清酒 (Sake) 原料為米及水，透過米麴菌 (Aspergillus oryzae) 及酵母菌 (Saccharomyces cerevisiae) 進行釀造，過程可分為酒母 (shubo) 及發酵 (fermentation) 階段。釀造時製備的麴飯是影響品質的一大關鍵，麴飯製備時將冷凍的麴菌直接培養在蒸飯上會使污染機率大增，品質控管難度上升。因此本研究將麴菌培養在可食用的培養基活化後再製作麴飯，以該麴飯釀造清酒，並觀察後續發酵過程的菌相組成及變化與成品品質的影響，和先前研究數據進行比較。本研究針對發酵階段前十八天的採樣進行比較，透過次世代定序 (next generation sequencing, NGS) 分析發酵時期的菌相變動。計算各樣本的 Shannon 指數後，發現本研究的菌相多樣性一進入發酵階段即開始上升，避免過去在第十天才有大幅度變化的延遲現象。接著再透過 non-metric multidimensional scaling (NMDS) 及hierarchical clustering analysis (HCA) 分析的結果得知，本次研究中的菌相會在第七天至第九天間出現明顯的變化導致樣本依相似度分成兩大群，而先前研究則是在第十天至第十五天才有明顯變化而分成兩群，顯示本研究的菌相較先前研究更快達到菌相的穩定。再透過菌相組成分析變化關聯微生物，發現兩次研究的優勢物種皆為乳桿菌目，分別是本研究中第零天樣本中佔比 33% 的乳球菌屬 (Lactococcus) 與先前研究中第零天樣本佔比 43% 的明串珠菌屬 (Leuconostoc)。這兩種乳酸菌的佔比在發酵過程中有最大的改變，但本次研究中的菌相皆緩慢變化，惟先前研究中會在第十天出現劇烈變動。再以熱點圖進行功能預測，發現在相同時間中兩次發酵細菌相在整個發酵過程中表現出的功能大致上相同，但相同的功能會在不同時期表現。將成品進行氣相層析分析，發現兩次釀出的清酒化學成分差異不大，惟本研究的清酒酒精濃度較高，可達 15.7 %。以目前資料推測在本研究中的清酒發酵菌相雖然顯著不同於上次釀造時之組成，但是這些不同的細菌仍然能夠提供同樣的功能，因此兩次研究中的清酒成品化學成分非常相似。此外，改良後的麴菌活化過程使清酒在發酵時菌相變化相對穩定，可能因而讓成品的酒精濃度得以提高。

Sake is brewed through sake yeast Saccharomyces cerevisiae, and the process can be divided into shubo and fermentation stages. During the preparation of koji rice, culture the frozen Aspergillus oryzae on steamed rice will greatly increase the risk of contamination. Therefore, in this study, koji rice was made after activation of Aspergillus oryzae on agar, and investigated the subsequent bacterial composition, microflora changes and quality of product, then compared to the previous research data. In this study, bacteria DNA will be extracted until 18 days, and the microflora will be analyzed through next generation sequencing (NGS). Through shannon index, non-metric multidimensional scaling (NMDS) and hierarchical clustering analysis (HCA), it is found that microflora in both studies will change from the initial phase to stable phase. The bacteria in this study reached the stable phase faster than the previous study. The analysis of bacterial composition showed that the microflora between the two studies are very different, the dominant species is still the lactic acid bacteria. Although the microflora is very different, the heat map revealed that microflora exhibited the same function in the fermentation stage in two studies. The product was analyzed by gas chromatography (GC). It was found that the chemical composition of the sake in the two studies was similar, and the alcohol concentration in this study was higher than previous study. Therefore, it can be thought that the improved koji rice can help the yeast fermentation effectively. From this study, the results showed that the improvement of koji rice caused a substantial change in the microflora of sake, but these different bacteria can still provide the same function, so the chemical composition of the sake in the two studies is similar. At the same time, the improvement of koji rice makes the microflora change relatively stable during the fermentation of sake, so the sake yeast can ferment stably, lead to the increase of the alcohol concentration.

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