Metabolomics analysis of cocoa bean (Theobroma cacao) fermentation to reveal conditions related to GABA (gamma-aminobutyric acid) levels
Laboratory of Bioresource Engineering, Fukusaki Lab
Abu Hanifah
Chapter 1: General Introduction
GABA (gamma-aminobutyric acid) is a naturally occurring non-proteinogenic amino acid found in various food sources. Research has demonstrated the potential benefits of GABA, including its role as an anti-insomnia, anti-depression, and anti-hypertension agent. Many plant-derived foods serve as rich sources of GABA. Fermented cocoa beans and chocolates are examples of such sources, with GABA content ranging from 31.7 to 101.2 mg/100 g and from 11.1 to 32.5 mg/100 g, respectively. The variation in GABA levels among cocoa beans is influenced by factors such as genotype, growth conditions, and the post-harvest method, particularly fermentation. Utilizing low environmental temperatures during fermentation is a natural approach to enhance GABA levels in food, which is generally well-received by consumers. However, the impact of low environmental temperature on GABA levels in cocoa beans during fermentation remains unknown. Furthermore, the process of chocolate-making results in the loss of some GABA. This loss occurs due to the occurrence of the Maillard reaction, which takes place during heating at temperatures ranging from 140°C to 165°C. The Maillard reaction contributes to the flavor, aroma, and brown color of chocolate by facilitating reactions between reducing sugars and amino acids. It is established that the origin of cocoa beans and the fermentation process can influence the metabolite profile, leading to variations in sugar composition. Nevertheless, whether the rate of GABA loss differs when different fermented beans are used in chocolate production remains to be investigated.
Chapter 2: GABA levels in cocoa beans are correlated with low environmental temperature
Indonesia, one of the countries known for its cocoa production, offers a diverse range of climates according to the Köppen climate classification. This characteristic makes it an ideal location for conducting this study. Raw and fermented cocoa beans obtained from various environmental temperatures were subjected to GC/MS-based analysis, which revealed differences based on the place of origin of the bean. Subsequently, the relationship between the metabolite profile and the environmental temperature of the place of origin was investigated using orthogonal projection to latent structure regression (OPLS-R) analysis. The OPLS-R analysis identified four metabolites, namely GABA, glycerol, ethanolamine, and succinic acid, that exhibited a negative correlation with the minimum and average temperatures. This indicates that lower minimum and average temperatures were associated with higher levels of these metabolites. Further observation of GABA reveals higher levels both before and after fermentation, suggesting that low environmental temperatures affect the GABA levels of cocoa beans during fermentation.
Chapter 3: Optimum fermentation condition to increase GABA levels
This chapter aims to refine the variables based on previous findings for smooth implementation in the chocolate industry. Daily observations of fermentation procedures of cocoa clones were conducted to monitor GABA level changes. The clones were grown in two distinct environmental temperatures (high and low) and fermented accordingly, followed by swapped fermentation. The swapping aim is to investigate the GABA level when beans are moved prior to fermentation. Results show that low environmental temperature during fermentation increases GABA levels, particularly in cocoa beans fermented in Malang (lower temperature), for both moved and not. The highest GABA levels were recorded on day 5 of fermentation, possibly due to increased decarboxylation of glutamic acid under low environmental temperature conditions. Further analysis revealed higher levels of organic acids in cocoa bean fermentation under low environmental temperatures, suggesting greater activity in the TCA cycle. This is supported by the lower intensity of trehalose and succinic acid in low environmental temperature conditions. GABA synthesis in plants is closely linked to the TCA cycle, indicating that cocoa bean fermentation in low environmental temperatures may promote TCA cycle activity and higher GABA levels.
Chapter 4: The sugar composition of fermented cocoa beans affects the GABA levels of SOC (single-origin chocolate)
Fermented cocoa beans play a crucial role as an ingredient in the chocolate industry, but they are not the final product. In this chapter, we collected fermented cocoa beans from five different origins in Indonesia to produce SOC. The chocolate-making process was carefully controlled to ensure uniformity in the treatment of each cocoa bean. Subsequently, we observed that GABA levels decrease at varying rates after the chocolate-making process. Pearson correlation analysis revealed a relationship between GABA loss and reducing sugars in fermented cocoa beans. Notably, sucrose exhibited a significant negative correlation (-0.829) with a GABA loss of 41%. On the other hand, mannose, the C-2 epimer of glucose, displayed a significant positive correlation (0.761) with a GABA loss of 78%, whereas glucose itself did not show a significant correlation. Therefore, these findings suggest that the GABA/mannose interaction is the primary factor contributing to the high GABA loss during the chocolate-making process.
Chapter 5: Conclusion and Future Perspective
This study uses metabolomics to investigate the relationship between cocoa bean fermentation conditions and GABA levels. Low environmental temperatures during fermentation significantly increase GABA levels in cocoa beans, regardless of their origin. This increase is likely due to higher TCA cycle activity under lower environmental temperature conditions. Farmers in tropical highlands can adopt this method to enhance the value of their fermented cocoa beans. However, chocolate production leads to GABA loss during roasting. Chocolate makers seeking high GABA levels can consider the sugar composition of fermented cocoa beans to minimize the loss. Future research should focus on identifying optimal conditions for artificial low temperature fermentation and improving GABA production using specific GABA-producing microorganisms, as well as selecting cocoa clones with desired sugar compositions.
List of publication:
Hanifah, A., Firmanto, H., Putri, S. P., and Fukusaki, E.: Unique metabolite profiles of Indonesian cocoa beans from different origins and their correlation with temperature, J. Biosci. Bioeng., 134, 125-132 (2022). https://doi.org/10.1016/j.jbiosc.2022.05.001.