臺灣博碩士論文加值系統

有許多研究報導，相較於單一菌種發酵，利用非釀酒酵母（non-Saccharomyces yeast）和釀酒酵母（Saccharomyces spp.）的混合發酵更能有效提升葡萄酒中香氣的含量與多樣性。本研究從水果果皮篩選可以適應釀酒環境之產香菌，並進行混合發酵之產香條件最適化研究。實驗利用添加氯黴素（Chloramphenicol）的YPDA培養基以及調整pH、葡萄糖濃度與酒精濃度的YPD培養基作為篩選平台，依外型挑選菌落進行分離純化。菌種鑑定以聚合酶連鎖反應擴增酵母菌之rDNA的ITS片段，經過定序後將定序結果與NCBI基因資料庫進行BLAST比對，以得知菌種。本實驗挑選Saccharomyces cerevisiae（A5）和 Pichia anomala（F8）進行反應曲面法實驗，實驗因子選擇菌株順序接種間隔時間、發酵溫度與初始葡萄糖濃度。根據RSM實驗結果，在發酵溫度21.36oC，初始糖度25.86%，接種間隔時間32.48小時具有最高乙酸乙酯產量85.77 ppm。儀器分析結果表明，經過乙酸乙酯最適化條件釀製的葡萄酒，在Ethyl acetate、1-propanol、Isobutanol、2-Methyl-1-Butanol、Ethyl octanoate、2,3-Butanediol、2-Phenylethanol以及Tyrosol等葡萄酒香氣成分的濃度上，均顯著高於未經過乙酸乙酯最適化條件釀製的葡萄酒。感官品評結果表明，在乙酸乙酯最適化條件下製作的葡萄酒在總體喜好、風味、口感和餘味的品評分數均顯著高於未最適化的葡萄酒和兩種商業葡萄酒。本研究證實最適化之混合發酵確實能提升葡萄酒的香氣含量，並擁有更好的風味，期待本研究成果能提供更多在釀酒工業上的應用。

Recently, many studies have reported that mixed fermentation of Saccharomyces yeast and non-Saccharomyces yeast has an effect on enhancing the content and complexity of wine aroma compared to single strain fermentation. In this study, yeasts were screened from fruit peels to adapt to the wine fermentation environment, and the optimum mix-fermentation conditions for ethyl acetate were studied. The experiment used YPDA medium which added chloramphenicol as a selecting platform, and YPD medium which adjusted pH, glucose concentration and alcohol concentration to mimic wine fermentation condition. Saccharomyces cerevisiae (A5) and Pichia anomala (F8) were selected for RSM (response surface methodology) experiments because of the high ethyl acetate production potential. Sequential inoculation interval time, temperature, and initial sugar concentration was chosen as the RSM factor to obtain the maximum ethyl acetate yield. After the RSM experiment, instrumental analysis and sensory evaluation using 9-Point hedonic scale method was carried out. According to the RSM experiment, the highest ethyl acetate yield of 85.77 ppm can be obtained at a temperature of 21.36oC, an initial sugar concentration of 25.86%, and an interval inoculation time of 32.48 h. Instrumental analysis shows an increase concentration of Ethyl acetate, 1-propanol, Isobutanol, 2-Methyl-1-Butanol, Ethyl octanoate, 2,3-Butanediol, 2-Phenylethanol and Tyrosol in optimized wine compare with un-optimized wine. In sensory evaluation experiment, five kinds of wine including three kinds of commercial wine and two kinds of self-made wine fermented under un-optimized condition and optimized condition was served to the judgers. The result indicates that the wine made under optimized condition was rated higher than the un-optimized wine and two commercial wine in overall liking, flavor, mouthfeel and aftertaste. This study confirms that the optimal mix-fermentation does enhance the aroma concentration of the wine and has a better flavor. It is expected that the research results will provide more applications in the wine industry.

謝誌 ii
摘要 iii
Abstract iv
圖目錄 ix
表目錄 xi
List of Figures xii
List of Tables xiv
一、前言 1
二、文獻回顧 3
2.1葡萄及葡萄酒的特性和發展 3
2.1.1葡萄及葡萄酒的營養價值 3
2.1.2葡萄酒產業的歷史和發展現狀 3
2.1.3葡萄酒中的微生物 5
2.1.4葡萄酒的製作流程 7
2.1.5臺灣金香葡萄 7
2.2釀酒酵母與非釀酒酵母的混合發酵之研究 10
2.2.1葡萄酒釀造中的非釀酒酵母 12
2.2.2非釀酒酵母在葡萄酒釀造中的應用 12
2.3葡萄酒的香氣成分與分析方法 13
2.3.1葡萄酒主要之香氣 13
2.3.1.1一級香氣 16
2.3.1.2二級香氣 18
2.3.1.2.1醇類香氣 18
2.3.1.2.2酯類香氣 20
2.3.1.2.3脂肪酸 20
2.3.1.2.4硫化物和揮發性酚類 20
2.3.1.3三級香氣 22
2.3.1.4葡萄酒中的乙酸乙酯 22
2.3.1.5葡萄酒之相關法規 22
2.3.2葡萄酒香氣之分析方法 23
2.3.2.1儀器分析 23
2.3.2.2感官分析 23
2.4反應曲面法（Response surface methodology, RSM） 24
三、研究目的與架構 29
3.1研究目的 29
3.2研究架構 30
四、材料與方法 31
4.1實驗材料 31
4.1.1酵母菌 31
4.1.2葡萄原料 31
4.2實驗主要儀器，試劑和培養基 33
4.2.1實驗主要之儀器 33
4.2.2實驗主要之試劑 34
4.2.3實驗主要之培養基 35
4.3實驗方法 35
4.3.1葡萄榨汁 35
4.3.2酵母菌株之篩選與鑑定 35
4.3.2.1酵母菌之篩選 35
4.3.2.2酵母菌的繼代 37
4.3.2.3酵母菌的保存 37
4.3.2.4 rDNA之ITS片段 37
4.3.2.5電泳分析 37
4.3.2.6基因庫比對 38
4.3.2.7生化特性分析 38
4.3.2.7.1酵母菌可利用之碳氮源的測定 38
4.3.2.7.2酵母菌之生長溫度及滲透壓的測定 38
4.3.3酵母菌發酵參數的測定 40
4.3.3.1酵母菌的生長曲線測定 40
4.3.3.2酵母菌之標準曲線的製作 40
4.3.3.3酵母菌之發酵之消耗糖份與產酒精能力實驗 40
4.3.3.4酵母菌發酵之pH變化 41
4.3.3.5發酵液GC分析之樣品前處理 41
4.3.4最適化發酵條件之研究 42
4.3.4.1接種間隔時間 42
4.3.4.2發酵糖度 42
4.3.4.3發酵溫度 42
4.3.4.4反應曲面法 43
4.3.5利用最適化之發酵條件釀製葡萄酒實驗 45
4.3.6指標芳香物質之測定與分析 45
4.3.6.1分析條件 45
4.3.7官能品評 45
4.4統計分析 46
五、結果與討論 47
5.1菌株鑑定 47
5.2後續實驗之菌株的選擇 47
5.2.1菌株發酵產乙酸乙酯之結果 47
5.2.2選擇後續實驗菌株之原因 50
5.3菌株之菌落型態 50
5.4菌株之生長曲線與檢量線 50
5.5菌株發酵之消耗糖份與產酒精能力實驗 56
5.6菌株發酵之pH變化 56
5.7菌株之生化特性分析實驗 61
5.7.1菌株之碳氮源同化實驗 61
5.7.2菌株之生長溫度與滲透壓實驗 61
5.8菌株混合發酵最適化產乙酸乙酯實驗 64
5.8.1反應曲面法實驗因子評估實驗 64
5.8.1.1釀酒酵母與非釀酒酵母順序接種間隔時間 64
5.8.1.2發酵初始糖度 64
5.8.1.3發酵溫度 64
5.8.2乙酸乙酯產量最適化實驗 68
5.9以最適化產乙酸乙酯條件釀製葡萄酒實驗 72
5.10葡萄酒之儀器分析實驗 74
5.11官能品評實驗 76
六、結論與展望 80
七、參考文獻 82
八、附錄 xv

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