Identification and cholesterol lowering effect of Lactic Acid Bacteria from Kimchi : 김치에서 분리한 유산균의 분리 동정과 콜레스테롤 저하 효과에 대한 연구

국문 초록 (Abstract)

고농도의 cholesterol은 심혈관계 질환을 일으키는 중요한 요인 중의 하나이다. 특히 사람의 혈중 높은 cholesterol 수치는 사람을 죽음에까지 이르게 하는 심장 마비와 같은 심혈관계 질환의 주요 원인으로 밝혀 졌다. 이로 최근에 콜레스테롤을 저하하는 유용한 기능이 있는 유산균에 대한 관심이 증가 하고 있다. 특히 유산균은 한국의 발효 김치의 우종균으로 알려져 있다. 이 실험을 통해 5 종류의 김치로부터 유산균을 분리 하였고 각각의 형태학적인 특징과, 물리적인 특징에 따라서 분리하였다. 이를 더욱 정확하게 동정하기 위하여 repetitive element sequence-based polymerase chain reaction (rep-PCR), randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR), and 16S rDNA gene sequencing의 유전형 동정 방법을 사용하였다. In vitro 실험으로 8종류의 LAB가 broth 상에서 cholesterol 저하 기능이 있는 것으로 나타났으며, HFI 58, HFI 40, Yeoulmu E 이 세 종류의 strains이 유의성 있는 cholesterol 저하 효과를 보였다. 위의 세 균을 선택하여 우진 B&G 회사를 통해 사료와 한 후 닭을 대상으로 in vivo test를 실시한 결과 HFI 58을 처리한 집단에서 유의성 있는 달걀의 cholesterol 수치 저하가 관측되었다.

다국어 초록 (Multilingual Abstract)

High levels of cholesterol are considered to be a major risk factor for vascular disease including coronary heart disease. Lipid research clinic program in USA reported that the higher total serum cholesterol is in humans, the greater is the risk for the emergence of coronary heart disease which cause human death. Much attention is recently being given to further elucidate beneficial functions of lactic acid bacteria for human health as probiotic function like reduction of cholesterol. Lactic acid bacteria are dominant microflora in fermented kimchi. In this study, lactic acid bacteria were isolated from 5 kinds of fermented kimchi and determined their physiologic property. Total 31 of LAB were isolated from kimch samples. In order to identify the LAB more accurately, were done repetitive element sequence-based polymerase chain reaction (rep-PCR), randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR), and 16S rDNA gene sequencing. 8 of them were selected by their ability to lower cholesterol level in vitro test. HFI 58, HFI 40, and Yeulmu E4 strains showed significant cholesterol lowering effect in vitro test. These were made of special feed of chicken by WooJin B&G. A Diet was tested during 5 weeks and feed of supplement with HFI58 strain groups had significant lower cholesterol concentration in egg yolk.

목차 (Table of Contents)

• Abstract = ⅰ
• 국문초록 = ⅲ
• CONTENTS = ⅳ
• List of Figure = ⅶ
• List of Table = ⅷ
• Ⅰ. Introduction = 1
• Ⅱ. Materials and Methods = 3
• 1. Collection of samples = 3
• 2. Microbial enumeration and isolation = 3
• 3. Phenotypic characterization = 4
• 3.1 Initial Characterization = 4
• 3.2 Lactic acid configuration = 4
• 3.3 Identification of meso-diaminopimelic acid (meso-DAP) = 5
• 3.4 Carbohydrate fermentation assay = 6
• 3.5 Hydrolysis of Arginine = 6
• 4. Genotypic characterization = 7
• 4.1 DNA Isolation = 7
• 4.2 RAPD-PCR analysis = 8
• 4.3 REP-PCR analysis = 9
• 4.4 16S rDNA sequencing = 9
• 5. BSH activity, Tolerance, and Reduction of cholesterol = 10
• 5.1 Measurement of Bile salt hydrolase activity = 10
• 5.2 Low pH tolerance and Bile salt tolerance = 10
• 5.3 In vitro test of cholesterol lowering effect by LAB = 11
• 5.4 In Vivo Test of Cholesterol reducing effect in Chicken egg yolk = 12
• 5.5 Statistical Analysis = 13
• Ⅲ. Results = 14
• 1. Isolation of Lactic Acid Bacteria = 14
• 2. Clustering of LAB by phenotypic characterization = 14
• 3. Genotypic characterization = 18
• 3.1 Rep-PCR fingerprinting analysis = 18
• 3.2 16S rDNA sequencing = 19
• 4. Cholesterol lowering property = 27
• 4.1 Low pH tolerance, Bile salt tolerance, and Bile salt hydrolase activity = 27
• 4.2 In vitro cholesterol-lowering test = 27
• 4.3 In vivo feeding trial = 28
• Ⅳ. Discussion = 34
• Ⅴ. Reference = 36