The effect of fermentation and roasting on free amino acids profile in Criollo cocoa (Theobroma cacao L.) grown in Venezuela

Abstract The development of cocoa flavor and aroma is a complex phenomenon that depends on how the fermentation and roasting processes are carried out. During fermentation, the formation of some compounds so-called “aroma and flavor precursors” takes place, which are finally expressed during the roasting stage. Therefore, the evaluation of aroma precursors such as the amino acids formed during fermentation is crucial in order to determine the quality of the cocoa. In this context, we developed and validated a method for the study of these compounds in cocoa samples. The amino acids were quantitatively converted into their trimethylsilyl derivatives before their determination by gas chromatography with mass spectrometry detection. The results were verified performing precision and accuracy studies. The inter and intra assay coefficients of variation (C.V, n = 5) were lower than 4.7% and 4%, respectively. The analytical recoveries (95% to 108% with C.V < 4.2, n = 5) demonstrated the high performance of the extraction procedure. The method was successfully applied to the analysis of the amino acids in 110 samples of Venezuelan Criollo cocoa during the three days of fermentation and roasting (110 °C for 25 min). All samples had an appreciable content of free amino acids ranging between 3.87 and 5.97 g/kg in the absence of fermentation. We observed degradation of the acidic amino acids during the first day of fermentation, while the rest of amino acids increased progressively during the fermentation process with a predominance of the hydrophobic ones, mainly leucine, phenylalanine, valine, alanine and isoleucine. Additionally, during the roasting stage a fraction of the amino acids, especially the hydrophobic ones, was partially degraded through Maillard reaction to form the compounds associated with the cocoa aroma and flavor.

摘要
可可风味与香气的形成是一类复杂过程，其表现取决于发酵与烘焙工艺的具体实施参数。发酵阶段会生成一类被称为“香气与风味前体”的化合物，此类前体最终会在烘焙阶段转化为特征性的香气与风味物质。因此，对发酵过程中生成的氨基酸等香气前体物质开展检测与评估，对可可品质的判定至关重要。
基于此研究背景，本研究开发并验证了一种可用于分析可可样品中此类化合物的检测方法：先将氨基酸定量转化为三甲基硅基衍生物，随后采用气相色谱-质谱联用法进行检测。通过精密度与准确度实验对方法可靠性进行了验证：批内与批间变异系数（C.V, n = 5）分别低于4.7%与4%；方法回收率处于95%~108%区间，且对应变异系数小于4.2%（n = 5），证明萃取流程具备优异的分析性能。
本方法成功应用于110份委内瑞拉克里奥罗（Criollo）可可豆样品的氨基酸分析，覆盖发酵3天与110℃、25分钟烘焙的全流程样本。未发酵样品的游离氨基酸含量可观，区间为3.87~5.97 g/kg。实验观察到，发酵首日酸性氨基酸发生降解，其余氨基酸则在发酵过程中逐步积累，其中以疏水氨基酸为主，主要包括亮氨酸、苯丙氨酸、缬氨酸、丙氨酸与异亮氨酸。此外，在烘焙阶段，部分氨基酸（尤其是疏水氨基酸）会通过美拉德反应发生部分降解，生成与可可香气和风味相关的特征化合物。