김치로부터 분리한 Leuconostoc mesenteroides subsp. mesenteroides가 생산하는 Bifidobacterium 성장 자극 물질의 분리 및 특성 = Isolation and Characterization of Bifidogenic Growth Stimulator Produced by Leuconostoc mesenteroides subsp. mesenteroides Isolated from Kimchi

다국어 초록 (Multilingual Abstract)

Bifidobacterium is known as a beneficial bacteria in the human gut, and some strains of the genus Bifidobacterium are used as probiotics. The purpose of this study is to find out bifidogenic growth stimulator (BGS)-producing lactic acid bacteria (LAB) that stimulate the growth of Bifidobacterium. Seven hundred three strains, isolated from kimchi, were used BGS screening. We selected the K748 strain, which showed the highest BGS activity of 320 AU/ml. This strain was identified as Leuconostoc mesenteroides subsp. mesenteroides using 16s rDNA sequencing and carbohydrate fermentation test, and the strain was named as L. mesenteroides K748. Measurements of the growth and BGS activity of L. mesenteroides K748 in the MRS medium showed BGS activity from 12 hours after incubation and maintained maximum activity (320 AU/ml) after 15 hours. Thus, BGS production of L. mesenteroides K748 was begun after the stationary phase. When incubating B. longum, addition of L. mesenteroides K748 culture supernatant increased significantly the growth of B. longum. Also, ethyl acetate extract of L. mesenteroides K748 culture supernatant increased significantly the growth of B. longum. Based on this result, ethyl acetate extract was used for HPLC and LC-MS analysis. DHNA, known as one of the BGS, was used as a standard material. Through HPLC analysis, both of DHNA and ethyl acetate extract of L. mesenteroides K748 were confirmed peaks at retention time of 7.5 minutes. Through LC-MS analysis, the molecular weight of DHNA and ethyl acetate extract of L. mesenteroides K748 was found to be 204.1. Collectively, we confirmed that L. mesenteroides K748 produced DHNA which stimulates the growth of Bifidobacterium.

목차 (Table of Contents)

• Ⅰ. 서 론 1
• Ⅱ. 재료 및 방법 4
• 1. Bifidogenic Growth Stimulator (BGS) 생산 유산균의 분리 및 동정 4
• 1-1. 김치 유산균의 분리 4
• 1-2. 균주 상등액 준비 4
• 1-3. BGS 생산 유산균 스크리닝 4
• 1-4. 16s rDNA sequencing 5
• 1-5. 탄수화물 발효능 확인 6
• 2. BGS 생산 유산균의 특성 7
• 2-1. 생육 곡선 7
• 2-2. BGS 활성 측정 방법 7
• 2-3. 흡광도를 이용한 BGS 활성 측정 7
• 3. BGS 물질의 특성 8
• 3-1. 용매별 분획 및 농축 8
• 3-2. 용매별 추출에 따른 BGS 활성 측정 8
• 3-3. High-performance liquid chromatography (HPLC) 9
• 3-4. Liquid chromatography-mass spectrometry (LC-MS) 10
• Ⅲ. 결과 및 고찰 12
• 1. BGS 생산 유산균의 분리 12
• 1-1. 1차 스크리닝 결과 12
• 1-2. 2차 스크리닝 결과 19
• 2. BGS 생산 유산균의 동정 21
• 2-1. 16s rDNA sequencing 결과 21
• 2-2. 탄수화물 발효능 결과 23
• 3. Leuconostoc mesenteroides K748의 BGS 활성 27
• 3-1. L. mesenteroides K748의 생육에 따른 BGS 활성 측정 27
• 3-2. 흡광도를 이용한 L. mesenteroides K748의 BGS 활성 측정 29
• 4. L. mesenteroides K748이 생산하는 BGS 물질의 특성 31
• 4-1. 용매별 추출에 따른 BGS 활성 측정 31
• 4-2. HPLC 분석 34
• 4-3. LC-MS 분석 37
• Ⅳ. 요약 및 결론 40
• Ⅴ. 참고 문헌 42