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

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Research work done by my IB student Grace Lee. Please cite and give proper referencing to her on her work if you use this material.

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

  1. 1. Extended Essay - Biology Shin-Ae Lee International Baccalaureate Extended Essay BiologyIn Vitro Study of the Effect of pH and Salt Concentration on thegrowth 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
  2. 2. Extended Essay - Biology Shin-Ae LeeAbstract Doenjang (Korean fermented soybean paste) is a traditional fermented soybean foodin Korea. It not only has been consumed as food, but has also been used as folk medicine foremergency treatments. This is due to lactic acid bacteria (LAB) and probiotic mold indoenjang. This research investigated the in vitro effect of pH and salt on the microbial growthof LAB/mold in doenjang using the standard viable plate count method. This research wasdivided into two parts: incubating doenjang extract at different pH and at various saltconcentrations. Doenjang extracts were incubated separately at pH 1, 3, 5, 7, 11, 14, and in0%, 10%, 20%, 30% and 40% salt concentrations in 4°C for 2 days. After incubation the pHand 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. Thebacteria 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 pH7 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 decreaseddrastically 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 levelsexcept for pH 14.(Word Count: 279) Page 2 of 51
  3. 3. Extended Essay - Biology Shin-Ae Lee Table of ContentsAbstract ................................................................................................................................... 2Table of Contents ................................................................................................................... 31. Introduction .......................................................................................................................... 5 1.1 About Doenjang.......................................................................................................... 5 1.2 Rationale of Study ...................................................................................................... 5 1.3 About Bacteria in Doenjang ....................................................................................... 6 1.4 Aim ............................................................................................................................. 72. Variables ............................................................................................................................... 8 2.1 Independent Variable.................................................................................................. 8 2.2 Controlled Variable .................................................................................................... 8 2.3 Dependent Variable .................................................................................................... 83. 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 .......................................................................................................... 154. 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 ...................................................................................................... 325. 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 ................................. 356. Discussion............................................................................................................................ 38 Page 3 of 51
  4. 4. Extended Essay - Biology Shin-Ae Lee 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 ................................................................................................ 417. Conclusion .......................................................................................................................... 438. References ........................................................................................................................... 449. Appendix ............................................................................................................................. 46 Page 4 of 51
  5. 5. Extended Essay - Biology Shin-Ae Lee1. Introduction1.1 About Doenjang Doenjang (Korean fermented soybean paste) is a traditional fermented soybean foodthat was developed in Korea along with other processed soybean foods (1). Because of its richprotein source as well as its taste for enhancing foods, doenjang has been an essential part ofKorean food from history. Doenjang has also been used as a folk medicine for emergencytreatments, believed to remove toxins from insect or snake bites, or simply for stoppingbleeding, 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 astheir tastes: the traditional type gives a strong, stinging smell with a salty taste, while theimproved 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 Koreanfermented soybean paste, doenjang, from my grandparents. In the early 1900‟s when medicalwasn‟t well developed yet in Korea, doenjang took place as medicines in many ways. Whenmy grandfather got a bruise from bumping his head on the edge of the desk, his motherpasted a spoonful of doenjang on the bruise to arrest bleeding. My grandmother used to pastedoenjang 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 5 of 51
  6. 6. Extended Essay - Biology Shin-Ae LeeI remember my mother fixing me doenjang soup when I had stomach troubles. This sparkedmy curiosity, wondering, „how can a food have such medical efficacy.‟ As a biology student, Isoon became interested in the chemical elements of doenjang and whether it really has somesort of medical function. Then after few days, I found a thought-provoking journal2 writtenby few Korean researchers about the microbial communities in doenjang. Interestinglyenough, it was written that “several types of lactic acid bacteria [LAB] including L.mesenteroides, T. halophilus, and E. faecium were observed as the predominant bacterialspecies” in doenjang (3). Knowing that LAB is pro-biotic, I found it worthy to furtherinvestigate to what extent this characteristic is preserved in affect to different pH levels andsalt concentration.1.3 About Bacteria in Doenjang According to previous researches, several microorganisms were identified indoenjang. These include molds such as Aspergillus Mucor and Rhizopus species that weredetected in meju3. Recently in 2007, Korean researchers have investigated the microbialcommunities in doenjang and announced an unexpected observation that Staphylococcusequorum and some lactic acid bacteria are the dominant species in doenjang, instead ofprevious founding that Bacillus. subtilis is the primary bacteria in doenjang (3). In addition tothese microorganisms, several fungi and yeast species were also found to be present indoenjang (3).2 See Appendix 2.3 Meju is a dried, fermented soybean block, which is further fermented with salt water to become doenjang. Page 6 of 51
  7. 7. Extended Essay - Biology Shin-Ae Lee1.4 Aim The aim of this investigation is to explore the effect of different pH levels and saltconcentration in doenjang, and whether LAB/mold will be viable in extreme pH and saltconditions. 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 andin different salt concentrations. These investigations are made possible by plating out onMRS4 agar, which cultivates LAB/mold.4 See Appendix 3. Page 7 of 51
  8. 8. Extended Essay - Biology Shin-Ae Lee2. Variables2.1 Independent Variable2.1.1 pH Doenjang is incubated at different pH level in the refrigerator for 2 days and furtherincubated 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 ofsodium chloride dissolved in 100 ml of distilled water. Salt concentration will be varied by0%, 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; thetime of incubation in the refrigerator and in the incubator; the pH and volume of MRS agarplate; 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 iscounted 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 countedwill 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 ofdistilled water. Page 8 of 51
  9. 9. Extended Essay - Biology Shin-Ae Lee3. Procedures3.1 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 Sterilizing3.3.1 Essential Apparatus to sterilize:Cheese cloth 10% NaCl Solution (w/v) Distilled WaterMRS Agar Solution Beakers Page 9 of 51
  10. 10. Extended Essay - Biology Shin-Ae Lee3.3.2 Method of sterilizing Sterilizing all solutions and instruments to be used in the experiment, including theessential apparatus listed above, is a significant step in this investigation, as it deals withbacteria. This will be done by using a pressure autoclave.63.4 Preparation of Aqueous Doenjang Extract at Different pH Levels andSalt Concentration3.4.1 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 10 of 51
  11. 11. Extended Essay - Biology Shin-Ae Lee 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 petridish, which gives difficulty in counting the bacterial population. After 2 days of incubation inthe refrigerator, leave the microcentrifuges of aqueous doenjang extract at different pH levelsand 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 11 of 51
  12. 12. Extended Essay - Biology Shin-Ae LeeDiagram 3.5.1: Procedure for serial dilution of doenjang at different pH and saltconcentrationTable 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. Page 12 of 51
  13. 13. Extended Essay - Biology Shin-Ae LeeTable 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 Plate3.6.1 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 therefrigerator 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 daysin 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 13 of 51
  14. 14. Extended Essay - Biology Shin-Ae LeeDiagram 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 ofcontamination. Plate out 50 μL of sterilized distilled water and 50 μL of 10% NaCl using thesame method of plating on MRS agar.3.7 IncubationA total of 44 petri dishes are placed in an electronic incubator upside down.10 Adjust thetemperature 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 14 of 51
  15. 15. Extended Essay - Biology Shin-Ae Lee3.8 Bacteria Count According to previous researches, it is expected to see bacteria colonies and moldson 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 15 of 51
  16. 16. Extended Essay - Biology Shin-Ae Lee4. Data Collection4.1 Raw Data for pH and DoenjangTable 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 2A = Number of CFU of LAB (per 50 μL dilution plated)B = Number of CFU of molds (per 50 μL dilution plated) Page 16 of 51
  17. 17. Extended Essay - Biology Shin-Ae LeeTable 4.1.2: Dilution Plates of doenjang extract at pH 3 pH 3 -2 10 Dilution Plates A B 10-3 Dilution Plates A B 52 38 10 2 42 30 14 6(*) = Replicate 1(**) = Replicate 2A = 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
  18. 18. Extended Essay - Biology Shin-Ae LeeTable 4.1.3: Dilution Plates of doenjang extract at pH 5 pH 5 -2 10 Dilution Plates A B 10-3 Dilution Plates A B 61 29 15 8 63 26 16 4(*) = Replicate 1(**) = Replicate 2A = Number of CFU of LAB (per 50 μL dilution plated)B = Number of CFU of molds (per 50 μL dilution plated) Page 18 of 51
  19. 19. Extended Essay - Biology Shin-Ae LeeTable 4.1.4: Dilution Plates of doenjang extract at pH 7 pH 7 -2 10 Dilution Plates A B 10-3 Dilution Plates A B 79 50 10 1 71 52 19 4(*) = Replicate 1(**) = Replicate 2A = Number of CFU of LAB (per 50 μL dilution plated)B = Number of CFU of molds (per 50 μL dilution plated) Page 19 of 51
  20. 20. Extended Essay - Biology Shin-Ae LeeTable 4.1.5: Dilution Plates of doenjang extract at pH 11 pH 11 -2 10 Dilution Plates A B 10-3 Dilution Plates A B 60 24 10 3 50 30 8 0(*) = Replicate 1(**) = Replicate 2A = Number of CFU of LAB (per 50 μL dilution plated)B = Number of CFU of molds (per 50 μL dilution plated) Page 20 of 51
  21. 21. Extended Essay - Biology Shin-Ae LeeTable 4.1.6: Dilution Plates of doenjang extract at pH 14 pH 14 -2 10 Dilution Plates A B 10-3 Dilution Plates A B 0 0 0 0 0 0 0 0(*) = Replicate 1(**) = Replicate 2A = Number of CFU of LAB (per 50 μL dilution plated)B = Number of CFU of molds (per 50 μL dilution plated) Page 21 of 51
  22. 22. Extended Essay - Biology Shin-Ae LeeTable 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 on 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 6a pH 7 10-2 79 71 75 50 52 51 10-3 10 19 14.5 1 4 2.5pH 11 10-2 60 50 55 24 30 27 10-3 10 8 9 3 0 1.5pH 14 10-2 - - - - - - 10-3 - - - - - -b Distilled Water -(-) = no activity(b) = Negative Control(a) = Positive Control Page 22 of 51
  23. 23. Extended Essay - Biology Shin-Ae Lee4.2 Raw Data for Salt concentration and DoenjangTable 4.2.1: Dilution plates of doenjang extract at 0% NaCl 0% NaCl -3 10 Dilution Plates A B 10-4 Dilution Plates A B 20 6 23 0 15 7 0 2(*) = Replicate 1(**) = Replicate 2A = Number of CFU of LAB (per 50 μL dilution plated)B = Number of CFU of molds (per 50 μL dilution plated) Page 23 of 51
  24. 24. Extended Essay - Biology Shin-Ae LeeTable 4.2.2: Dilution Plates of doenjang extract at 10% NaCl 10% NaCl -3 10 Dilution Plates A B 10-4 Dilution Plates A B 26 12 5 1 27 13 35 2(*) = Replicate 1(**) = Replicate 2A = Number of CFU of LAB (per 50 μL dilution plated)B = Number of CFU of molds (per 50 μL dilution plated) Page 24 of 51
  25. 25. Extended Essay - Biology Shin-Ae LeeTable 4.2.3: Dilution plates of doenjang extract at 20% NaCl 20% NaCl -3 10 Dilution Plates A B 10-4 Dilution Plates A B 14 10 1 6 29 13 0 0(*) = Replicate 1(**) = Replicate 2A = Number of CFU of LAB (per 50 μL dilution plated)B = Number of CFU of molds (per 50 μL dilution plated) Page 25 of 51
  26. 26. Extended Essay - Biology Shin-Ae LeeTable 4.2.4: Dilution Plates of doenjang extract at 30% NaCl 30% NaCl -3 10 Dilution Plates A B 10-4 Dilution Plates A B 32 15 0 1 30 23 0 0(*) = Replicate 1(**) = Replicate 2A = Number of CFU of LAB (per 50 μL dilution plated)B = Number of CFU of molds (per 50 μL dilution plated) Page 26 of 51
  27. 27. Extended Essay - Biology Shin-Ae LeeTable 4.2.5: Dilution Plates of doenjang extract at 40% NaCl 40% NaCl -3 10 Dilution Plates A B 10-4 Dilution Plates A B 4 11 1 0 22 12 3 1(*) = Replicate 1(**) = Replicate 2A = Number of CFU of LAB (per 50 μL dilution plated)B = Number of CFU of molds (per 50 μL dilution plated) Page 27 of 51
  28. 28. Extended Essay - Biology Shin-Ae LeeTable 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 mlNaCl Dilution Plate 1 Plate 2 Average Plate 1 Plate 2 Averagea 0% 10-3 20 15 17.5 6 7 6.5 10-4 23 0 11.5 0 2 110% 10-3 26 27 26.6 12 13 12.5 10-4 5 35 20 1 2 1.520% 10-3 14 29 21.5 10 13 11.5 -4 10 1 0 0.5 6 0 330% 10-3 32 30 31 15 23 19 10-4 - - - 1 0 0.540% 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 28 of 51
  29. 29. Extended Essay - Biology Shin-Ae Lee4.3 Data Processing for Calculating CFU Calculations for CFU/ml of doenjang solution - Number of CFU at 10 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 100 = 2 5For example, in doenjang at pH 1, the average number of CFU at 10-2 dilution plate per 0.05ml is 20.5. Number of CFU at 10  dilution plate (per 0.05 ml)  100  10Number of CFU/ml = 2 5 20.5 100 10 2 = 2 5 205,000 = 2 5 = 82,000 Page 29 of 51
  30. 30. Extended Essay - Biology Shin-Ae Lee = 8.2 104 CFU/mlTable 4.3.1: Number of CFU/ml in original concentration of doenjang affected bydifferent pH levels Doenjang at A B (a) C different pH LAB Mold LAB Mold buffer 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 Number of CFU at 10 dilution plate (per 0.05 ml)  100  10(a) = Calculation refers to 2 5(b) = Controlled valueA = Average number of CFU at 10-2 dilution plates per 0.05 ml for duplicate samplesB = Number of CFU/ml in pure doenjang × 104C = Total number of CFU/ml of LAB and molds at in pure doenjang × 104 Page 30 of 51
  31. 31. Extended Essay - Biology Shin-Ae LeeTable 4.3.2: Number of CFU/ml in original concentration of doenjang affected bydifferent salt concentrations Doenjang at A B* C different salt LAB Mold LAB Moldconcentration / % 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 Number of CFU at 10 dilution plate (per 0.05 ml)  100  10(a) = Calculation refers to 2 5(b) = Controlled valueA = Average number of CFU at 10-3 dilution plates per 0.05 ml for duplicate samplesB = Number of CFU/ml in pure doenjang × 105C = Total number of CFU/ml of LAB and molds at in pure doenjang × 105 Page 31 of 51
  32. 32. Extended Essay - Biology Shin-Ae Lee4.4 Data Presentation Graph 4.4.1: Number of CFU/ml in Doenjang at different pH Buffer Levels LAB 55 Mold 50.4 50 Total (LAB+Mold) 45 Number of CFU/ml × 104 40 35.8 35 32.4 30 30.8 30 24.8 25 20.4 18.8 20 20 13.6 15 10.4 11 10.8 10 8.2 5 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). Page 32 of 51
  33. 33. Extended Essay - Biology Shin-Ae Lee Graph 4.4.2: Number of CFU/ml in Doenjang at different Salt Concentrations (%) LAB 25 Mold 22.5 Total (LAB+Mold) 20 20Number of CFU/ml × 105 17.5 15.6 15 13.2 12.4 12.5 10.6 9.6 9.8 10 8.6 7 7.6 7.5 5 4.6 5.2 4.6 5 2.6 2.5 0 0 (Control) * 10 20 30 40 Salt Concentration / % * = 0% NaCl is interpreted as a controlled value as it was substituted with distilled water (neutral medium). Page 33 of 51
  34. 34. Extended Essay - Biology Shin-Ae Lee5. Data Analysis5.1 Observation of the Effect of pH on Doenjang5.1.1 LAB Generally, the growth of LAB was dominant over the growth of mold in doenjang atall pH levels, excluding pH 14. According to graph 4.3.1, the number of LAB started todevelop 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 cellgrowth in pH 14, an extreme alkaline solution. In comparison to the controlled value—pH 7— viable cell counts decreased from (30to 8.2)×104 CFU/ml with a 73% decrease in the number of LAB in doenjang at pH 1 (11). InpH 3, the number decreased by approximately 37%, and in pH 5, by 17%. This general trendsuggests 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. Thisindicates 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 atextreme 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 abroad range of pH conditions (10).5.1.2 Mold Generally, according to graph 4.3.1, the viable cell counts of mold at different pHlevels were always less than the number of LAB in doenjang. Moreover, the growth of moldin doenjang at different pH levels showed no consistency. From this, we can hypothesize thatthe growth of mold in doenjang is not affected by the acidity nor the alkalinity of the solution, Page 34 of 51
  35. 35. Extended Essay - Biology Shin-Ae Leeexcept 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 withan 89% decrease from the controlled value. Furthermore, there was no viable growth of moldin pH 14. This suggests that acidity and alkalinity somehow affects the growth of mold indoenjang.5.1.3 Total (LAB/Mold) The general trend of the growth of LAB/mold is following the trend of the growth ofLAB in doenjang at different pH levels. There is an increase growth of LAB/mold as theacidity decreases. When pH 1 was compared to pH 7, there was a 79% decrease in the viablecell 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% frompH 5 to pH 7. Then after, as the alkalinity increased from pH 7, the total number ofLAB/mold decreased from (50.4 to 30.8)×104 CFU/ml. In pH 14 LAB/mold were both unableto survive in extreme alkaline concentration. Overall, this general trend supports thatLAB/mold are tolerant in extreme acidic level, but not towards extreme alkaline level. Inother words, the survivability of LAB/mold proves to be absent in extreme alkaline solution.5.2 Observation of the Effect of Salt Concentration on Doenjang5.2.1 LAB According to graph 4.3.2, LAB in doenjang has the ability to survive under a broadrange of salt concentration from 0% to 40%. This graph also suggests that the optimum viablecell count of LAB is found in 30% salt concentration, with approximately 56% increase in Page 35 of 51
  36. 36. Extended Essay - Biology Shin-Ae Leecomparison to the controlled value—0% salt concentration. The viable cell count of LAB in0% to 30% salt concentration all showed development. However, in an extreme 40% saltconcentration, a rapid decrease was shown with approximately 26% decrease and the leastviable 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 itwas 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 saltconcentrations, but were less than the growth of LAB at different salt concentration. Thecontrolled value showed the least viable mold with 2.6 CFU/ml×105. Growth of mold wasstill shown in the extreme, 40% salt concentration, but decreased about 39% from the growthof mold in 30% salt concentration. It also showed no difference in the number of growth from20% salt concentration. The maximum growth was observed in 30% salt concentration with a66% increase compared to the controlled value. Predominantly, it is displayed in graph 4.3.2that mold is survivable at all salt concentrations, but can‟t conclusively state which is theoptimum 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 in30% salt concentration, with a 52% increase in comparison to the controlled value. Thoughthere was a decrease of viable cell counts from 10% to 20% salt concentration, 15% decreaseseems insignificant in this trend as the number increased by 34% from 20% to 30%, anddecreased 51% from 30% to 40% salt concentration. Overall, the graph shows that LAB is Page 36 of 51
  37. 37. Extended Essay - Biology Shin-Ae Leedominant over the growth of mold, and that LAB/mold at 30% salt concentration showed themost growth. It is significant to notice that at 40% salt concentration, the growth ofLAB/mold decreased in a high percentage, but were viable to survive in all saltconcentrations, including the extreme 40% concentration. Page 37 of 51
  38. 38. Extended Essay - Biology Shin-Ae Lee6. Discussion6.1 Effect of pH on doenjang In cellular respiration, which is the breakdown of organic compounds, a chain ofreaction—glycolysis—takes place that converts glucose into a substance called pyruvate.Fermentation is the process of this pyruvate being broken down anaerobically, producingeither lactate (lactic acid) or ethanol (alcohol) and CO2 (7).Figure 6.1.1 Process of glycolysis and anaerobic fermentationImage taken from: Prentice Hall Biology Textbook In figure 6.1.1, it is shown that 2 NADH adds with 2 hydrogen molecules to convertto 2 NAD+. This process in anaerobic fermentation is crucial as 2 NAD+ is being convertedto 2 NADH in the glycolysis. Thus, hydrogen ion concentration is very significant in theoverall 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 hydrogenion concentration is being changed11. Hydrogen ions have the potential to disrupt the bondsthat maintain the tertiary shape of the enzymes. These bonds are primarily hydrogen bonds11 See appendix 6. Page 38 of 51
  39. 39. Extended Essay - Biology Shin-Ae Leeand 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 longermaintains its original shape. Thus, since this catalysis is what causes the metabolic reactionsto 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 evidentlyshown 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 extremeconcentration of alkalinity in the solution. pH 14 consists of 1/10,000,000 hydrogen ionconcentration in comparison to distilled water. Thus, this concentration could affect the LABmetabolism pathway that disrupts the survivability of LAB/mold due to the enzyme beingdenatured. Though pH 1, another extreme pH buffer level, has the least viable LAB/mold, thegrowth of LAB/mold itself in such an acidic level is showing that doenjang is high acidtolerant. This may suggest that there are acid tolerant mechanisms in LAB/mold in doenjangthat prevent the enzyme from being denatured. This leads to a hypothesis that thesemechanisms 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 isthat the enzymes involved in metabolic or fermentation pathway are resistant towards acidiccondition. In pH 14, LAB/mold in doenjang wasn‟t able to survive through the extreme alkalineconcentration. This might be due to enzymes which are very susceptible towards highhydroxide ion concentration. At extreme alkaline environment, the enzymes which areinvolved in fermentation or metabolic pathway can be easily denatured due to the highhydroxide ion concentration, by disrupting the tertiary structure of the enzyme. Anotherpossible reason is that LAB/mold doesn‟t possess any alkaline tolerant mechanism to pump Page 39 of 51
  40. 40. Extended Essay - Biology Shin-Ae Leeout hydroxide ions that get into the cell. Thus, it can be deduced that enzyme and membranebound 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 notyeasts (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 aresusceptible to pH solution. This founding also directs us to the fact that NaCl improved thequality of fermentation. This premise reflects on graph 4.3.2 as the viable mold in doenjang atdifferent salt concentrations is generally greater than the controlled value. The ability to survive in all salt concentrations show that LAB in doenjang ishalophilic—survivable in environments with high salt concentration. It also shows thatsalinity promoted the growth of the useful bacteria while inhibiting the growth of theunfavorable bacteria. This might suggest that LAB/mold have mechanism which canmaintain the osmolarity of the cell and prevent them from dehydration. Possible mechanismslike membrane bound protein pump, which pumps in water from its surrounding might helpto maintain its osmolarity. Another possible mechanism might be due to the presence ofNa+/K+, which are embedded on the plasma membrane and help to regulate the movement ofNa+ ions into the cell. This pump will remove any accessible Na+ that diffuse in, and thusmaintain its osmolarity, enabling LAB/mold to survive under all different salt concentrations. There might be osmotic regulation performed by enzymes which are osmotolerantand are able to function at high salt concentration (12). Outer membrane bound protein forLAB might have transport mechanisms which function as osmoregulants that help LAB to Page 40 of 51
  41. 41. Extended Essay - Biology Shin-Ae Leesurvive in high osmotic stress. Their outer membrane structure and phospholipid bilayercomposition 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 tocount the exact number of LAB/mold due to its similar appearance after a certain stage ofgrowth. Another uncertainty is that the number of CFU for LAB is difficult to determine asthe growth of mold will tend to cover up LAB. Thus, this bacteria cell count is biased anduncertain. Period of incubation should be shortened, so that the mold will not overgrow onthe MRS agar, covering the LAB. Due to time constrain, duplicate trial was done for this investigation. Thus, the resultis only limited to the data collected from the two trials. Isolating and identifying the dominant LAB strain could not be done due to lack ofexpertise 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, thenumber of CFU/ml of LAB/mold cannot be conclusively stated that they are actuallyLAB/mold. Selective agar medium like Nitrite Actidione Polymyxin (NAP) or Raka RayAgar 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 forfurther investigation. Identifying this strain could lead to producing favorable characteristics,and thus giving commercial values of the necessary production. Growing together theprobiotics of other food can provide a solution for people who are in need of certain nutrients. Page 41 of 51
  42. 42. Extended Essay - Biology Shin-Ae LeeWith this concept of synergism, culturing these probiotics can give rise to a production withfavorable characteristics in it. The exact optimum condition of pH and salt concentration for the growth ofprobiotic in doenjang can be further investigated. Finding out the optimum condition couldmaximize the growth of the beneficial bacteria in doenjang. This could both support theidentified probiotic, as well as maximize their viability for their growth in a beneficialenvironment. Page 42 of 51
  43. 43. Extended Essay - Biology Shin-Ae Lee7. Conclusion The initial aim of this research was to investigate the effect of pH levels and saltconcentration on the growth of LAB/mold in doenjang, and whether LAB/mold are stillviable in those extreme conditions. Results showed that LAB/mold survived in all tested pHlevels (pH 1, 3, 5, 7, 11, and 14), except in pH 14. The optimum growth occurred in pH 7, thecontrolled value of the experiment. This possibly suggests that LAB/mold in doenjang consistsome sorts of acid tolerant mechanisms that support the growth even in extreme acidicconditions. 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 indoenjang that helps maintain the osmolarity of the cell. Thus, this research shows that LAB/mold are viable in acidic medium and high saltconcentrations. Page 43 of 51
  44. 44. Extended Essay - Biology Shin-Ae Lee8. 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
  45. 45. Extended Essay - Biology Shin-Ae Lee 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 45 of 51
  46. 46. Extended Essay - Biology Shin-Ae Lee9. Appendix1. Method of making Doenjang The traditional doenjang first starts with the preparation of meju, which is a naturallyfermented 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 cookedfor 4 hours at 100°C. The enzymes in the fermentation of soybeans are mainly from themicroorganisms of meju. These soybean blocks are dried for 3 days in the air, tied up withrice straw, and then traditionally hung at the edge of an eave for 1 to 2 months to initiatenatural fermentation, which involves Bacillus sp., molds, and yeasts on the outside of themeju (1). It is in this process of fermentation that Bacillus subtilis, a type of bacteria indoenjang, reproduce, consuming soybean protein and water in the meju. When the process offermentation is finished, these bacteria are transformed into spores and endospores, which isthe cause of the unpleasant ammonia smell produced during the fermentation. After the wholeprocess of fermentation, the meju are put into large opaque pottery jars with brine and left tofurther ferment. It is at this stage of fermentation that various beneficial bacteria transformthe mixture into a further vitamin-enriched substance (2). Once the fermentation process isdone, the liquids and solids are separated. This solid part is the Korean fermented soybeanpaste, doenjang. There are mainly two different kinds of doenjang: one made by theconventional type, and one by the improved type. These two doenjangs differ in taste: thetraditional type gives a strong, stinging smell with a salty taste, while the improved type isn‟tas extreme. This peculiar taste is produced by a bacterium called Bacillus, which is inferredto have antibiotic characteristics. Page 46 of 51
  47. 47. Extended Essay - Biology Shin-Ae Lee2. Journal: Analysis of microbial communities in doenjang, a Koreanfermented soybean paste, using nested PCR-denaturing gradient gelelectrophoresis Page 47 of 51
  48. 48. Extended Essay - Biology Shin-Ae Lee3. MRSLactobacilli MRS Agar are used to isolate, enumerate, and cultivate Lactobacillus species.They are based on the formulations of deMan, Rogosa, and Sharpe. (4) The expectedappearances 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 48 of 51
  49. 49. Extended Essay - Biology Shin-Ae Lee4. 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 49 of 51
  50. 50. Extended Essay - Biology Shin-Ae Lee5. Type of Doenjang Used“Traditional, commercial, ripen fermented doenjang without preservatives. Didn‟t apply anysort of heat to preserve enzymes in doenjang.” Page 50 of 51
  51. 51. Extended Essay - Biology Shin-Ae Lee6. pH ScalepH is in a logarithmic scale, and thus a change of one pH unit becomes a factor of 10 inhydrogen 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 Page 51 of 51

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