柳然禹 이승현 2005 249 https://aurora.ajou.ac.kr/handle/2018.oak/16377 학위논문(석사)--아주대학교 대학원 :분자과학기술학과 / 생체소재전공,2005 치마버섯(Schizophyllum commune)의 균사체 배양조건을 최적화하여 유효성분인 β-glucan의 생성을 극대화시키고자 하였다. 치마버섯 균사체 배양에서 탄소원과 질소원의 종류 및 농도를 검토한 결과, 배지조성은 glucose 5%(w/v), yeast extract 0.3%(w/v), malt extract 0.3%(w/v), KH₂PO₄ 0.05%(w/v), MgSO₄· 7H₂O 0.05%(w/v)가 최적이었다. 배양 조건은 pH 5.5, 28℃, 1 vvm, 300 rpm에서 균사체의 생육과 β-glucan의 생성이 가장 효과적이었다. 세포 외 β-glucan의 생성을 증가시키기 위해 초기 glucose의 첨가량을 3%로 낮추어 glucose에 의한 균사체 성장 저해작용을 최소화하는 한편, 배양이 70시간에 이르렀을 때에 glucose 3%, yeast extract 0.1%, malt extract 0.1%의 추가 배지를 첨가하여 배양함으로써 batch 배양에 의한 4.7 g/L의 β-glucan 생산에 비해 35%정도 향상된 7.3 g/L의 세포 외 β-glucan을 얻을 수 있었다. 또한 균사체 내의 세포벽 β-glucan을 효과적으로 추출하기 위해 세포벽분해 효과가 있는 복합효소인 cellulase와 단백분해효소인 bromelain의 연속적인 효소반응으로 추출량이 3.0 g/L로 증가하여 균사체의 열수 추출에 의한 추출량인 0.4 g/L에 비해 약 7.5배의 추출 수율이 향상되었다. 치마버섯 β-glucan의 조건별 특성을 조사한 결과, 치마버섯 β-glucan 0.1%(w/v) 용해액 무게평균 분자량은 3,379,000 Da이고, 점도는 122 cps이었다. 점도는 농도가 증가할 수록 선형적으로 증가하여 1%(w/v) 용해액에서는 1800 cps로 측정되었다. β-glucan 0.1%(w/v) 용해액을 온도별 처리한 결과는 120℃에서 초기 점도의 20% 정도로 떨어졌고 20~100℃에서는 온도가 증가할 수록 약간의 감소가 이루어진 것으로 볼 때 열에 비교적 안정한 구조 인 것을 알 수 있었다. Ultrasonication 처리에서는 시간별로 점도와 분자량이 현격하게 감소하였고, gamma ray 처리에서도 흡수선량에 따라 일정한 폭의 감소가 이루어졌다. 목차 LIst of Tables = Ⅰ List of Figures = Ⅱ 국문 요약 = Ⅳ 제1부. 서론 = 1 1. 치마버섯의 개요 = 1 2. 치마버섯 β-glucan의 생산 = 2 3. 치마버섯 β-glucan의 특성과 생리활성 = 3 제2부. 재료 및 방법 = 7 1. 미생물 및 배양조건 = 7 1-1. 균주 및 종균배양 = 7 1-2. β-glucan 생산을 위한 배양최적화 = 7 2. 배양액 분석 및 추출 = 8 2-1. 시료의 제조 = 8 2-2. 시료의 당 및 단백질 분석 = 8 2-3. β-glucan의 정량분석 = 9 2-4. 효소를 이용한 세포 내 β-glucan 추출 = 10 3. β-Glucan의특성 = 11 3-1. β-Glucan의 분자량 측정 = 11 3-2. β-glucan의 농도별 점도 측정 = 12 3-3. 온도 및 pH에 따른 β-glucan의 점도 측정 = 12 3-4. Ultrasonication 처리에 따른 β-glucan의 특성 변화 = 12 3-5. Gamma ray 처리에 따른 β-glucan의 특성 변화 = 12 제3부. 결과 및 고찰 = 14 1. 미생물 및 배양 조건 = 14 1-1. 탄소원의 영향 = 14 1-2. 질소원의 영향 = 14 1-3. 무기염류의 영향 = 15 1-4. 배양조건의 영향 = 15 1-5. 배지 feeding = 16 2. 배양액 분석 및 추출 = 28 2-1. 시료의 분석 = 28 2-2. 효소반응에 의한 β-glucan 추출의 영향 = 28 3. β-glucan의 특성 = 33 3-1. β-glucan의 분자량 측정 = 33 3-2. β-glucan의 농도별 점도 측정 = 33 3-3. 온도 및 pH에 따른 β-glucan의 점도 측정 = 33 3-4. Ultrasonication 처리에 따른 β-glucan의 특성 변화 = 33 3-5. 감마선 처리에 따른 β-glucan의 특성 변화 = 34 제4부. 결론 = 41 제5부. 참고 문헌 = 43 Abstract = 48|Table 1. Effect of carbon sources on the mycelial growth and β-glucan production = 18 Table 2. Effect of glucose concentration on the mycelial growth and β-glucan production = 19 Table 3. Effect of nitrogen sources on the mycelial growth and β-glucan production = 20 Table 4. Optimized medium composition and culture conditions for submerged culture = 26 Table 5. Culture conditions and optimized medium composition for submerged culture and semi-fed batch = 27 Table 6. β-glucan and protein contents in mycelia and culture broth = 30|Figure 1. Fruit body and mycelia of Schizophyllum commune = 5 Figure 2. Structure of β-glucan oreinted from Schizophyllum commune = 6 Figure 3. Effect of pH on the mycelial growth and β-glucan production = 21 Figure 4. Effect of temperature on the mycelial growth and β-glucan production = 22 Figure 5. Effect of aeration on the mycelial growth and β-glucan production = 23 Figure 6. Time course of the mycelial growth, β-glucan production and glucose comsumption by the cultivation of Schizophyllum commune = 24 Figure 7. Time course of the mycelial growth, β-glucan production and glucose consumption by the cultivation of Shizophyllum commune with feeding at the point of 70 hours = 25 Figure 8. Effect of enzyme treatment for the extraction of β-glucan from mycelia = 31 Figure 9. Effect of mixed enzyme treatment for the production of β-glucan from mycelia = 32 Figure 10. Average molecular weight of β-glucan's 0.1% solution = 35 Figure 11. Viscosity of β-glucan's solution of concentration 0.1% to 1.0% = 36 Figure 12. Viscosity of β-glucan's 0.5% solution from 20℃ to 100℃ = 37 Figure 13. Viscosity of β-glucan's 0.5% solution from pH 2 to pH 10 = 38 Figure 14. Effect of ultrasonication treatment on the β-glucan's 1% solution = 39 Figure 15. Effect of Gamma ray's treatment on the β-glucan's 0.5% solution = 40 kor The Graduate School, Ajou University 아주대학교 논문은 저작권에 의해 보호받습니다. 치마버섯 균사체 배양을 통한 β-glucan 생산 최적화와 β-glucan의 특성 Optimization of β-glucan Production from the Mycelial Culture of Shizophyllum commune and Characterization of β-glucan Thesis 아주대학교 일반대학원 일반대학원 분자과학기술학과 2005. 2 Master http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000000249 β-Glucan has been efficiently produced with high yield by the optimization of micelial liquid cultivation conditions oriented from schizophyllum commune Fries. The result which has investigated the type and a concentration of carbon source and nitrogen source from micelial cultivation, the optimal composition of medium for batch culture was 5%(w/v) of glucose as a carbon source, 0.3%(w/v) of yeast extract and 0.3%(w/v) of malt extract as a nitrogen source, 0.05%(w/v) of KH₂PO₄ and 0.05%(w/v) MgSO₄· 7H₂O, which had been the base medium for determination of other conditions. Cultivation condition was pH 5.5, 28℃, 1 vvm for aeration and 300 rpm for agitation. Due to maximize the production of extracellular β-glucan and to minimize the inhibition effect of glucose on the initial growth of mycelia, we have reduced the initial glucose feed to 3%(w/v) and added 3%(w/v) of glucose, 0.1%(w/v) of yeast extract and 0.1%(w/v) of malt extract, at the point of 70 hr from the initial feed. As feeding medium, β-glucan's productions increased 35% degree in 7.3 g/L compared with β-glucan's productions of 4.7 g/L from a batch cultivation. Moreover, the serial treatment of a cellulase and bromelain to the mycelia was effective for extraction of the cell wall bound β-glucan. The yield of β-glucan extraction by the enzyme treatment was 3.0 g/L, which was almost 7.5 times higher than 0.4 g/L by hot-water extraction. The result which has investigated β-glucan's qualities as conditions, weight average molecular weight of 0.1%(w/v) β-glucan's solution was 3,379,000 Da and viscosity was 122 cps. According to increasing β-glucan's concentration viscosity increased in direct proportion, viscosity of 1%(w/v) β-glucan's solution measured 1800 cps. Viscosity of 0.1%(w/v) β-glucan's solution decreased 20% degree of intial viscosity in 120℃, according to increasing temperature from 20℃ to 100℃ viscosity decreased a little. We have known character of β-glucan from schizophyllum commune is relatively stable from heat. The viscosity and molecular weight considerably fell in treatment of ultrasonication as hour and in treatment of gamma ray also decreased uniformly according to absorption