect of pH and salt on growth of lactic acid bacteria in doenjang

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


Page 2 of 51

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)


Page 3 of 51

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 4 of 51

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


Page 5 of 51

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.


Page 6 of 51

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.


Page 7 of 51

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.


Page 8 of 51

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.


Page 9 of 51

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


Page 10 of 51

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.


Page 11 of 51

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.


Page 12 of 51

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.


Page 13 of 51

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.


Page 14 of 51

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.


Page 15 of 51

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.


Page 16 of 51

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)


Page 17 of 51


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




Page 18 of 51


pH 5 10



61 29

15 8

63 26

16 4

(*) = Replicate 1

(**) = Replicate 2




Page 19 of 51


pH 7 10



79 50

10 1

71 52

19 4

(*) = Replicate 1

(**) = Replicate 2




Page 20 of 51


pH 11 10



60 24

10 3

50 30

8 0

(*) = Replicate 1

(**) = Replicate 2




Page 21 of 51


pH 14 10



0 0

0 0

0 0

0 0

(*) = Replicate 1

(**) = Replicate 2




Page 22 of 51

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


Page 23 of 51

4.2 Raw Data for Salt concentration and Doenjang

Table 4.2.1: Dilution plates of doenjang extract at 0% NaCl

0% NaCl 10



20 6

23 0

15 7

0 2

(*) = Replicate 1

(**) = Replicate 2




Page 24 of 51

Table 4.2.2: Dilution Plates of doenjang extract at 10% NaCl

10% NaCl 10



26 12

5 1

27 13

35 2

(*) = Replicate 1

(**) = Replicate 2




Page 25 of 51

Table 4.2.3: Dilution plates of doenjang extract at 20% NaCl

20% NaCl 10



14 10

1 6

29 13

0 0

(*) = Replicate 1

(**) = Replicate 2




Page 26 of 51


30% NaCl 10



32 15

0 1

30 23

0 0

(*) = Replicate 1

(**) = Replicate 2




Page 27 of 51


40% NaCl 10



4 11

1 0

22 12

3 1

(*) = Replicate 1

(**) = Replicate 2




Page 28 of 51

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


Page 29 of 51

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


Page 30 of 51

= 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


Page 31 of 51

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


Page 32 of 51

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).


Page 33 of 51

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).


Page 34 of 51

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 35 of 51

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 36 of 51

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


Page 37 of 51

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.


Page 38 of 51

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.


Page 39 of 51

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 40 of 51

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 41 of 51

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 42 of 51

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.


Page 43 of 51

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.


Page 44 of 51

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 45 of 51

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.


Page 46 of 51

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.


Page 47 of 51

2. Journal: Analysis of microbial communities in doenjang, a Korean

fermented soybean paste, using nested PCR-denaturing gradient gel

electrophoresis


Page 48 of 51

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.


Page 49 of 51

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


Page 50 of 51

5. Type of Doenjang Used

“Traditional, commercial, ripen fermented doenjang without preservatives. Didn‟t apply any

sort of heat to preserve enzymes in doenjang.”


Page 51 of 51

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

Effect of pH and salt on growth of lactic acid bacteria in doenjang

Education

effect of ph

salt concentrations

doenjang extracts

incubating doenjang

different ph levels

cfu of labmold

phand doenjang solutions

bacteria count