Functional study of Lactobacillus sakei and Leuconostoc mesenteroides isolated from Kimchi

다국어 초록 (Multilingual Abstract)

The Kimchi samples were acquired from Seoul, Gwangju, Andong, Suncheon, and Damyang. From Kimchi liquid sampled from each Kimchi, 114 strains were separated from Lactibacillus sakei and 98 strains were separated from Leuconostoc mesenteroides. Several experiments were held in steps with the separated strains, and the outstanding ones were selected among the strains.
The first experiment was held to find if the separated strains grow well in a condition similar with human body. So the testing environment was set as 37℃ and oxygen-free. In the experiment with Lactobacillus sakei, 3 strains could not grow at 37℃, and 19 strains could not grow in oxygen-free circumstance. In the experiment with Leuconostoc mesenteroides, 4 strains could not grow at 37℃ while all strains grew in oxygen-free circumstance.
The selected strains underwent tests to figure out antioxidant, antitumor effect, and anti-inflammatory. The result of antioxidant test did not show any significant difference with the test result of LGG strains that were used as control strains. Yet, most of the strains showed more than 60% of the free radical scavenging ability. In the antitumor effect test, the strains were found to hinder the cell lines growth in HT-29, MCF 7, and HeLa cell line. Lastly, in the anti-inflammatory test using RAW 264.7 cell line, Lactobacillus sakei and Leuconostoc mesenteroides were found to be effective at lowering nitric oxide.
After selecting 10 strains from each species that have outstanding vitality through the previous experiments. As the RAPD test was intended to identify the dissimilarity of DNA sequence, the selected strains present different DNA sequence even though their species are same. In the antibacterial activity test, the degree of vitality was identified by the size of inhibition zone that forms by agar-well diffusion. Lactobacillus sakei was found to have good antibacterial activity. The degree of adipocyte differentiation was identified through Oil-red-O staining that comes out after differentiating Lactobacillus sakei and Leuconostoc mesenteroides together during the adipocyte differentiation process of C3H10T1/2 cell line. In the experiment with Lactobacillus sakei, the differentiation of AM2 and ADM14 strains was controlled the most. In the experiment with Leuconostoc mesenteroides, the differentiation of S-YRWM27 and S-YRWM28 strains was controlled the most. AM2 and ADM14 decreased the expression of the genes compared with the controls. The growth curve of the 4 strains was studied at 15℃, 20℃, 30℃, and 37℃. Since ADM14 showed outstanding growth at every temperature, ADM14 is expected to have highest industrial use availability.

목차 (Table of Contents)

• Ⅰ. Introduction
• Ⅱ. Materials and Method
• 1. Sampling and LAB isolation 12
• 2. DNA extraction/purification and 16S rRNA gene sequence analysis 12
• 3. Temperature and Anaerobic test 13
• 4. Measurement of anti-oxidative activity 13
• 4.1 LAB preparation and culture 13
• 4.2 Radical scavenging activity 13
• 5. Measurement of anti-cancer activity 14
• 5.1 LAB preparation and culture 14
• 5.2 Cell culture 14
• 5.2.1 Medium and reagent 14
• 5.2.2 Cell line 14
• 5.2.3 Cell culture 14
• 5.3 MTT assay 15
• 6. Measurement of Anti-inflammatory activity 15
• 6.1 LAB preparation and culture 16
• 6.2 Cell culture 16
• 6.2.1 Medium and reagent 16
• 6.2.2 Cell line 16
• 6.2.3 Cell Culture 16
• 6.3 NO (Nitric oxide) assay 17
• 7. RAPD (Random amplification of polymorphic DNA) 17
• 8. Antibacterial activity 18
• 9. Adipocyte differentiation 18
• 9.1 LAB culture medium and cytoplasmic fraction 19
• 9.2 Cell culture and adipogenic differentiation 19
• 9.3 Mesurement of intracellular lipid accumulation 19
• 9.4 Mesurement of adipocyte gene expression 20
• 10. Growth curve 21
• 11. Statistical analysis 21
• Ⅲ. Result
• 1. Strain selection and identification 22
• 2. Temperature and Anaerobic test 24
• 3. Measurement of anti-oxidative activity 24
• 3.1 Radical scavenging activity 24
• 4. Measurement of anti-cancer activity 32
• 4.1 MTT assay 32
• 5. Measurement of Anti-inflammatory activity 54
• 5.1 NO assay 54
• 6. RAPD (Random amplification of polymorphic DNA) 62
• 7. Antibacterial activity 65
• 8. Adipocyte differentiation 68
• 8.1 Effect of Lactobacillus sakei and Leuconostoc mesenteroides strains on adipocyte differentiation 68
• 8.2 Effect of the cytoplasmic fraction of Lactobacillus sakei and Leuconostoc mesenteroides strains on adipogenic-specific gene expression 71
• 9. Growth curve 73
• Ⅳ. Discussion
• Ⅴ. References