咖啡是世界上最廣泛的飲料之一，也是重要的經濟作物。精品咖啡 (Specialty coffee) 是在咖啡產業中的第三波咖啡浪潮 (Wave of coffee) 中提出的新概念，在特別的氣候與地理條件下可培育出具有獨特風味的咖啡豆，但特殊風味的咖啡豆難以維持每批次生產的品質穩定。精品咖啡利用淺烘焙方式以凸顯其香氣，但淺烘焙過程中因溫度較低及時間較短，若咖啡豆遭赭麴毒素 (Ochratoxin) 汙染則不易經由焙炒而降解，造成食品安全風險。隨咖啡產業規模提升，咖啡副產物的產量也隨之增加，咖啡果肉是咖啡加工過程中需去除之部分，但大量的咖啡果肉副產物會對環境造成負擔。本研究目標係透過篩選可產生特殊風味及香氣的野生酵母菌，控制發酵條件，以開發具特殊風味及香氣且能兼顧食品安全之精品咖啡。探討以酵母菌發酵咖啡果肉之產物抑制赭麴真菌孢子萌芽的能力。另評估副產物咖啡果肉再利用可能性，期能達成永續與潔淨生產的目標。實驗設計利用篩選野生酵母菌及標準菌株，以厭氧方式發酵新鮮咖啡漿果，並以氣相層析儀 (Gas chromatography，GC) 進行咖啡豆的香氣物質分析，以確認微生物接種對芳香物質的影響。咖啡果肉以野生酵母菌及標準菌株進行厭氧發酵，並探討其發酵產物對赭麴真菌孢子萌芽抑制之影響，並利用高效能液相層析儀 (High performance liquid chromatography，HPLC) 分析有機酸 (Organic acid) 的含量，探討其對於降低食品安全風險之潛力。另以反應曲面法 (Response surface methodology，RSM) 探討咖啡果肉副產物替代培養次世代益生菌 (Next-generation probiotic) 的潛力。野生酵母菌及次世代益生菌的篩選及定序結果主要為 Pichia kudriavzevii、Candida glabrata 及 Akkermansia muciniphila。焙炒後咖啡豆之芳香化合物則以丙酮醇 (Hydroxyacetone) 及呋喃甲醇 (Furfuryl alcohol) 為主，使其具有焦糖、甜味、焦香味及奶油味。其中，乳酸乙酯 (Ethyl lactate) 具有奶油及果香，只在 MC741 (Pichia kudriavzevii) 組別中檢測出。Control (無接菌) 組檢測到巨豆三烯酮 (Megastigmatrienone)，其具有菸草味，另四組微生物接種後則沒有檢測出。酵母菌發酵咖啡果肉產物抑制孢子萌發試驗結果顯示，Saccharomyces cerevisiae 菌株發酵產物減少孢子萌發的能力較佳，檸檬酸 (Citric acid) 可能為主要抑制孢子萌發之有機酸類物質。Akkermansia muciniphila 利用咖啡果肉替代培養基可利用反應曲面法挑選出最適培養條件。綜上所述，本研究篩選出之酵母菌株在厭氧條件下發酵咖啡漿果除具有抑制 Aspergillus niger BCRC 33485 孢子萌芽的能力外，經淺中焙之咖啡豆具有醛 (Aldehyde)、酮 (Ketone)、吡嗪 (pyrazine) 及吡啶 (Pyridine) 等香氣物質，且咖啡製程中接種不同微生物產生之香氣組合皆不一樣。咖啡加工之副產物咖啡果肉也具有再利用潛力，除能降低食品安全風險外還能應用於次世代益生菌之培養，期未來達成永續與潔淨生產的目標。

Coffee is one of the most widely consumed beverages globally and serves as a crucial economic commodity. Specialty coffee, a new coffee culture introduced during the third wave of the coffee industry, is cultivated under specific climate and geographical conditions to produce unique coffee beans with distinct flavors. However, the production of consistently high-quality batches of coffee beans with special flavors remains a challenge. Specialty coffee uses a light roasting method to highlight its inherent aroma. However, during the light roasting process, the lower temperature and shorter duration result in a reduced ability to degrade ochratoxin if contamination occurs. With the expansion of the coffee industry's scale, the yield of coffee by-products has also increased accordingly. Coffee pulp is a byproduct that needs to be removed during coffee processing. The large quantities of coffee pulp can burden the environment. However, it is rich in carbohydrates and can be extracted and reuse. The aim of this study is to develop specialty coffee with unique flavors and aromas that also ensure food safety by screening wild yeast strains capable of producing distinctive flavors and aromas, and controlling fermentation conditions. The study investigates the ability of yeast fermentation products from coffee pulp to inhibit the germination of Aspergillus niger BCRC 33485 spores. Additionally, the potential for reusing the by-product, coffee pulp, evaluate in order to achieve the goals of sustainability and clean production. The experimental design involves the selection of wild yeast strains and standard strains to anaerobically ferment fresh coffee cherries. The aroma compounds of the coffee beans analyze using gas chromatography (GC) to confirm the impact of microbial inoculation on aromatic substances. Coffee pulp will be anaerobically fermented with wild yeast strains and standard strains, and investigate the effect of the fermentation products on the inhibition of A. niger BCRC 33485 spore germination. The content of organic acids analyzes using high-performance liquid chromatography (HPLC) to explore their potential for reducing food safety risks. Additionally, response surface methodology (RSM) uses to investigate the potential of coffee pulp as a substitute medium for culturing next-generation probiotics. The selection and sequencing results of wild yeast strains and next-generation probiotics mainly identified Pichia kudriavzevii, Candida glabrata, and Akkermansia muciniphila. The aromatic compounds of roasted coffee beans were primarily hydroxyacetone and furfuryl alcohol, imparting caramel, sweet, burnt, and buttery flavors. Ethyl lactate, which has buttery and fruity odor, was only detected in the MC741 (Pichia kudriavzevii) group. Megastigmatrienone, which has a tobacco-like aroma, was detected in the control (no inoculation) group but not in the other four microbial inoculation groups. The spore germination inhibition tests of yeast-fermented coffee pulp metabolites showed that the fermented products of the Saccharomyces cerevisiae strain had a better ability to reduce spore germination, with citric acid likely being the main organic acid responsible for spore germination inhibition. Using response surface methodology, the optimal culture conditions for A. muciniphila utilizing coffee pulp as a substitute medium were identified. In summary, the yeast strains selected in this experiment, when fermenting coffee cherries under anaerobic conditions, not only have the ability to inhibit spore germination but also produce coffee beans with aldehyde, ketone, pyrazine, and pyridine aroma compounds after cinnamon roasting. The aromatic profiles vary depending on the microorganisms used. Additionally, the by-product of coffee processing, coffee pulp, shows potential for reuse. It can reduce food safety risks and be applied in culturing next-generation probiotics, aiming to achieve sustainability and clean production in the future.

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