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손바닥 선인장 열매 고분자 점질물을 분해하는 진균의 분리 및 동정

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Alternative Title
Isolation and Identification of a Fungus Degrading Mucilage Polymers from Fruit of Opuntia ficus-indica var. Saboten
Abstract
The prickly fruit of Opuntia ficus-indica cultivated in JeJu Island is rich with high molecular weight mucilage, which has high viscosity and hinders its use in food industry. A fungus that produced the mucilage-degrading enzyme was isolated and identified in this study.
From prickly pears viscous mucilage was extracted and purified. Carbohydrates were detected in the mucilage preparations by the Molisch method and its total sugar concentration by the phenol-sulfuric acid method using maltose as a standard was 64.85 mg/mL in 0.5% aqueous solution. Ninhydrin-positive reaction was observed in 0.5% aqueous mucilage solution and our results showed that the mucilage preparations have amino acids or peptides.
Many strains of fungi isolated from soil were grown on M9 medium containing congo red using the mucilage as the sole carbon source and the fungus with the best decolorizing ability of congo red was selected. Its pure culture was obtained through repeated subculturing on M9 medium containing the mucilage as the sole carbon source and symbol Bx was assigned to the purified fungal strain. Crude enzyme was prepared from culture filtrate of the fungus Bx.
To confirm the fungal enzymatic degradation of the mucilage, analysis by TLC, HPLC and liquid gel permeation chromatography were carried out. The degradation products at Rf 0.19 was observed by TLC of solution after incubation of the mucilage with fungal crude enzyme. All mucilage solution, fungal enzyme preparation and solution after incubation of the mucilage with heat-inactivated crude enzyme showed no degradation product. The spot at Rf 0.19 clearly showed that mucilage was degraded by the fungal enzyme. When the degradation products were tested for its coloring reaction with ninhydrin or phenol-sulfuric acid as a spry reagent, only phenol sulfuric acid made brown color development. This coloring reaction suggested the presence of carbohydrates without amino acids or peptides within degradation products of the mucilage.
Analysis by HPLC of the mucilage after incubation with fungal crude enzyme showed that high molecular weight fractions(molecular weight 2x10^(6)) shifted to low molecular weight fractions(less than molecular weight 1x10⁴).
Analysis by gel permeation chromatography on Sephadex G-100 also showed molecular weight decreased of the mucilage resulting from incubation with fungal culture filtrate.
These results showed that high molecular weight mucilage fractions were degraded by the crude enzyme from the fungus Bx.
To test whether the crude enzyme from the fungus Bx influences on antioxidative activity of prickly pear, total polyphenol contents, flavonoide contents, DPPH radical scavenging activity, hydroxy radical scavenging activity, hydrogen peroxide scavenging activity, ACE inhibition activity were investigated. Both contents of polyphenol and flavonoid in prickly pear extract after incubation with fungal crude enzyme were higher than those without incubation with fungal crude enzyme. Both hydrogen peroxide scavenging activity and ACE inhibition activity of the extract increased through preincubation of prickly pear with the crude enzyme.
Fungus Bx on microscope(×400) was filamentous and had fusiform spore. The spores of fungus Bx were scattered or gregarious, with two or more apical appendages and consisted of 2-several layers of brown. The ITS regions of 5.8S rDNA sequences from the isolate fungus Bx showed higher similarity than 99% to those of Pestalotiopsis aquatica.
This could be the first report concerning the isolate and identification of a microorganism degrading high molecular weight mucilage from prickly fruit of O. ficus-indica.
Author(s)
허윤희
Issued Date
2008
Awarded Date
2008. 2
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000004226
Alternative Author(s)
Huh, Yoon-Hee
Affiliation
제주대학교 대학원
Department
대학원 식품공학과
Advisor
고영환
Table Of Contents
Ⅰ. 서론 = 1
Ⅱ. 연구사 = 6
1. 손바닥 선인장 열매 점질물 = 6
2. 손바닥 선인장 열매 색소 = 9
3. 손바닥 선인장의 효능 = 11
4. 진균이 생산하는 당분해 효소 = 13
5. 균주(진균) 동정 = 14
Ⅲ. 재료 및 방법 = 16
1. 재료 = 16
1) 선인장 열매 점질물 분리 = 16
2) 균주 분리용 토양시료 = 16
3) Congo red 용액 = 17
4) 시판 선인장 열매 동결건조 분말 = 17
2. 선인장 열매 점질물의 이화학적 특성 = 18
1) 닌하이드린 시험 = 18
2) Molisch 시험 = 18
3) 페놀-황산법에 의한 총당 정량 = 19
3. 선인장 열매 점질물 분해 균주 및 효소 분리 = 20
1) 선인장 열매 점질물 분해 균주 분리 = 20
2) 분리 균주에서 분해효소 생산 = 22
4. 분리 균주 효소에 의한 선인장 열매 점질물의 분해 확인 = 23
1) 선인장 열매 점질물 효소 반응 = 23
2) Thin layer chromatography(TLC) 분석 = 24
3) High performance liquid chromatography(HPLC) 분석 = 24
4) Liquid gel permeation chromatography 분석 = 26
5. 분리 균주 효소 처리한 시판 선인장 열매 동결건조 분말의 항산화활성 변화 = 27
1) 시판 선인장 열매 동결건조 분말의 항산화 활성 시료 = 27
2) 총폴리페놀 함량 분석 = 28
3) 플라보노이드 함량 분석 = 29
4) 1,1-Diphenyl-2-pricrylhydrazyl(DPPH) 라디칼 소거활성 = 30
5) Hydroxyl 라디칼 소거 활성 = 31
6) Hydrogen peroxide 소거 활성 = 32
7) Angiotensin converting enzyme(ACE) 저해 활성 = 33
6. 분리 균주 동정 = 34
1) 분리 균주의 형태학적 동정 = 34
2) Internal transcribed spacer(ITS)-5.8S rDNA sequencing = 34
Ⅳ 결과 및 고찰 = 35
1. 선인장 열매 점질물의 이화학적 특성 = 35
2. 선인장 열매 점질물 분해 미생물 분리 = 37
3. 분리 균주 효소에 의한 선인장 열매 점질물의 분해 확인 = 39
1) Thin layer chromatography(TLC) 분석 = 39
2) High performance liquid chromatography(HPLC) 분석 = 41
3) Liquid gel permeation chromatography 분석 = 45
4. 분리 균주 효소 처리한 시판 선인장 열매 동결건조 분말의 항산화활성 변화 = 47
1) 총폴리페놀과 플라보노이드 함량 = 47
2) 1,1-Diphenyl-2-pricrylhydrazyl(DPPH) 라디칼 소거활성 = 49
3) Hydroxyl 라디칼 소거 활성 = 50
4) Hydrogen peroxide 소거 활성 = 52
5) Angiotensin converting enzyme(ACE) 저해활성 = 53
5. 분리 균주 BX의 동정 = 55
1) 분리 균주 Bx의 형태학적 동정 = 55
2) Internal transcribed spacer(ITS) 5.8S rDNA sequencing = 58
Ⅴ 요약 = 63
참고문헌 = 65
Degree
Doctor
Publisher
제주대학교 대학원
Citation
허윤희. (2008). 손바닥 선인장 열매 고분자 점질물을 분해하는 진균의 분리 및 동정
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General Graduate School > Food science and Engineering
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