Table_3_Unraveling the Role of Acetic Acid Bacteria Comparing Two Acetification Profiles From Natural Raw Materials: A Quantitative Approach in Komagataeibacter europaeus.DOCX

The industrial production of vinegar is carried out by the activity of a complex microbiota of acetic acid bacteria (AAB) working, mainly, within bioreactors providing a quite specific and hard environment. The “omics” sciences can facilitate the identification and characterization analyses of these microbial communities, most of which are difficult to cultivate by traditional methods, outside their natural medium. In this work, two acetification profiles coming from the same AAB starter culture but using two natural raw materials of different alcoholic origins (fine wine and craft beer), were characterized and compared and the emphasis of this study is the effect of these raw materials. For this purpose, the composition and natural behavior of the microbiota present throughout these profiles were analyzed by metaproteomics focusing, mainly, on the quantitative protein profile of Komagataeibacter europaeus. This species provided a protein fraction significantly higher (73.5%) than the others. A submerged culture system and semi-continuous operating mode were employed for the acetification profiles and liquid chromatography with tandem mass spectrometry (LC-MS/MS) for the protein analyses. The results showed that neither of two raw materials barely modified the microbiota composition of the profiles, however, they had an effect on the protein expression changes in different biological process. A molecular strategy in which K. europaeus would prevail over other species by taking advantage of the different features offered by each raw material has been suggested. First, by assimilating the excess of inner acetic acid through the TCA cycle and supplying biosynthetic precursors to replenish the cellular material losses; second, by a previous assimilation of the excess of available glucose, mainly in the beer medium, through the glycolysis and the pentose phosphate pathway (PPP); and third, by triggering membrane mechanisms dependent on proton motive force to detoxify the cell at the final moments of acetification. This study could complement the current knowledge of these bacteria as well as to expand the use of diverse raw materials and optimize operating conditions to obtain quality vinegars.
Clinical Trial Registration:[www.ClinicalTrials.gov], identifier [PXD031147].

食醋的工业化生产主要依靠醋酸菌（acetic acid bacteria, AAB）组成的复杂微生物群落的代谢活动，该过程主要发生在具备特定严苛环境的生物反应器中。
组学（omics）技术可简化这类微生物群落的鉴定与表征分析——其中多数菌种在脱离其自然培养基的条件下，难以通过传统培养方法获取。
本研究针对两组醋化过程展开表征与对比：两组过程均源自同一醋酸菌发酵剂菌种，但分别采用两种不同酒精来源的天然原料，即优质葡萄酒与精酿啤酒。本研究的核心重点为原料对醋化过程的影响。
为此，本研究采用宏蛋白质组学（metaproteomics）技术，分析了两组醋化过程全程存在的微生物群落组成与自然行为，重点聚焦于欧洲葡糖醋杆菌（Komagataeibacter europaeus）的定量蛋白质谱。该菌种的蛋白质组分占比显著高于其他菌种，达73.5%。
本研究采用深层培养体系与半连续操作模式开展醋化实验，并通过液相色谱-串联质谱（liquid chromatography with tandem mass spectrometry, LC-MS/MS）进行蛋白质组分析。
结果显示，两种原料均未对微生物群落组成产生显著影响，但可改变不同生物过程中的蛋白质表达模式。研究提出了一套分子策略：欧洲葡糖醋杆菌可通过利用不同原料提供的差异化特性，在群落中占据竞争优势。具体机制包括三方面：其一，通过三羧酸循环（TCA cycle）同化胞内过量乙酸，并提供生物合成前体以补充细胞物质损耗；其二，在啤酒原料体系中，通过糖酵解与磷酸戊糖途径（pentose phosphate pathway, PPP）预先同化过量的游离葡萄糖；其三，在醋化后期触发依赖质子动力势的细胞膜机制，以实现细胞解毒。
本研究可完善当前对这类醋酸菌的认知，并为拓展多元原料的应用、优化工艺条件以获得高品质食醋提供理论支撑。
临床试验注册：[www.ClinicalTrials.gov]，标识符[PXD031147]。