Aspergillus oryzae와 당분해효소를 이용한 인삼의 생물전환 = Biotransformation of Korean Ginseng by aspergillus oryzae and commercial glucosidases

다국어 초록 (Multilingual Abstract)

Biotransformation of Korean Ginseng by Aspergillus oryzae and Commercial Glucosidase


Kim Bo-Hye

Department of Microbiology and Immunology,
College of Medicine, Pusan National University, Pusan, Korea


Abstract

When Ginseng is taken orally as a crude drugs, the metabolites of Ginseng saponin(ginsenoside) is absorbed from the intestines. The clinical basis of Ginseng treatment may be partly associated with differences in intestinal microflora of hosts. Protopanaxadiol-type ginsenosides such as Rb1, Rb2, and Rc are metabolized by intestinal bacteria after oral administration to their final derivative 20-O-β-D-flucopyranosyl-20-(S)-protopanaxadiol (compound K, M1, IH901). The in vivo anti-metastatic effect of the ginsenosides was primarily based on their metabolite compound K.
It has been assumed that the glycosides cannot be absorbed from the small intestine and cleavage of the β-glycoside linkage will not occur until the compounds reach the microflora in the large intestine. Biological activity depends on the presence or absence of the glycoside residue. The position and nature of the sugar residue may increase the uptake of the compound in the small intestine. The aglycone is likely to have a greater biological effect than the glycoside.
Therefore, Ginseng was fermented by Aspergillus oryzae to get metabolites with increased biological activity, higher digestiveness, and modified structure. Then new biotransformed compounds (WG2-2-1, WG2-2-2) were detected and isolated through several chromatographic techniques. And WG2-2-2 was confirmed to biologically active compound K by TLC, HPLC, and mass spectrum. Also biological activity of biotransformed WG2-2-1 and WG2-2-2 were investigated on antibacterial, cytotoxic, and DPPH radical scavenging assay. WG2-2-2 was more active on aboved biological assays. It suggests that WG2-2-1 is intermediate metabolite transforming to final WG2-2-2 (compound K). Also biotransformation of ginseng saponin or ginseng products by pectinex was performed. Then compounds (PG-1, PG-2, PG-3) were biotranformed from ginseng. And those compounds were isolated by several chromatographic techniques. Finally PG-3 was identified as compound K, which was transformed by Aspergillus oryzae in this research.
Also its biological activity was observed to be active on DPPH free radical scavenging and proliferation of tumor cells. But minor biotransformed metabolites were under the investigation to obtain a lot of compounds by scale-up fermentation and enzyme reaction to identify those of structure and observe those of biological activity.

목차 (Table of Contents)

• 목 차
• Ⅰ. 서 론 1
• Ⅱ. 재료 및 방법 8
• 1. 인삼 재료 8
• 2. 사용 균주 및 효소 8
• 2.1. 사용균주 8
• 2.2. 사용효소 9
• 3. 사용시약 및 사용기기 9
• 3.1. 사용시약 9
• 3.2. 사용기기 10
• 4. 생균제를 이용한 인삼의 발효 11
• 4.1. 생균제의 발효 및 Aspergillus oryzae를 이용한 대량배양 11
• 4.2. 배양액의 추출 및 용매분획 11
• 5. 당분해효소를 이용한 인삼의 전환 12
• 5.1. 처리조건 12
• 5.2. 배양액의 추출 및 용매분획 12
• 6. 배양산물의 분석 13
• 6.1. Thin Layer Chromatography (TLC) 13
• 6.2. High performance liquid chromatography (HPLC) 13
• 6.3. 전환산물의 분리 및 동정 14
• 7. 생리활성 분석 14
• 7.1. 항균활성 측정 14
• 7.2. DPPH radical 소거 활성 측정 15
• 7.3. 세포독성 저해 활성 측정 15
• Ⅲ. 결 과 17
• 1. 생균제를 이용한 수삼의 발효 17
• 1.1. 발효물의 추출 및 분석 17
• 1.2 수삼 발효를 위한 생균제의 선정 19
• 1.3. Aspergillus oryzae KCTC6292에 의한 수삼발효의 대량배양 22
• 1.4 전환물질의 분리 및 정제 26
• 1.5 전환물질의 동정 29
• 1.6 생리활성 측정 31
• 1.6.1 항균활성 측정 31
• 1.6.2 DPPH free radical 소거활성 측정 33
• 1.6.3 세포독성 측정 35
• 2. 당분해효소에 의한 인삼의 전환 38
• 2.1 전환물의 추출 및 분석 38
• 2.2 효소의 선정 38
• 2.3 인삼 시료의 선정 42
• 2.4 효소 최적 반응조건 선정 45
• 2.4.1 효소의 농도 45
• 2.4.2 효소의 반응온도 및 반응시간 49
• 2.4.3 효소의 최적 반응조건 51
• 2.5 분리 및 정제 51
• 2.6 전환물의 분석 54
• 2.7 생리활성 분석 56
• Ⅳ. 결론 및 활용방안 58
• 1. Aspergillus oryzae에 의한 수삼의 전환 58
• 2. Pectinex에 의한 인삼의 전환 59
• 3. 활용방안 59
• 참고문헌 62
• Abstract 68
• List of Tables
• Table 1. Fermentation Condition of Undried Ginseng by Probiotics. 20
• Table 2. Fermentation of Undried Ginseng by Aspergillus oryzae KCTC6292. 24
• Table 3. Antibacterial Activity of Ginseng Fermented by Probiotics. 32
• List of Figures
• Figure 1. Structure of Ginsenosides. 7
• Figure 2. Quantitative Analysis of Compound K. 18
• Figure 3. TLC Analysis of Fermented Ginseng by Probiotics. 21
• Figure 4. Reverse Phase TLC Analysis of Undried Ginsengs Fermented by
• Probiotics. 21
• Figure 5. Culture Profiles of Fermented Ginseng by Aspergillus oryzae
• KCTC6292. 24
• Figure 6. Cultural Profile of Undried Ginseng by Aspergillus oryzae. 25
• Figure 7. Purification Scheme of WG2-2-1 and WG2-2-2 from Fermented
• Undried Ginseng. 27
• Figure 8. Purification of WG2-2-1 from Fermented Undried Ginseng. 28
• Figure 9. LR-MS Spectrum of WG2-2-2. 30
• Figure 10. DPPH Radical Scavenging Activity of WG1, WG2, and WG3. 34
• Figure 11. Cytotoxicity of WG1, WG2, and WG3 on PC-3 Cells. 36
• Figure 12. Cytotoxicity of WG1, WG2, and WG3 on MCF-7 Cells. 36
• Figure 13. Cytotoxicity of WG2-2, WG2-2-1, and WG2-2-2 on PC-3 Cells. 37
• Figure 14. Ginseng Saponin Biotransformed by AMG, Pectinex, and
• Viscozyme. 40
• Figure 15. HPLC Chromatograms of Biotransformed Saponin by Enzymes. 41
• Figure 16. Ginseng Extract Biotransformed by Pectinex 43
• Figure 17. Ginseng Biotransformed by Pectinex. 44
• Figure 18. TLC Analysis of Biotransformed Tail Ginseng by Pectinex. 46
• Figure 19. TLC Analysis of Biotransformed Undried Ginseng by Pectinex. 47
• Figure 20. TLC Analysis of Biotransformed Red Ginseng Powder by Pectinex. 48
• Figure 21. Effect of Time and Temperature on Biotransformation of Ginseng. 50
• Figure 22. Purification Scheme of PG-1, PG-2 and PG-3 53
• Figure 23. TLC Analysis of PG-1, PG-2, and PG-3 55
• Figure 24. LR-MS Spectrum of PG-3 55
• Figure 25. Cytotoxicity of PG-1, PG-2, and PG-3 on PC-3 Cells 57