Díaz-Muñoz Cristian, Van de Voorde Dario, Comasio Andrea, Verce Marko, Hernandez Carlos Eduardo, Weckx Stefan, De Vuyst Luc
Research Group of Industrial Microbiology and Food Biotechnology, Faculty of Sciences and Bioengineering Sciences, Vrije Universiteit Brussel, Brussels, Belgium.
Laboratorio de Calidad e Innovación Agroalimentaria, Escuela de Ciencias Agrarias, Universidad Nacional de Costa Rica, Heredia, Costa Rica.
Front Microbiol. 2021 Jan 11;11:616875. doi: 10.3389/fmicb.2020.616875. eCollection 2020.
Starter culture-initiated cocoa fermentation processes can be applied to improve the quality of cured cocoa beans. However, an accurate monitoring of the microbial strains inoculated in fresh cocoa pulp-bean mass to assess their contribution to the cocoa bean curing process is still lacking. In the present study, eight different cocoa fermentation processes were carried out with Trinitario cocoa in vessels in Costa Rica to assess the contribution of two candidate yeast starter culture strains, namely IMDO 050523 and IMDO 020508, inoculated in combination with IMDO 0611222 and IMDO 0506386. A multiphasic approach, consisting of culture-dependent selective plating and incubation, rRNA-PCR-DGGE community profiling of agar plate washes, and culture-independent high-throughput amplicon sequencing, combined with a metabolite target analysis of non-volatile and volatile organic compounds (VOCs), was performed on samples from the fermentation and/or drying steps. The different starter culture mixtures applied effectively steered the cocoa fermentation processes performed. Moreover, the use of an amplicon sequence variant (ASV) approach, aligning these ASVs to the whole-genome sequences of the inoculated strains, allowed the monitoring of these inoculated strains and their differentiation from very closely related variants naturally present in the background or spontaneous fermentation processes. Further, traits such as malolactic fermentation during the fermentation step and acetoin and tetramethylpyrazine formation during the drying step could be unraveled. Finally, the yeast strains inoculated influenced the substrate consumption and metabolite production during all starter culture-initiated fermentation processes. This had an impact on the VOC profiles of the cured cocoa beans. Whereas the strain produced a wide range of VOCs in the cocoa pulp, the strain mostly influenced the VOC composition of the cured cocoa beans.
起始培养物引发的可可发酵工艺可用于提高发酵可可豆的品质。然而，目前仍缺乏对新鲜可可果肉 - 豆团中接种的微生物菌株进行准确监测，以评估它们对可可豆发酵过程的贡献。在本研究中，在哥斯达黎加的容器中对特立尼达可可进行了八种不同的可可发酵工艺，以评估两种候选酵母起始培养菌株，即IMDO 050523和IMDO 020508，与IMDO 0611222和IMDO 0506386组合接种的贡献。对来自发酵和/或干燥步骤的样品采用了多相方法，包括依赖培养的选择性平板接种和培养、琼脂平板洗涤液的rRNA - PCR - DGGE群落分析以及不依赖培养的高通量扩增子测序，并结合对非挥发性和挥发性有机化合物（VOCs）的代谢物目标分析。所应用的不同起始培养物混合物有效地引导了所进行的可可发酵过程。此外，使用扩增子序列变体（ASV）方法，将这些ASV与接种菌株的全基因组序列进行比对，能够监测这些接种菌株，并将它们与背景或自发发酵过程中自然存在的密切相关变体区分开来。此外，还可以揭示发酵步骤中的苹果酸 - 乳酸发酵以及干燥步骤中乙偶姻和四甲基吡嗪形成等特征。最后，接种的酵母菌株在所有起始培养物引发的发酵过程中影响了底物消耗和代谢物产生。这对发酵可可豆的VOC谱产生了影响。虽然[具体菌株]在可可果肉中产生了多种VOCs，但[具体菌株]主要影响了发酵可可豆的VOC组成。
