Tigrero-Vaca Joel, Villavicencio-Vásquez Mirian, Coronel Jonathan, Cevallos-Cevallos Juan Manuel
Escuela Superior Politécnica del Litoral, ESPOL, Centro de Investigaciones Biotecnológicas del Ecuador (CIBE), Guayaquil, Ecuador.
Escuela Superior Politécnica del Litoral (ESPOL), Facultad de Ingeniería Mecánica y Ciencias de la Producción (FIMCP), Centro de Biotecnología (CIBE), Guayaquil, Ecuador.
Front Bioeng Biotechnol. 2025 Aug 26;13:1630515. doi: 10.3389/fbioe.2025.1630515. eCollection 2025.
Cacao fermentation is a spontaneous process in which microorganisms play a key role in the development of distinctive chocolate flavors. The microbiota acting during cacao fermentation has been routinely characterized by culture-based techniques and next-generation sequencing using Illumina's platform. However, the potential of sequencing technologies to monitor microbial dynamics during cacao fermentation has not been assessed. In this study, cacao bean samples were collected at 0, 24, 48, 72, and 96 h after the start of the fermentation. Total DNA was extracted, and sequencing libraries were prepared for further sequencing using Illumina's and Nanopore's MinION sequencing platforms. Additionally, microorganisms were isolated using traditional culture-based methods. At the order and family taxonomic levels, Illumina and MinION sequencing revealed similar microbial composition in the samples. However, discrepancies were observed at the genus and species levels. In this sense, Illumina sequencing revealed a predominance of , , , and , while MinION sequencing showed a prevalence of , , , , and during fermentation. The three methods were consistent in detecting the major yeast (), lactic acid bacteria (, , , , and ) and acetic acid bacteria () species during fermentation. Functional analysis based on a hybrid assembly of Illumina and MinION data revealed the roles of lactic acid bacteria and acetic acid bacteria in the metabolism of carbohydrates, amino acids, and secondary metabolites such as polyphenols and theobromine. This study represents the first report assessing the applicability of MinION sequencing for the characterization of microbial populations during cacao fermentation, demonstrating its potential as a complementary tool to established sequencing platforms.
可可发酵是一个自发过程，在此过程中微生物在独特巧克力风味的形成中发挥关键作用。传统上，利用基于培养的技术和使用Illumina平台的新一代测序技术对可可发酵过程中起作用的微生物群进行了表征。然而，尚未评估测序技术监测可可发酵过程中微生物动态变化的潜力。在本研究中，在发酵开始后的0、24、48、72和96小时收集可可豆样本。提取总DNA，并使用Illumina和Nanopore的MinION测序平台制备测序文库以进行进一步测序。此外，还使用传统的基于培养的方法分离微生物。在目和科的分类水平上，Illumina测序和MinION测序显示样本中的微生物组成相似。然而，在属和种的水平上观察到差异。从这个意义上说，Illumina测序显示 、 、 、 和 占优势，而MinION测序显示在发酵过程中 、 、 、 、 和 的流行率较高。这三种方法在检测发酵过程中的主要酵母（ ）、乳酸菌（ 、 、 、 、 和 ）和醋酸菌（ ）种类方面是一致的。基于Illumina和MinION数据的混合组装的功能分析揭示了乳酸菌和醋酸菌在碳水化合物、氨基酸以及多酚和可可碱等次生代谢物代谢中的作用。本研究是评估MinION测序在可可发酵过程中微生物群体表征方面适用性的首份报告，证明了其作为现有测序平台补充工具的潜力。
