群落定向驯化：基于关键碳源构建的白酒发酵乙酸乙酯代谢强化菌剂与模拟应用

Community directional domestication: construction and simulated application of ethyl acetate synthesis-enhancing microbial agents for Baijiu based on key carbon sources

摘要:

【背景】 乙酸乙酯(ethyl acetate)作为酱香型白酒中关键的香气成分之一，其含量对酒体风味特征有着直接且关键的影响。【目的】 攻克单菌种强化在提升乙酸乙酯应用中所面临的功能退化难题，从群落水平出发，通过定向驯化实现微生物代谢乙酸乙酯水平的提升。【方法】 通过对基因表达丰度进行深入分析，解析得到微生物代谢底物中营养成分的差异化特性，并紧密结合微生物代谢乙酸乙酯的途径，确定了葡萄糖、果糖和阿拉伯糖这3种关键碳源营养物质。以3种碳源营养对菌群进行定向驯化。【结果】 微生物积累生物量(S1: 1.37 mg/mL, S2: 1.40 mg/mL, S3: 1.57 mg/mL)均低于CN对照组(1.87 mg/mL)。经过14轮定向驯化后，菌群多样性呈现下降趋势，而所形成真菌结构相似。其中，异常威克汉姆酵母(Wickerhamomyces anomalus)的相对丰度由初始的0.04%显著提升至65%及以上(P < 0.05)。研究发现，发酵微生物在连续稀释传代培养后，其代谢乙酸乙酯的能力下降约44%。而经过定向驯化的微生物群落能够有效避免乙酸乙酯代谢能力的降低，并能在不影响乙醇产量的前提下，提升20%-70%的乙酸乙酯代谢能力。将驯化后的微生物群落与原始大曲进行模拟发酵实验，结果表明，无论是采用未经微生物提取的半合成方式(EG1)，还是提取后再混合的全合成方式所制备的强化发酵菌剂(EG2)，在发酵过程中均能使乙酸乙酯的产量提升210%以上。【结论】 成功通过定向驯化策略构建出能够高效合成乙酸乙酯的微生物群落。

Abstract:

[Background] Ethyl acetate is a crucial aroma component in Maotai-flavor Baijiu, and its content directly impacts the flavor characteristics. [Objective] To overcome the functional degradation of single strains for enhancing ethyl acetate synthesis in the practical application, we employed directional domestication to enhance the ethyl acetate synthesis of microorganisms at the community level. [Methods] We thoroughly analyzed the gene expression abundance to identify the differential nutrients in substrates for microbial metabolism. According to the pathway of microbial synthesis of ethyl acetate, we determine three key carbon sources: glucose, fructose, and arabinose, which were then used for the directional domestication of the microbial community. [Results] The microbial biomass in groups S1 (1.37 mg/mL), S2 (1.40 mg/mL), and S3 (1.57 mg/mL) was lower than that in the CN control group (1.87 mg/mL). After 14 rounds of directional domestication, the microbial diversity showed a downward trend and the structure of the formed fungal community was similar. The relative abundance of Wickerhamomyces anomalus increased remarkably, surging from 0.04% to 65% and even higher (P < 0.05). The ability of fermentation microorganisms to synthesize ethyl acetate decreased by about 44% after continuous dilution and subculture. The microbial community after directional domestication can avoid the reduction of ethyl acetate synthesis and increase the ethyl acetate accumulation by 20%-70% without affecting ethanol production. The domesticated microbial community was then blended with the original Daqu for simulated fermentation. The results showed that the microbial fermentation agent prepared by the semi-synthetic method without microbial extraction (EG1) or the total synthesis method after extraction and mixing (EG2) increased the yield of ethyl acetate by more than 210% during the fermentation process. [Conclusion] The microbial community capable of efficiently synthesizing ethyl acetate was successfully constructed by the directional domestication strategy.