Antigenotoxic Activity of Lactic Acid Bacteria from Korean Fermented Kimchi and Identification of Selected Strains : 김치에서 분리한 유산균들의 항유전독성 활성과 이들의 동정에 대한 연구

국문 초록 (Abstract)

식이와 관련하여 암이 높은 발생률을 보인다는 관점에서, 하나의 중요한 연구 목표로 유전독성을 가지는 발암원들과 그것들의 활성을 방해하는 잠재력을 지닌 영양적 요인들의 동정을 말할 수 있다.
본 연구에서는 김치로부터 우세 발효 유산균주를 분리하고 직접발암물질인 N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)를 이용하여 이들이 갖는 항유전독성 활성을 조사하였다. 단일 세포 겔 전기영동 기술(코멧법) 은 여러 포유동물 세포들에 있어 DNA 손상을 측정하는 방법이다. 코멧법은 단일 세포들에 존재하는 DNA 절편을 감지하는데에 민감하고 빠른 기술이다. 그래서 이 기술을 이용하여 6종류의 김치들로부터 분리한 30종의 우세 발효 유산균주들의 항유전독성 활성을 측정함으로 이것들이 가지는 암예방 잠재력을 조사하였다. 먼저 3T3 세포주를 이용하여 각종 김치로부터 분리한 30종의 우세 발효 유산균주들의 항유전독성 활성을 검색한 뒤, 효과가 뛰어난 8종을 선발하여 Spraque-Dawley rat의 대장 세포에서 이들의 항유전독성 활성을 조사하였다. 또한 이들을 동정한 결과 이들은 Leuconostoc mesente sub. mesenteroides, Lactobacillus plantarum, Lactobacillus brevis 등으로 확인되었다.
김치로부터 분리한 총 30종의 유산균주 중 약 25~30% 정도인 8종의 유산균주들(부추김치에서 분리한 B-1, B-5, B-6, 오이김치에서 분리한 O-4, O-5, 백김치에서 분리한 M-2, 열무김치에서 분리한 Y-5 그리고 포기김치에서 분리한 P-1)은 MNNG에 대하여 유의할만한 항유전독성 활성을 나타내었고, 특히 B-1과 B-6는 Spraque-Dawley rat의 primary colon cells에서도 매우 강한 항유전독성 활성을 보였다.
결론을 말하자면, 이 연구는 김치로부터 분리한 우세 발효 유산균주들 중 일부가 3T3 세포와 Spraque-Dawley rat의 대장 세포 모두에 있어 MNNG에 의한 유전독성을 강하게 저해하며 그 기작은 알 수 없지만, 특히 부추김치로부터 분리한 B-1(Lactobacillus plantarum)과 B-6(Lactobacillus brevis)는 강한 암예방 잠재력을 가진다고 말할 수 있다.

다국어 초록 (Multilingual Abstract)

In view of the high incidence of dietary-related tumors, one important research goal is to identify the participating genotoxic carcinogens and the nutritional factors that may counteract their activities.
In this study, dominant lactic acid bacteria strains were isolated and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a directly acting carcinogen, was treated to investigate the antigenotoxic activity of these isolates.
Single cell gel electrophoresis technique (in other words, comet assay) is a relatively simple, rapid and sensitive method for measuring DNA strand breaks at the level of single cells, and is widely used to measure DNA damage in various mammalian cells. Therefore, I used comet assay to examine the antigentoxic activity of 30 different isolates of dominant lactic acid bacteria strains which were extracted from 6 other kinds of Kimchi, and furthermore investigated the potential of cancer prevention of these isolates. First, the antigenotoxic activity of 30 different isolates of dominant lactic acid bacteria strains were screened using 3T3 cell line. Next, among these isolates, 8 isolates which showed strong antigenotoxic activity were selected and their antigenotoxic activities were examined in primary colon cells of Spraque-Dawley rat. Further identification of these isolates classified them into strains of Leuconostoc mesente sub. mesenteroides, Lactobacillus plantarum and Lactobacillus brevis.
Among 30 strains isolated from several kimchi, about 25 to 30 percent of them, 8 strains (B-1, B-5, B-6 isolates from buchu kimchi, O-4, O-5 from cucumber kimchi, M-2 from baik kimchi, Y-5 from yeolmoo kimchi and P-1 from baechu kimchi) showed antigenotoxic activity. Especially B-1 and B-6 isolates revealed significantly strong antigenotoxic activity in primary colon cells of Spraque-Dawley rats.
In conclusion, several isolates of dominant lactic acid bacteria strains which were isolated from Kimchi strongly inhibited the genotoxic effect induced by MNNG treatment in both 3T3 cell line and primary colon cells from Spraque-Dawley rat. Although the mechanisms have yet to be revealed, it is sufficient to conclude through this study that these isolates, especially B-1(Lactobacillus plantarum) and B-6(Lactobacillus brevis) isolated from buchu kimchi have strong potential of cancer prevention.

목차 (Table of Contents)

• Abstract = ⅰ
• 국문 초록 = ⅱ
• CONTENTS = ⅳ
• List of Figures = ⅵ
• List of Tables = ⅵ
• Ⅰ. Introduction = 1
• Ⅱ. Materials and Methods = 4
• 1. General reagents and chemicals = 4
• 2. Kimchi for experiments = 4
• 3. Test compound = 4
• 4. Isolation and culture of lactic acid bacteria = 4
• 4.1 Isolation and culture of lactic acid bacteria = 4
• 4.2 Treatment of MNNG and lactic acid bacteria = 5
• 5. Cell culture and preparation for experiments = 5
• 6. Animals and isolation of primary colon cells = 6
• 6.1 Animals = 6
• 6.2 Isolation of primary colon cells of Spraque-Dawley rats = 6
• 7. Comet assay = 7
• 7.1 Preparation of slides = 7
• 7.2 Coating the gel with cell extracts = 7
• 7.3 Lysis, unwinding, electrophoresis and neutralization = 8
• 7.4 Staining and analysis of slides = 8
• 7.5 Statistical analysis = 10
• 8. Isolation of lactic acid bacteria having antigenotoxic activity = 10
• 8.1 Utilization of fructooligosaccharides = 11
• 8.2 Hydrophobicity assay = 11
• 8.3 Lactic acid configuration = 11
• 8.4 Identification of meso-diaminopimeric acid (meso-DAP) = 12
• 8.5 Biogenic amines production = 12
• 8.6 Nitrate reduction = 12
• Ⅲ. Results = 14
• 1. Determination of the optimum concentration of MNNG treatment on cells of 3T3 cell line and primary colon cells from Spraque-Dawley rats = 14
• 1.1 Determination of the optimum concentration of MNNG treatment on cells of 3T3 cell line = 14
• 1.2 Determination of the optimum concentration of MNNG treatment on primary colon cells from Spraque-Dawley rats = 16
• 2. Antigenotoxic acitivity of lactic acid bacteria strains from kimchi in cells = 18
• 2.1 Antigenotoxic acitivity of lactic acid bacteria strains from kimchi in cells of 3T3 cell line = 18
• 2.2 Antigenotoxic acitivity of lactic acid bacteria strains from kimchi in primary colon cells of Spraque-Dawley rats = 20
• 3. Identification of lactic acid bacteria strains from kimchi showing strong antigenotoxic acitivity = 23
• Ⅳ. Discussion = 27
• Ⅴ. References = 28