유산균 분리 방법

2. Materials and methods

 

2.1 Bacterial culture condition

1. Lactic acid bacteria는 MRS배지로 37℃에서 24시간 배양하였다.

1. Lactic acid bacteria was cultured to MRS broth(BD Difco, france) for 24 h at 37℃.

2. MRS배지의 조성은 glucose 20 g, peptone No.3 10 g, beef extract 10 g, sodium acetate 5 g, ammonium citrate 2 g, potassium, phosphate dibasic (K2HPO4) 2 g, Tween 80 1 mL, FeSO4⦁7H2O 35 mg, MgSO4 575 mg, MnSO4⦁7H2O 120 mg per liter 이다.

2. The MRS medium ingredients is glucose 20 g, peptone No.3 10 g, beef extract 10 g, sodium acetate 5 g, ammonium citrate 2 g, potassium, phosphate dibasic (K2HPO4) 2 g, Tween 80 1 mL, FeSO4⦁7H2O 35 mg, MgSO4 575 mg, MnSO4⦁7H2O 120 mg per liter.

3. Steptococcus iniae, Vibrio sp.는 BHI+1% NaCl와 Marin배지로 25℃에서 24시간 배양하였다.

3. Streptococcus iniae, Vibrio sp. was cultured to Marine medium(BD Difco, France) and 1%NaCl addition BHI medium(BD Difco, France) for 24h at 25℃.

4. BHI배지의 조성은 calf brains(infusion from 200 g) 7.7 g, beef heart(infusion from 250 g) 9.8 g, proteose peptone 10 g, dextrose 2 g, sodium chloride 5 g, disodium phosphate 2.5 g plus 1% NaCl fer liter 그리고 Marine배지의 조성은 peptone 5 g, yeast extract 1 g, ferric citrate 0.1 g, sodium chloride 19.45 g, magnesium chloride 5.9g, magnesium sulfate 3.24 g, calcium chloride 1.8 g, potassium chloride 0.55 g, sodium bicarbonate 0.16 g, potassium bromide 0.08 g, strontium chloride 34 mg, boric acid 22 mg, sodium sillicate 4 mg, sodium fluoride 2.4 mg, ammonium nitrate 1.6 mg, disodium phosphate 8 mg fer liter.

4. The BHI medium ingredients is calf brains(infusion from 200 g) 7.7 g, beef heart(infusion from 250 g) 9.8 g, proteose peptone 10 g, dextrose 2 g, sodium chloride 5 g, disodium phosphate 2.5 g fer liter and Marine medium ingredients is peptone 5 g, yeast extract 1 g, ferric citrate 0.1 g, sodium chloride 19.45 g, magnesium chloride 5.9g, magnesium sulfate 3.24 g, calcium chloride 1.8 g, potassium chloride 0.55 g, sodium bicarbonate 0.16 g, potassium bromide 0.08 g, strontium chloride 34 mg, boric acid 22 mg, sodium sillicate 4 mg, sodium fluoride 2.4 mg, ammonium nitrate 1.6 mg, disodium phosphate 8 mg fer liter.

 

2.2 Screening for isolation of lactic acid bacteria

1. 본 실험에서는 신선한 닭내장과 김치에서 유산균을 분리하였습니다.

1. In this study, lactic acid bacteria was isolated to fresh chicken intestine and kimchii.

2. 닭내장은 코니컬튜브에 담아 아이스박스에 보관하였다.

2. Chicken intestine was keep in ice box to put conical tube.

3. 닭내장은 멸균된 나이프와 핀셋으로 조각냈다.

3. Chicken intestine were cut into sterilized knife and pincers.

4. 조각낸 닭내장을 0.85% NaCl 용액 100 mL에 넣어 섞어 주었다.

4. The cutting chicken intestine was mixed at 0.85% NaCl solution 100mL.

5. 그 다음에 MRS배지에 희석법을 사용해 도말하였다.

5. Then was spread to MRS medium agar by using serial dilutions method.

6. 도말된 MRS배지는 30℃, 37℃에서 2일간 배양하였다.

6. The spreading MRS agar were cultured for 48h at 30℃ and 37℃.

7. 이때 MRS배지에 보이는 백색콜로니의 균주를 분리하였다.

7. At this time was isolated visible white colony strains to MRS agar medium.

8. 분리 균주들은 16S RNA sequence으로 동정하였다.

8. Isolated strains were identification as 16S RNA sequence.

9. 분리된 Lactobacillus spp., Lactococcus spp., Leuconostoc spp.는 20% glycerol으로 -70℃에서 보관하였다.

9. Isolated Lactobacillus sp., Lactococcus sp., Leuconostoc sp. were stored at - 80℃ in the presence of 20%(v/v) glycerol.

microbiota : 토양, 물, 대기, 또는 생체표면, 기관 등 미생물의 서식권에 있는 미생물의 종류, 양, 혼합비등의양상.

2.3 16S RNA 동정 (Molecular identification)

1. 본 실험에서는 16S ribosomal RNA sequencing을 제노텍에서 실시하였다.

1. In this study, 16S ribosomal RNA sequencing were performed on Genotech, Co. (Daejeon, Republic of Korea).

2. 선택균주는 일반적인 primer(9F/1512R)를 사용하여 PCR하였다.

2. The isolated strains were polymerase chain reaction using universal primer(9F, 5'-AGA GTT TGA TCC TGG CTC AG-3' / 1512R, 5'-ACG GCT ACC TTG TTA CGA CTT-3').

3. PCR이 끝나면 10분 동안 전기영동 한다.

3. The PCR finish after was electrophoresis for 10 minute.

4. 전기영동 후에 PCR product purification kit(Genotech)으로 정제한다.

4. After electrophoresis was purify of PCR product purification kit.(Genotech).

5. 정제한 PCR product를 518F, 800R, 27F, 1492R primer(선택3개)의 사용으로 sequencing 반응을 진행한다.

5. Purified PCR product were performed sequencing reaction of using 518F, 800R, 27F, 1492R primer(select 3).

6. PCR이 끝나면 형광물질을 ddNTP제거-EtOH down방법으로 깨끗이 정제한다.

6. PCR finishes was purified fluorescent material as ddNTP remove-EtOH down method.

7. 정제 후 D.W나 HDF(Hi-Di-Formamide)에 녹이고, 3730xl sequencer(ABI)로 염기서열을 확인한다.

7. Purification finishes was dissolved in D.W or HDF(Hi-Di-Formamide), and base sequence to 3730xl sequencer(ABI) were identified.

8. 확인된 염기서열은 phred-phrap-consed pakage 프로그램으로 어셈블리 후에 최종 분석된 서열을 얻었다.

8. Identified base sequence got final identified sequence after assembly as phred-phrap-consed pakage program.

9. 최종 염기서열은 “GeneBank”나 DB로 비교 후에 확인했습니다.

9. Final base sequence was identification after comparing to 'genebank' or DB.

 

2.4 Stress response assay

1. 분리균주의 스트레스 반응분석은 동물의 장내생육 가능성에 대하여 확인하기 위해 진행하였다.

1. Stress response assay in isolated strains were performed for identified about intestines growth potential.

2. 소금, 산, 담즙에 대한 내성실험을 진행하였다.

2. Tolerance test proceed for salt, bile and acid.

3. 먼저 MRS배지에 소금농도는 0~2M으로, 산은 pH2.0~5.0으로 담즙산은 0~5%로 첨가해 만들었다.

3. First of all, MRS medium has making as salt 0~2M, bile 0~5% and acid pH 2.0~5.0 concentration .

4. 종균을 1% 접종하여 각 온도에서 24h동안 배양한다.

4. The Seed was incubation during 24h on each temperature by 1% inoculate.

5. 단계희석법을 이용해 대조군에 비교하여 내성을 측정하였다.

5. Using serial dilution method has measured tolerance in comparison at control group.

 

2.5 Antibiotics tolerance

 

1. 항생제 내성실험은 BBL Sensi-disk(Becton-Dickinson)를 사용해 확인하였다.

1. Antibiotics tolerance test using BBL Sensi-disk(Becton-Dickinson) had identify.

2. 본실험에서 사용한 항생제는 어류양식에 흔히 사용하는 것으로 선택했다.(Table 1)

2. In this study used antibiotics has selection as commonly using to fish culture(Table 1).the

3. 먼저, 균주를 MRS broth에 배양한다.

3. First, strains was cultured to MRS broth.

4. 배양된 균주를 MRS agar에 분주한다.

4. Cultured strains had spread to MRS agar.

5. 균주를 분주한 MRS agar에 항생제 disk를 올린다.

5. Cultured strains spread to MRS agar, and put up a antibiotics disk.

6. 24h 배양 한 후 Disk 주변의 투명환 크기를 측정한다.

6. After 24 h incubation, clear zone size of disk edge was measured.

7. BBL Sensi-disc Method를 이용해 감수성 및 내성을 확인한다.

7.Using the BBL Sensi-disc Method was identification in susceptibility and resistance.

Table 1. used antibiotics

Antibiotics	Content
Kanamycin	30 ug
Neomycin	30 ug
Streptomycin	10 ug
Amocicillin/Clavulanic acid	30 ug
Ampicillin	10 ug
Penicillin	10 IU/IE/UI
Tetracycline	30 ug
Erythromycin	15 ug
Lincomycin	2 ug

 

 

2.6 Antibacterial activitiy fish pathogens

1. 분리균주의 항균활성을 측정하기 위해 어류병원균을 사용했다.

1. Antibacterial activity of isolated strains had used in fish pathogen for measure. .

2. Vibrio anguilarum, Vibrio vulnificus, Vibrio ichthyoenteri, Edwardsiella ictaluri, Streptococcus iniae, Streptococcus parauberis 균주는 KCTC로부터 분양받았다.

2. Vibrio anguilarum (KCTC 2711), Vibrio vulnificus (KCTC 2962), Vibrio ichthyoenteri (KCTC12725), Edwardsiella ictaluri (KCTC12264) was obtained from the Korean Collection for Type Culture (Daejeon, Korea).

3. 항균활성은 disk diffusion assay 방법으로 측정하였다.

3. Antibacterial activity was measured as disk diffusion assay method.

4. 먼저, 배양된 균주를 Marine, NB+0.5% NaCl agar에 100 uL 도말하여 준비한다.

4. First of all, cultured fish pathogens prepare spreading 100 uL to Marine, NB+0.5% NaCl agar.

5. 그다음 paper disk (6mm)에 유산균배양액 100 uL를 흡수시킨다.

5. Then lactic acid bacteria culture medium 100 uL have absorb to paper disk(6 mm).

6. 어류병원균이 도말된 배지에 유산균배양액이 흡습된 paper disk를 올린다.

6. Lactic acid bacteria culture medium absorbed paper disk were put to fish pathogen spreading medium.

7. 24시간 배양 후 paper disk 주변의 투명환 크기를 측정한다.

7. After 24h incubation, paper disk edge is measured clear zone size.