표제지
국문초록
목차
I. 서론 16
II. 재료 및 방법 24
1. 재료 24
1.1. 균주 24
1.2. 발효제 및 원료 24
1.3. 시약 24
2. 막걸리 양조효모 실험방법 25
2.1. 입국 당화액 제조 25
2.2. 효모균제 접종량 25
2.3. 알코올 생성능 및 향 생성능력 25
2.4. 최적 발효온도 설정 25
3. 막걸리 양조 25
4. 막걸리 양조효모 특성 분석 27
4.1. 형태학적 특성 27
4.2. 분리 미생물의 동정 27
4.3. 알코올 내성 및 내당성 28
4.4. 침강성 28
4.5. 탄소 이용능 29
4.6. 균체 지방산 29
4.7. 효소활성 30
4.8. In vitro 생리활성 32
5. 막걸리의 이화학적 특성 분석 33
5.1. 알코올 함량 33
5.2. pH 및 고형분 함량 34
5.3. 총산 34
5.4. 환원당 34
5.5. 유기산 34
5.6. 휘발성 화합물 35
5.7. 관능특성 분석 35
6. 막걸리 대사체 분석 35
6.1. 대사체 추출 35
6.2. 대사체 분석 36
7. 통계처리 36
III. 결과 및 고찰 38
1. 막걸리 양조효모 선발 38
1.1. 막걸리 양조효모 분리 38
1.2. 효모 Y197-13의 발효특성 40
1.3. 효모 Y197-13의 발효온도 설정 42
1.4. 효모 Y197-13의 양조특성 44
2. 균주 P. anomala Y197-13의 특성 48
2.1. 형태학적 성질 48
2.2. 분리 미생물의 동정 50
2.3. 내당성, 내알코올성, 침강성 55
2.4. 탄소 이용능 59
2.5. 균체 지방산 조성 63
2.6. 효소활성 65
2.7. In vitro 생리활성 67
3. 균주 Pichia anomala Y197-13의 막걸리 양조적성 69
3.1. 발효제별 양조적성 69
3.2. 온도별 양조적성 78
3.3. 급수량별 양조적성 93
4. 균주 Pichia anomala Y197-13 막걸리 특성 106
4-1. 이화학 및 관능특성 106
4-2. 휘발성 향기성분 113
4-3. 지방산 조성 115
4-4. In vitro 생리활성 117
4-5. 발효기간 동안 대사체 변화 119
4-6. 발효기간 중 확인된 대사체의 정성분석 129
4-7. 발효기간 중 확인된 대사체의 정량분석 135
IV. 결론 139
1. 균주 P. anomala Y197-13의 형태학적 특성 139
2. 균주 P. anomala Y197-13의 내알코올성, 내당성, 침강성 139
3. 균주 P. anomala Y197-13의 탄소원 이용능 140
4. 균주 P. anomala Y197-13의 지방산 구성 140
5. 균주 P. anomala Y197-13의 효소활성 및 in vitro 생리기능성 140
6. 균주 P. anomala Y197-13의 막걸리 양조적성 140
7. 균주 P. anomala Y197-13 막걸리의 지방산 및 in vitro 생리활성 142
8. 균주 P. anomala Y197-13 막걸리의 대사체 분석 142
9. 균주 P. anomala Y197-13 막걸리의 발효기간 중 대사체 정량분석 142
V. 참고문헌 144
Abstract 161
Appendix 165
Table 1. Gradient analysis condition for UPLC-Q-TOF MS 37
Table 2. 1st Brewing characteristics of the wild yeast strains for Makgeolli 41
Table 3. 2nd Brewing characteristics of the wild yeast strains for Makgeolli 47
Table 4. Morphological characteristics of the type strain(ATCC 24858) and the wild yeast strains(Y98-5 and Y197-13) 49
Table 5. Identification of the wild yeast strains Y98-5 and Y197-13 52
Table 6. Flocculence of the type strain(ATCC 24858) and the wild yeast strains(Y98-5 and Y197-13) 58
Table 7. Phenotype characteristics of the wild type yeast strains Y98-5 and Y197-13 61
Table 8. Fatty acid profiles of the wild yeast strains Y98-5 and Y197-13 64
Table 9. Enzyme activity of the wild yeast strains Y98-5 and Y197-13 66
Table 10. In vitro physiological activity of the wild yeast strains Y98-5 and Y197-13 68
Table 11. Organic acid contents of Makgeolli using Y98-5 and Y197-13 brewed with different fermentation agents 75
Table 12. Preference test for Makgeolli using Y98-5 and Y197-13 brewed with different fermentation agents 77
Table 13. Chemical contents of Makgeolli brewed using yeast strains Y98-5 and Y197-13 depending on the temperature 83
Table 14. Organic acid contents of Makgeolli brewed using yeast strains Y98-5 and Y197-13 depending on the temperature 85
Table 15. Volatile compounds of Y98-5 Makgeolli depending on the temperature 86
Table 16. Volatile compounds of Y197-13 Makgeolli depending on the temperature 87
Table 17. Preference test for Makgeolli brewed using yeast strains Y98-5 and Y197-13 depending on the temperature 92
Table 18. Chemical characteristics of Makgeolli brewed using yeast strains Y98-5 and Y197-13 depending on the water-grist ratio 98
Table 19. Organic acid contents of Makgeolli brewed using yeast strains Y98-5 and Y197-13 depending on the water-grist ratio 99
Table 20. Volatile compounds of Y98-5 Makgeolli depending on the water-grist ratio 101
Table 21. Volatile compounds of Y197-13 Makgeolli depending on the water-grist ratio 102
Table 22. Preference test for Makgeolli brewed using yeast strains Y98-5 and Y197-13 depending on the water-grist ratio 105
Table 23. Chemical characteristics of alcoholic beverage using Y98-5 and Y197-13 109
Table 24. Chemical characteristics of Makgeolli brewed using yeast strains Y98-5 and Y197-13 110
Table 25. Organic acid content of Makgeolli brewed using yeast strains Y98-5 and Y197-13 111
Table 26. Preference test for Makgeolli brewed using strains Y98-5 and Y197-13 112
Table 27. Volatile compounds of Makgeolli brewed using yeast strains Y98-5 and Y197-13 114
Table 28. Fatty acids of Makgeolli brewed using yeast strains Y98-5 and Y197-13 116
Table 29. In vitro physiological functionality of Makgeolli using yeast strains Y98-5 and Y197-13 118
Table 30. Identity and fold change of Makgeolli metabolite using yeast strains Y98-5 and Y197-13 during fermentation period 128
Table 31. Identification of Makgeolli metabolite in positive and negative mode by UPLC-Q-TOF MS 132
Table 32. Quantitative analysis of major matabolite in Y98-5 Makgeolli during fermentation time by UPLC-Q-TOF MS 137
Table 33. Quantitative analysis of major metabolite in Y197-13 Makgeolli during fermentation time by UPLC-Q-TOF MS 138
Fig. 1. Metabolite flux distributions in P. anomala 20
Fig. 2. Relationships of selected members of the Saccharomycetales 22
Fig. 3. Flow diagram of the steps used to brew Makgeolli 26
Fig. 4. Flow diagram of the steps used to select the wild yeast strains for Makgeolli 39
Fig. 5. Alcohol fermentation capacity of wild yeast strains at different fermentation temperatures 43
Fig. 6. Changes of pH(A) and soluble solid(B) in mash during fermentation 45
Fig. 7. Contig alignment coverage of the wild yeast strains Y98-5 AND Y197-13 sequences. F. ITS1 forward primer, R, ITS4 reverse primer 51
Fig. 8. Clustal multiple alignment of Pichia anomala Y197-13 and type strain AY046221. 54
Fig. 9. Phylogenetic tree of Pichia anomala Y197-13. 54
Fig. 10. Ethanol tolerance(A) and glucose tolerance(B) of the type strain(ATCC 24858) and the wild yeast strains(Y98-5 and Y197-13) 57
Fig. 11. Alcohol content(bars) and soluble solids(symbols) of Makgeolli brewed using yeast strains Y98-5 and Y197-13 with different fermentation agents. 70
Fig. 12. pH(bars) and total acids(symbols) of Makgeolli brewed using yeast strains Y98-5 and Y197-13 with different fermentation agents. 72
Fig. 13. Reducing sugar content of Makgeolli brewed using yeast strains Y98-5(A) and Y197-13(B) with different fermentation agents. 73
Fig. 14. Changes in pH of mash brewed using yeast strains Y98-5(A) and Y197-13(B) depending on the temperature. 79
Fig. 15. Changes in soluble solid of mash brewed using yeast strains Y98-5(A) and Y197-13(B) depending on the temperature. 80
Fig. 16. Alcohol contents of alcoholic beverage brewed using strains Y98-5 and Y197-13 depending on the temperature. 82
Fig. 17. Principal component analysis(PCA) of the 30 volatile compounds of Makgeolli brewed using yeast strains Y98-5 and Y197-13 depending on the temperature. 90
Fig. 18. Changes in pH of mash brewed using yeast strains Y98-5(A) and Y197-13(B) depending on the water-grist ratio. 94
Fig. 19. Changes in soluble solid of mash brewed using yeast strains Y98-5(A) and Y197-13(B) depending on the water-grist ratio. 95
Fig. 20. Alcohol contents of alcoholic beverage brewed using yeast strains Y98-5 and Y197-13 depending on the water-grist ratio. 96
Fig. 21. Principal component analysis(PCA) of the 30 volatile compounds of Makgeolli brewed using yeast strains Y98-5 and Y197-13 depending on the water-grist ratio. 103
Fig. 22. Changes in pH(A) and soluble solid(B) of mash during fermentation time. 107
Fig. 23. Total metabolite of aligned compounds of MassHunter mass profiler prefessional(MPP). 120
Fig. 24. Total ion chromatogram(TIC) of alcoholic beverage using yeast strains Y98-5(A) and Y197-13(B) during fermentation period(1, 2, 3, 6, 7, 8 days). 121
Fig. 25. TIC of Koji, Makgeolli using yeast strains Y98-5 and Y197-13, and amino acid standard mixture. 122
Fig. 26. Validate model of Makgeolli using yeast strains Y98-5 and Y197-13 for PLS-DA. 124
Fig. 27. The score plot of PLS-DA for Makgeolli using yeast strains Y98-5(A), Y197-13(B) and Y98-5 with Y197-13(C) during fermentation period. 125
Fig. 28. Volcano plot of Makgeolli metabolite using yeast strains Y197-13 vs. Y98-5 during fermentation period. 126
Fig. 29. Mass fragmentation of identified metabolite. 133