IB EE on the effect of pH and salt on the growth of Lactobacillus in meju

Extended Essay Jaeeun Kwon

1

Extended Essay

Biology

Effect of salt concentration and pH on the growth of

lactic acid bacteria and mold in meju (Korean

fermented soy bean)

3826 Words

Name: Jaeeun Kwon

Candidate Number: 002213-054

School: Taejon Christian International School


2

Abstract

Meju (a brick of dried fermented soybeans) serves as a base for condiments in

Korean cuisine. Fermented products are popular in Korean cuisine because they enhance non-

specific immune responses and disease resistance. They are also known to enhance growth

performances, feed efficiency and digestibility. The beneficial mold, Aspergillus oryzae,

contained in meju has been reported to produce extra-cellular enzymes.

This research examined the effect of salt and pH on the growth of lactic acid bacteria

and mold in meju. The meju was exposed to various concentrations of NaCl (10%, 20%, 30%,

40%) and pH (0, 2, 4, 7, 10, 14) to determine the optimal growth condition, and to observe

the range in which the bacteria can survive. After refrigerating the mixtures for seven days,

they were diluted to 10-5

. Then, they were spread on prepared Agar plates (MRS nutrient

agar). These plates were then incubated at approximately 37.5° C for 14 hours.

The experiment yielded results that showed that lactic acid bacteria and mold were

able to survive in all concentrations of salt. Nevertheless, the optimum growth was shown at

30% salt concentration, determined by the maximum colony-forming unit (CFU), a measure

of viable bacterial numbers. On the other hand, the results showed that lactic acid bacteria

were able to survive in all pH levels, but mold did not survive in pH 0. This was also

determined by CFU. The optimum growth occurred with pH 10 for bacteria and pH 2 for

mold.

In conclusion, lactic acid bacteria was able to survive in all salt concentrations and

all pH levels, and mold was able to survive in all salt concentrations and all pH levels but 0

pH.

(277 words)


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Table of Contents

Abstract ..................................................................................................................................... 2

Table of Contents ..................................................................................................................... 3

1. Introduction .......................................................................................................................... 4

1.1 Meju .............................................................................................................................................................. 4

1.2 Bacteria ......................................................................................................................................................... 5

1.3 Mold ............................................................................................................................................................. 6

1.4 Rationale ....................................................................................................................................................... 7

1.5 Aim ............................................................................................................................................................... 7

2. Variables ............................................................................................................................... 8

2.1 Independent Variable .................................................................................................................................... 8

2.2 Dependent Variable ....................................................................................................................................... 8

2.3 Controlled Variable ....................................................................................................................................... 8

3. Method .................................................................................................................................. 9

3.1 Preparation of Agar Plates ............................................................................................................................ 9

3.2 Preparation of Sodium Chloride Solution ................................................................................................... 10

3.3 Preparation of Aqueous Meju Extract ......................................................................................................... 10

3.4 Preparation of Solutions of Meju with different pH Levels ........................................................................ 11

3.5 Preparation fo Solutions of Meju with different Salt Concentration........................................................... 12

3.6 Serial Dilution ............................................................................................................................................. 12

3.7 Plating on Agar Plates ................................................................................................................................. 13

3.8 Incubation ................................................................................................................................................... 13

3.9 Counting Bacteria ....................................................................................................................................... 13

4. Data Collection .................................................................................................................... 15

4.1 Raw Data for Meju at controlled conditions ............................................................................................... 15

4.2 Raw Data for Meju at Different Salt Concentrations .................................................................................. 15

4.3 Raw Data for Meju at Different pH Levels ................................................................................................. 16

4.4 Qualitative Observation .............................................................................................................................. 17

4.5 Bacteria and Mold Count after 14 Hours .................................................................................................... 19

5. Data Processing ......................................................................................................................................... 21

6. Data Presentation ................................................................................................................ 24

7. Data Analysis ....................................................................................................................... 26

8. Conclusion. .......................................................................................................................... 28

9. Evaluation. ........................................................................................................................... 29

10. References .......................................................................................................................... 30


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1. Introduction

1.1 Meju

Meju (brick of dried fermented soybeans) is a traditional food that was developed in

Korea along with other processed soybean foods. All these fermented soybean foods are

produced through fermentation by micro-organisms originating from the surrounding

environment.1 Because of its rich protein source, such as protease and amylase, and its

unique taste and smell, meju has been an essential part of Korean cuisine since the Iron Age2

and the period of the Three Kingdoms3. Meju is made by fermenting beans, barley, wheat,

rice and such like. Although soybeans have been shown to alleviate metabolic syndromes, it

has been reported that fermented soybeans may have even greater effects. The unique taste is

produced by the presence of a bacterium called Bacillus4.

Figure 1 – Block of Meju

1 Information obtained from Source (4)

2 Iron Age is the period from 400 to 60 B.C.

3 Three Kingdoms is the period from 37 B.C. to 668 A.D.



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1.2 Bacteria

Lactic acid bacteria are a group of Gram-positive bacteria that produce lactic acid as

a result of carbohydrate fermentation. The microbes are generally used to produce fermented

food products. Their growth lowers the carbohydrate content of the foods they ferment and

the pH content because of lactic acid production. The pH may drop to 4.0 to inhibit the

growth of other microorganisms, including common human pathogens, allowing the foods

prolonged life. The acidity changes the texture of the food due to precipitation of some

proteins, and the biochemical conversions involved in growth enhance the flavor. The

fermentation is self-limiting because of the bacteria’s sensitivity to acidic pH5.

Some lactobacillus species are used in the production of fermented products,

including meju. The lactobacillus present in meju is considered to possess potential

beneficial characteristics, such as anti-inflammatory and anti-cancer activities. In addition,

meju contains several fungi and yeast species6.

Figure 2: Lactic Acid Bacteria




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1.3 Mold

All fermented soybean foods are produced through fermentation by micro-organisms

originating from the surrounding environment. Meju is inoculated with fungi, Aspergillus

oryzae, to degrade cooked soybeans with proteases, amylases, and lipases from the fungi.

This provides meju with its unique taste.7

Figure 3: Aspergillus oryzae

Characteristics of Aspergillus oryzae that make it so important in the fermentation of

soybeans are: growth, enzymes, aesthetics, and color. Favorable conditions for Aspergillus to

invade organic substrates include high moisture content and high temperature.


http://www.foodprocessing-technology.com/projects/kikkoman/


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1.4 Rationale

Koreans are very proud of their foods’ superior healthful benefits. My mother always

used to tell me that the billions of enzymes and bacteria in doenjang have functions such as

simple medicinal functions (such as soften the skin, aid digestion, and treat constipation and

diarrhea) and challenging functions (prevent cancer, dementia, and heart diseases). This made

me wonder, “How can food have medicinal effects?” I became interested in the chemical

elements of doenjang and whether it really has scientifically proven medical functions. I

researched more about it online, and I found an article reporting that fermented soybeans

have even greater effects. I became interested in meju, the preceding product before soybeans

are made into doenjang. Thus, I decided to investigate the characteristic of bacteria in meju,

and in what extreme and optimal conditions (of pH levels and salt concentrations) these

bacteria grow.

1.5 Aim

The aim of this investigation is to explore the effect of different salt concentrations

and pH levels on meju. This experiment will also investigate whether lactic acid bacteria and

mold will be able to survive in extreme pH and salt conditions. My precise research question

is:

How will changing the salt concentration and pH level affect the rate of growth of

Lactobacillus bacteria in meju?


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2. Variables

2.1 Independent Variable

Salt

Meju is incubated at different salt concentrations, prepared by the different

amount of sodium chloride dissolved in 100ml of distilled water. Salt

concentrations are: 0%, 10%, 20%, 30%, and 40% (saturated)8.

pH

Meju is incubated at different pH levels for 48 hours and for 14 hours after

plating in a MRS agar9. The pH levels are: 0, 2, 4, 7, 10, and 14.

2.2 Dependent Variable

Number of bacterial colonies forming on the surface of the MRS agar plate is counted in

colony forming unit (CFU)10

. The solutions will be diluted by serial dilution to avoid bacteria

from excessively covering the petri dish so that the bacterial count is between 30 to 500,

which is statistically acceptable.

2.3 Controlled Variable

Temperature of the room (25°C), refrigerator (4°C), and incubator (37.5°C)

The time of incubation of meju extract in the refrigerator (48 hours) and in the

incubator (14 hours)

The volume of MRS agar (approximately 25mL per plate)

The volume of plating (50μL per plate)

8 The solution reached the state at which the maximum amount of salt has been dissolved

9 MRS agar is a type of bacterial growth medium that favors the growth of Lactobacilli for lab study. The

sodium acetate in the medium prevents the growth of other competing bacteria 10

CFU (colony-forming unit) measures viable bacterial numbers


9

3. Method

3.1 Preparation of Agar Plates

1. Add 70g of MRS agar powder to a conical flask

2. Add 1000 cm3 of distilled water using a volumetric flask

3. Stir the mixture to dissolve the powder completely

4. Place a small beaker over the opening of the conical flask

5. Place the conical flask into the autoclave (Figure 4) to sterilize the agar solution

Figure 4: Stovetop autoclaves

6. Pour approximately 30 cm3 into each petri dish and cover the lids after it has been

hardened (shown by Figure 5)

Figure 5: Pouring agar plates

http://upload.wikimedia.org/wikipedia/en/6/69/Autoclave_stove_top.jpg


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3.2 Preparation of Sodium Chloride Solution

1. Add 10 g of sodium chloride to 100 cm3 of distilled water using a volumetric flask

(shown by Figure 6)

Figure 6: Preparing sodium chloride solution

2. Place on a magnetic stirrer and let the solution dissolve completely

3. Repeat step 1 and 2 by substituting 10 g with 20 g, 30 g, and 40 g for 10%, 20%,

30%, and 40% (saturated) NaCl Solution (shown by Figure 7)

Figure 7: Sodium Chloride Solutions

3.3 Preparation of Aqueous Meju Extract

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

3. Place approximately 50 g of meju in a mortar and pestle


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4. Add 50 mL of distilled water into the mortar and pestle

5. Begin grinding to form an aqueous solution (shown by Figure 8)

Figure 8: Grinding meju

6. Pour out approximately 30 g of the solution onto the cheese cloth

7. Squeeze out meju extract into a sterilized beaker

3.4 Preparation of Meju solution at different Salt Concentrations

1. Prepare the following sterilized salt concentration solution: (0%, 10%, 20%, 30%,

and 40%)

2. Label 5 micro-centrifuges tubes (shown by Figure 9) with the different salt

concentrations

Figure 9: Microcentrifuge tubes

3. Fill in 500 μL of pure aqueous meju extract using the micropipette

4. To maintain equilibrium, add the same amount of salt solution (500 μL) to the extract


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of the rightly labeled micro-centrifuge tube using the micropipette

5. Thoroughly mix each micro-centrifuge tube using the micropipette

6. Incubate these micro-centrifuges tubes at 4°C for 48 hours

3.5 Preparation of Meju solution at different pH levels

1. pH buffers 0, 2, 4, 7, 10, 14 from Carolina were used

2. Label 6 micro-centrifuges tubes with the different pH levels

3. Fill in 500 μL of pure aqueous meju extract using the micropipette

4. To maintain equilibrium, add the same amount of pH buffer (500 μL) to the extract of

the rightly labeled micro-centrifuge tubes using the micropipette

5. Thoroughly mix each micro-centrifuge tubes using the electronic vortex mixer.

6. Incubate these micro-centrifuge tubes at 4°C for 48 hours

3.6 Serial Dilution

Serial dilution technique is used to prevent too much bacteria from covering the petri dish

because this would create difficulty in counting the bacterial population. After 48 hours of

incubation, leave the treated meju in different salt and pH solutions are diluted down to 10-5

(shown by Figure 10), so that the CFU will be countable.

Figure 10: 10-fold Serial Dilution on treated meju (with different salt and pH)

Treated meju 10-1

10-2

10-3

10-4

10-5


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3.7 Plating on Agar Plates

Plate out meju extract with (0%, 10%, 20%, 30% and 40%) NaCl incubated for 48 hours on

MRS agar plate with dilution 10-4

and 10-5

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 correctly labeled

MRS agar plate.

3. Use a sterile cotton swab to swab the surface of the nutrient agar.

4. Close the lid of the petri dish

5. Repeat step 1 to 5 for different dilutions of pH and salt

3.8 Incubation

A total of 44 petri dishes are placed in an electronic incubator. Adjust the temperature of the

incubator to 37.5°C. Keep them in the incubator for 14 hours.

Figure 11: Lab incubator

3.9 Counting Bacteria

According to previous researches, it is expected for bacteria colonies and molds to appear on

the MRS agar plate after incubation.

1. After incubation for 14 hours, take out the MRS agar plates and leave it in room

temperature to cool down

2. Count the CFU of LAB by marking dots on the petri dish when a round bacteria is

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found

3. Count the CFU of mold by marking dots on the petri dish when a blurred colony

(mold) is found


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4. Data Collection

4.1 Raw Data for untreated meju

Figure 12: Untreated meju at controlled conditions

*10

-4dilution plate, replicate 1

** 10-4

dilution plate, replicate 2

# 10-5


## 10-5


4.2 Raw Data for treated meju at different Salt Concentrations

Treated meju at 10% Salt Concentration Treated meju at 20% Salt Concentration

Treated meju at 30% Salt Concentration Treated meju at 40%

11 Salt Concentration

Figure 13: Meju incubated at different percentage salt concentration

* 10

-4 dilution plate, replicate 1

** 10-5


# 10-5


## 10-5


11

saturated, salt concentration of 40%

* **

# ##

*

* *

* ** **

# #

# #

## ##

## ##

** **


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4.3 Raw Data for treated meju incubated at different pH Levels

Treated meju at pH 0 Treated meju at pH 2



Figure 14: Treated meju incubated at different pH levels

* 10

-4 dilution plate, replicate 1

** 10-5


# 10-5


## 10-5


* *

* *

* *

** **

** **

** **

# #

#

#

# #

## ##

##

##

## ##


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4.4 Qualitative observation

Conductivity

Meju 10% 20% 30% 40%

Conductivity

(TDS)

1510 1649 1650 1652 1652

Table 6: Conductivity of Meju and Salt solutions

Figure 15: Measuring the conductivity of untreated meju

pH of untreated meju solution: 7~9

Figure 16: Measuring the pH level of untreated meju

**


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The conductivity of the untreated meju was measured using the conductivity probe, as shown

in Figure 15. The measurements showed that the salt concentration of untreated meju seems

to be less than 10%, as the measure of conductivity (TDS) in untreated meju solution seems

less than that of the other salt solutions. Then, the pH level of the untreated meju was

measured using the pH probe, which yielded results that the untreated meju solution lay

between 7 and 9. This measurement is shown by Figure 16.


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4.5 Bacteria and Mold Count after 14 hours

Bacteria and Mold Count for Control

Bacteria Count Mold Count Total Count

Dilution at 10

-5

Trial 1 Trial 2 Average

(a) Trial 1 Trial 2 Average

(a) Total

(a)

Control(b) 180 160 170 2 1 2 172

Table 1: Bacteria and Mold Count for Control

Bacteria and Mold Count for different Salt Concentrations


Dilution at 10

-5

NaCl Concentration / % Trial 1 Trial 2 Average(a)

Trial 1 Trial 2 Average(a)

Total(c)

10 86 64 75 0 1 1 76

20 107 99 103 81 0 -(d)

-(d)

30 133 137 135 68 63 66 201

40 (saturated) 104 122 113 120 148 134 247

Table 2: Bacteria and Mold Count for different Salt Concentrations

__________________ (a)

Average for duplicate samples (b)

Untreated meju (c)

Average bacteria count + Average mold count (d)

Average couldn’t be obtained, due to experimental error


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Bacteria and Mold Count for different levels of pH


Dilution at 10

-5

pH Trial 1 Trial 2 Average(a)

Trial 1 Trial 2 Average(a)

Total(b)

0 1 5 3 0 0 0 3

2 47 49 48 94 95 95 143

4 99 98 99 68 82 75 174

7 83 87 85 75 71 73 158

10 150 158 154 1 8 5 159

14 12 2 7 10 4 7 14

Table 3: Bacteria and Mold Count for different levels of pH

______________________ (a)


Average bacteria count + Average mold count


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5. Data Processing

5.1 Calculating CFU

Controlled plate

Average number of CFU for LAB at 10-5

dilution plate: 170 colonies (per 0.05 ml)

Total LAB concentration at 10-5

dilution factor

Average number of mold at 10-5

dilution plate: 2 (per 0.05 ml)

Total mold concentration at 10-5

dilution factor

mold / ml

Using this same method of calculations, the number of CFU/ml and mold in original

concentration of meju can be found.


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Number of CFU for LAB and mold in original concentration of treated meju (at different percentage salt concentrations)

Table 4: Number of CFU for LAB and mold in original concentration of treated meju (at different percentage salt concentrations)

Number of CFU of LAB and mold in original concentration of meju(at different levels of pH)

pH

Average(a)

number of LAB at

10-5

dilution

Number of CFU at

original

concentration / 106

Average(a)

number

of mold at 10-5

dilution

Number of molds at

original

concentration / 106

Average number of

LAB and mold at 10-

5 dilution

Number of LAB and

mold at original

concentration / 106

0 3 6 0 0 3 6

2 48 97 95 190 143 287

4 99 198 75 150 174 348

7 85 170 73 146 158 316

10 154 308 5 100 159 408

14 7 14 7 14 14 28

Table 5: Number of CFU of LAB and mold in original concentration of meju (at different levels of pH)

__________________________ (a)


Average couldn’t be obtained, due to experimental error

Salt

concentration

Average(a)

number of LAB at

10-5

dilution

Number of CFU at

original

concentration /

106

Average(a)

number

of mold at 10-5

dilution

Number of molds at

original

concentration /

106

Average number

of LAB and mold

at 10-5

dilution

Number of LAB

and mold at

original

concentration /

106

10% 75 150 1 2 76 152

20% 103 206 -(b)

-(b)

-(b)

-(b)

30% 135 270 66 132 201 402

40% 113 226 134 268 247 494


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Percentage Reduction

Salt Concentration 10%

20% 30% 40%

Bacteria -55.88% -39.41% -20.59% -33.53%

Mold -50.00% -(b)

+3200% +6600%

Total -55.81% - +16.86% +43.60%

Table 7: Percentage decrease/increase of plates treated with salt percentage concentration(a)

pH 0 2 4 7 10 14

Bacteria -98.24% -71.47% -41.76% -50.00% -9.42% -95.88%

Mold -100% +4650% +3650% +3550% +150.0% +250.0%

Total -98.26% -16.57% +1.163% -8.139% -7.558% -91.86%

Table 8: Percentage decrease/increase of plates treated with pH(a)

___________________________ (a)

– represents a decrease and + represents an increase (b)

the result could not be obtained, due to experimental error


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6. Data Presentation

Graph 1: Number of CFU for LAB and Mold in Meju at different percentage salt concentrations after 14 hours of incubation

_____________ (a)

Untreated meju (b)

20% did not show the number of mold (and therefore total count) due to experimental error (c)

40% salt concentration is saturated

340

150

206

270

226

4 2

132

268

344

152

402

494

0

100

200

300

400

500

600

Control 10% 20% 30% 40%

Nu

mb

er o

f C

FU

/ml

x 1

06

Salt Concentration / %

Number of CFU for LAB and Mold in Meju at different percentage salt concentrations

LAB

Mold

Total

(b) (c) (a)


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Graph 2: Number of CFU for LAB and Mold in Meju at different pH Levels

_________________ (a)

Untreated meju

340

6

97

198

170

308

144 0

190

150 146

10 14

344

6

287

348

316 318

28

0

50

100

150

200

250

300

350

400

Control 0 2 4 7 10 14

Nu

mb

er o

f C

FU

/ml

x 1

06

pH Levels

Number of CFU for LAB and Mold in Meju at different pH Levels

LAB

Mold

Total

(a)


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7. Data Analysis

Observation of Plates treated with Salt

Generally, there was a positive correlation between the percentage salt concentration

and the number of bacteria and mold. This can be easily observed by the graph; the total

number of bacteria and mold shows an increase from 10% to 40% salt concentration. As the

concentration increased on the x-axis, the bars generally showed an overall increase.

There were more bacteria present than mold on the plates. Examining the number of

bacteria more specifically, the trend varied a little. Although the number of bacteria increased

from 10% to 30% salt concentration, there was a slight decrease for 40% salt concentration

(saturated). Therefore, the maximum number of bacteria occurred with the 30% salt

concentration. The slight decrease in number of bacteria from 30% to 40% salt concentration

may be due to the fact that the 40% concentration was saturated. In comparison to the number

of bacteria in the control plate, all plates treated with salt showed a decrease in number of

bacteria.

The number of mold showed an overall increase from 10% to 40%. Unfortunately the

plates for 20% salt concentration did not show any mold, due to experimental error. Therefore,

the total number of bacteria of mold could not be calculated for 20% salt concentration. The

increase in number of mold in plates treated with salt from 10% to 40% showed a more rapid

growth compared to that of bacteria. This may show that mold reacts more sensitively to

changes in the environment.

The bacteria and mold may have been able to survive in extreme salt concentrations

due to the altered permeability of the cell membrane and its reversal by sodium chloride, or

the maintenance of critical levels of other intracellular ions by sodium chloride.12



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Observation of Plates treated with pH

The solutions that were treated with pH 0 had very little bacteria and no mold. In fact,

although bacteria still survived, mold did not grow at all when treated with pH 0. This shows

that bacteria do not thrive, and mold does not survive at all in extremely acidic conditions.

There was still a positive correlation between the number of bacteria and the pH level

among pH 0, 2, and 4. However, there was a slight drop from pH 4 to 7 for the number of

bacteria. pH 10 showed the maximum number of bacteria, but fell drastically for pH 14. This

shows that bacteria cannot thrive in extremely basic conditions either.

Mold grew in plates treated with pH 2. However, there was a gradual drop from pH 2

to pH 7, and a drastic drop to pH 10. There was little difference in the number of mold in

plates treated with pH 10 and 14. The small number of mold in the plates treated with bases

shows that mold grows less in basic conditions.

Examining the plate treated with pH 2 showed that mold grew better than bacteria.

However, the plate treated with pH 4 showed the opposite result. Bacteria grew rapidly, but

mold showed a slight decrease. Then, the plate treated with pH 7 showed a slight decrease in

the number of both bacteria and mold. As the plate encountered basic environment, the

number of bacteria grew rapidly, but the number of mold fell drastically. This shows that

mold is very sensitive to the pH environment, and cannot thrive in basic conditions.

The total number of bacteria and mold increases from pH 0 to pH 4, gradually drops

with pH 7, then increases slightly for pH 10, and drops drastically for pH 14.

The bacteria, unlike mold, may have survived in extremely acidic condition due to

their ability to penetrate the cytoplasmic membrane, resulting in reduced intracellular pH. In

other words, the ability to regulate the pH inside the membrane may be the reason for the

survivability of the bacteria13



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8. Conclusion

This extended essay investigated the effect of pH and salt concentration on the

growth of lactic acid bacteria and mold in meju, a fermented cooked soybean product that is a

major ingredient in Korean traditional fermented foods.

Using the methods outlined above, the experiment yielded results that showed that

overall, bacteria and mold both showed a large range of survivability. Lactic acid bacteria

showed a generally stronger survivability in the sodium chloride medium, which is shown by

their rich growth in all concentrations of salt. The bacteria is halophilic because it is able to

survive in a wide range of salt concentrations. Thus, this result suggests that lactic acid

bacteria may have mechanisms that allow them to maintain the osmoloarity of the cell,

preventing dehydration.

On the other hand, mold did not show halophilic characteristics because it only

managed to thrive in high salt concentrations (30% and 40%). This difference can be

explained by mold’s inability to regulate the osmolarity of the cell, a mechanism likely to be

found in lactic acid bacteria.

Lactic acid bacteria showed strong survivability even in different pH conditions.

Changing the pH level is important in the process of fermentation because pH determines the

ion concentration of the medium, specifically hydrogen ions. These ions affect the structure

of the enzymes, which are responsible for the function of the enzyme. For example, any

change to the tertiary structure of an enzyme may lead to its denaturation, which means that

fermentation may not be carried out. The inability for both bacteria and mold to thrive in pH

0 and 14 is due to the extremely acidic and alkaline condition of the solution.

Therefore, this investigation shows that lactic acid bacteria and mold are able to

survive in a wide range of pH and salt conditions.


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9. Evaluation

The uncertainty implied by the counting of viable cells on the MRS plate serves as a

limitation to this experiment. After a certain stage of growth, the mold and the bacteria

displayed similar appearances, which made it difficult to distinguish between the two. In

addition, the amount of space that mold took up to grow sometimes prevented a clear display

of bacteria as the mold sometimes tended to cover up the bacteria.

In addition, the MRS agar supported the growth of a variety of LAB and other

bacterial strains. Also, the autoclave may not have been completely effective in getting rid of

all bacteria before the actual plating of the meju extract onto the agar plate. Therefore, it was

not possible to determine the dominant strain and all strains were counted together. In other

words, the results in this experiment took into count all strains of bacteria and mold.

Unfortunately, due to the lack of resources and expertise, the exact salt concentration

and pH level of the control sample (the meju extract itself) could not be determined. If this

information were obtained, its comparison to results obtained from this experiment could

yield the exact conditions needed for optimum condition for soybean fermentation.

Finally, the duplicate trial was a limitation to this experiment. Due to the time

constraint, only two results could be obtained for each variable. For this reason, sometimes

results were inaccurate as one trial did not produce reasonable results. Nevertheless, such

outliers were not taken into consideration when making conclusions.


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10. References

1. Alakomi, H.-L., E. Skytta, M. Saarela, T. Mattila-Sandholm, and K. Latva-Kala. “Lactic

Acid Permeabilizes Gram-Negative Bacteria by Disrupting the Outer Membrane.”

Applied and Environmental Microbiology. (2000): Print.

2. Britannica, Encyclopaedia. The New Encyclopaedia Britannica. Chicago: Encyclopaedia

Britannica, 2007.

3. Goldmn, Manuel, R.H. Deibel, and C.F. Niven, Jr. "Interrelationship between

Temperature and Sodium Chloride on Growth of Lactic Acid Bacteria Isolated

from Meat-Curing Brines." University of Chicago. (1962): Print.

4. Lee, J.H., T.W. Kim, H Lee, H.C. Chang, and H.Y. Kim. “Determination of microbial

diversity in meju, fermented cooked soya beans, using nested PCR-denaturing

gradient gel electrophoresis.” Letters in Applied Microbiology. (2010): Print.

5. "Meju." Doosan Encyclopedia. Seoul: Doosan, 2010. Web.

<http://100.naver.com/100.nhn?docid=61633>.

6. Mo, Ae-young, Bora Kwon, Seralathan Kamala-Kannan, Kui-Jae Lee, and Byung-Taek

Oh. "Isolation and characterization of Bacillus polyfermenticus isolated from

Meju, Korean soybean fermentation starter." World Journal of Microbiology and

Biotechnology. (2009): Web

<http://www.springerlink.com/content/c657200420l66t75/fulltext.html>.

7. Thomas, Peggy. Bacteria and Viruses. Lucent Books, 2004. Print.

IB EE on the effect of pH and salt on the growth of Lactobacillus in meju

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