鳳梨酒發酵過程中之微生物組成對其甲醇與乙醇 生成的影響

Abstract

甲醇具毒性，過量攝取會導致頭痛、失明甚至死亡，是水果酒安全監控的重要指標。在發酵過程中，甲醇由果膠在果膠甲基酯酶 (pectin methylesterase, PME) 作用下產生，其生成量受果膠含量、甲基酯化程度及 PME 活性影響，但目前尚未釐清哪一項為關鍵因素。本研究以鳳梨為材料，探討發酵期間果膠含量、甲基酯化度與甲醇生成之關聯，並以不同處理模擬植物與微生物PME的貢獻，搭配高通量定序 (NGS) 分析微生物群落變化與甲醇及乙醇濃度之關聯性。首先以台農2號與台農17號兩品種鳳梨之自然發酵特性，追蹤果膠、甲醇與乙醇含量變化。台農2號與台農17號初始果膠含量相近，但台農2號的甲基酯化度 (24.23%) 較台農17號 (13.97%) 高。發酵28天後，最終甲醇含量分別為台農2號64.43 ppm、台農17號68.18 ppm。乙醇濃度約4% 和 5%，換算成純乙醇基準的甲醇含量分別為1208.6 ppm與1499.5 ppm，均低於法定上限4000 ppm ，此結果顯示鳳梨的果膠含量並非決定鳳梨酒甲醇產量的唯一因素。為排除品種間差異，使用不同成熟度之台農17號進行實驗。在成熟度比較中，控制組 (C) 果膠初始含量與甲基酯化度較高，但其最終甲醇約50 ppm，而過熟組 (OR) 甲醇則高達96 ppm，乙醇濃度C組約5%，OR組約2%，換算成純乙醇基準後 OR 組甲醇濃度為4808.3 ppm，已超過法定上限，因此鳳梨酒中甲醇生成差異可能主要受到PME活性所致，而非原料之果膠含量及其甲基酯化程度。為釐清PME來源對甲醇生成的影響，模擬實驗結果顯示，微生物組 (59.74 ppm) 甲醇含量高於殺菁組 (41.06 ppm) 與植物組 (45.95 ppm) ，微生物來源之 PME 在發酵過程中對於甲醇生成具較高貢獻，菌落組成亦可能為關鍵因素。NGS 分析顯示，發酵初期 C 組真菌群落多樣性較高，隨發酵進行逐漸由 Hanseniaspora 成為優勢菌屬。OR 組則持續維持高度多樣性，最終以 Debaryomyces 為優勢菌，兩組菌相變化趨勢不同，此差異不僅造成最終乙醇濃度分別為5% 與 2%，亦可能影響微生物所產生之PME活性差異。為進一步驗證微生物對甲醇生成之影響，設計混合組 (CR)，將 C 組微生物接種至已去除微生物之 OR 果汁中。結果顯示，CR組鳳梨酒之甲醇含量45.73 ppm，接近C組鳳梨酒之 41.2 ppm，低於OR組鳳梨酒之63.73 ppm，再次印證發酵過的微生物組成對鳳梨酒的甲醇生成具關鍵性影響。本研究指出果膠含量與其酯化度對甲醇生成影響有限，微生物組成及其PME活性才是關鍵。此外，乙醇濃度不僅參與發酵穩定性，更是法規中甲醇換算基準，過低時會導致換算值超標，增加食品安全風險。未來若能進一步釐清微生物代謝特性，並透過功能性菌株選擇與發酵管理優化，可以控制發酵過程中的甲醇生成，建立更安全、穩定的水果酒製程。

Methanol is toxic to human, and excessive intake can lead to headaches, blindness, or even death. Therefore, methanol concentration is a critical indicator for fruit wine safety monitoring. Methanol is produced through the demethylation of pectin catalyzed by pectin methylesterase (PME) during fermentation. Its production is influenced by the pectin content, degree of methyl esterification, and the source and activity of PME. The precise contributions of each factor have not yet been fully elucidated. This study selects pineapple (Ananas comosus) as the experimental material and focus on investigating the relationship among pectin content, degree of methyl esterification, PME activity, and methanol production during fermentation. Accordingly, different treatments were applied to assess the contributions of plant- and microbe-derived PME, and changes in the microbial community, along with their correlations to methanol and ethanol concentrations, were analyzed using high-throughput sequencing (NGS). The spontaneous fermentation of two pineapple cultivars, Tainung 2 and Tainung 17, was monitored to track the changes in pectin, methanol, and ethanol contents. Although the initial pectin contents of Tainung 2 and Tainung 17 were similar, the degree of methyl esterification in Tainung 2 (24.23%) was higher than that of Tainung 17 (13.97%). After 28 days of fermentation, the final methanol contents were 64.43 ppm for Tainung 2 and 68.18 ppm for Tainung 17. Ethanol concentrations were 4% and 5%, respectively. When expressed on a pure ethanol basis, methanol levels were 1208.6 ppm and 1499.5 ppm, respectively, both falling below the legal limit of 4000 ppm. These results indicate that pectin content is not the major factor influencing methanol production during pineapple wine fermentation. To exclude the varietal differences, fermentation experiments were conducted using only Tainung 17 pineapples at different ripening stages. In this comparison, the control group (C), with higher initial pectin content and degree of methyl esterification, produced 50 ppm of methanol, while the overripe group (OR) yielded 96 ppm. Ethanol concentrations were 5% in C and 2% in OR. Based on pure ethanol equivalence, the methanol concentration in the OR group reached 4808.3 ppm, which exceeded the legal limit. This result suggested the methanol production was primarily driven by PME activity rather than by pectin content or degree of methyl esterification. To further elucidate the contributions of PME sources, simulated fermentation experiments were performed. Methanol levels were higher in the microbial group (59.74 ppm) than those in the blanching (41.06 ppm) and plant (45.95 ppm) groups, suggesting a more significant contribution of microbe-derived PME to the methanol production. The composition of the microbial community likely also played a critical role. NGS revealed that the fungal community in C wines rapidly shifted to Hanseniaspora dominance, whereas OR ones maintained high diversity before becoming dominated by Debaryomyces. These distinct succession patterns were associated with different final ethanol concentrations, with 5% in C and 2% in OR wines and might reflect variation in PME activity associated with microbial community structure. To further confirm the role of microorganisms in methanol production, a composite group (CR) was created by introducing microbiota from the C into sterilized OR juice. The methanol concentration in the CR group (45.73 ppm) was similar to that of the C group (41.2 ppm) and significantly lower than in the OR group (63.73 ppm), highlighting the crucial influence of microbial composition on methanol generation. This study demonstrates that pectin content and degree of methyl esterification have limited impact on methanol production, whereas microbial composition and PME activity are the primary factors. Ethanol concentration affects fermentation stability and serves as a basis for regulatory methanol limits. Low ethanol levels may cause methanol concentrations to exceed legal limits after adjustment, posing safety risks. Future research should focus on the microbial metabolism and fermentation optimization, including the selection of functional strains, to better control methanol formation and ensure safer, more consistent fruit wine production.