Table_1_Unraveling potential enzymes and their functional role in fine cocoa beans fermentation using temporal shotgun metagenomics.DOCX

Cocoa beans fermentation is a spontaneous process, essential for the generation of quality starting material for fine chocolate production. The understanding of this process has been studied by the application of high-throughput sequencing technologies, which grants a better assessment of the different microbial taxa and their genes involved in this microbial succession. The present study used shotgun metagenomics to determine the enzyme-coding genes of the microbiota found in two different groups of cocoa beans varieties during the fermentation process. The statistical evaluation of the most abundant genes in each group and time studied allowed us to identify the potential metabolic pathways involved in the success of the different microorganisms. The results showed that, albeit the distinction between the initial (0 h) microbiota of each varietal group was clear, throughout fermentation (24–144 h) this difference disappeared, indicating the existence of selection pressures. Changes in the microbiota enzyme-coding genes over time pointed to the distinct ordering of fermentation at 24–48 h (T1), 72–96 h (T2), and 120–144 h (T3). At T1, the significantly more abundant enzyme-coding genes were related to threonine metabolism and those genes related to the glycolytic pathway, explained by the abundance of sugars in the medium. At T2, the genes linked to the metabolism of ceramides and hopanoids lipids were clearly dominant, which are associated with the resistance of microbial species to extreme temperatures and pH values. In T3, genes linked to trehalose metabolism, related to the response to heat stress, dominated. The results obtained in this study provided insights into the potential functionality of microbial community succession correlated to gene function, which could improve cocoa processing practices to ensure the production of more stable quality end products.

可可豆发酵是一类自发过程，对于高品质巧克力生产所需的优质起始原料的制备至关重要。学界已通过应用高通量测序技术（high-throughput sequencing technologies）对该过程展开研究，以更精准地解析参与此次微生物演替的各类微生物类群及其功能基因。本研究采用宏基因组鸟枪法（shotgun metagenomics），对发酵过程中两类不同可可豆品种的微生物组所携带的编码酶类的基因进行鉴定。通过对各组、各采样时间点下丰度最高的基因进行统计分析，本研究得以明确参与不同微生物定植演替的潜在代谢通路。研究结果显示，尽管两类可可豆品种的初始（0小时）微生物组群落结构差异显著，但在发酵全程（24~144小时）中该差异逐渐消失，这表明存在群落选择压力。微生物组编码酶类基因的时序变化表明，发酵过程可划分为三个特征鲜明的阶段：24~48小时（T1）、72~96小时（T2）以及120~144小时（T3）。在T1阶段，丰度显著上调的编码酶类基因主要与苏氨酸代谢及糖酵解通路相关，这一现象可由培养基中丰富的糖类底物所解释。在T2阶段，与神经酰胺（ceramides）和藿烷类脂质（hopanoids lipids）代谢相关的基因占据主导地位，这类基因与微生物对极端温度及pH值的耐受能力密切相关。在T3阶段，与海藻糖（trehalose）代谢相关的基因成为优势类群，该代谢通路与微生物的热应激响应机制相关。本研究所得结果揭示了与基因功能相关的微生物群落演替潜在功能机制，该发现可用于优化可可豆加工工艺，以保障产出品质更为稳定的终端产品。