Dissecting fine-flavor cocoa bean fermentation through metabolomics analysis to break down the current metabolic paradigm

Cocoa fermentation plays a crucial role in producing flavor and bioactive compounds of high demand for food and nutraceutical industries. Such fermentations are frequently described as a succession of three main groups of microorganisms (i.e., yeast, lactic acid, and acetic acid bacteria), each producing a relevant metabolite (i.e., ethanol, lactic acid, and acetic acid). Nevertheless, this view of fermentation overlooks two critical observations: the role of minor groups of microorganisms to produce valuable compounds and the influence of environmental factors (other than oxygen availability) on their biosynthesis. Dissecting the metabolome during spontaneous cocoa fermentation is a current challenge for the rational design of controlled fermentations. This study evaluates variations in the metabolic fingerprint during spontaneous fermentation of fine flavor cocoa through a multiplatform metabolomics approach. Our data suggested the presence of two phases of differential metabolic activity that correlate with the observed variations on temperature over fermentations: an exothermic and an isothermic phase. We observed a continuous increase in temperature from day 0 to day 4 of fermentation and a significant variation in flavonoids and peptides between phases. While the second phase, from day four on, was characterized for lower metabolic activity, concomitant with small upward and downward fluctuations in temperature. Our work is the first to reveal two phases of metabolic activity concomitant with two temperature phases during spontaneous cocoa fermentation. Here, we proposed a new paradigm of cocoa fermentation that considers the changes in the global metabolic activity over fermentation, thus changing the current paradigm based only on three main groups of microorganism and their primary metabolic products.

Preliminary study on development of cocoa beans fermentation level measurement based on computer vision and artificial intelligence

The fermentation process is an important indicator of cocoa beans’ quality. The standard method used is the Magra test by splitting the cocoa beans and observing the color of the beans with the naked eye to estimate the degree of fermentation. Although, manual estimation systems require specific expertise, which can lead to inconsistency in predicting cocoa bean fermentation rate. This research aims to develop a classification model of two categories of cocoa, i.e., fermented and unfermented cocoa, using computer vision and a machine learning model. Image analysis has been carried out, and color features have been used to train and compare several classification models. After analyzing the data, it was found out that a model that can quantify the standard and accurate measurement of the degree of fermentation of cocoa beans using artificial neural network models so that it can segment, calculate, and grade classification by using color feature extraction, which is the average value of RGB and L*a*b. The Artificial Neural Network (ANN) Multilayer Perceptron (MLP) has been found to be superior compared to other models achieving training and validation accuracy of 94%.

Indigenous Yeast, Lactic Acid Bacteria, and Acetic Acid Bacteria from Cocoa Bean Fermentation in Indonesia Can Inhibit Fungal-Growth-Producing Mycotoxins

Cocoa bean fermentation is an important process in the manufacturing of cocoa products. It involves microbes, such as lactic acid bacteria, yeast, and acetic acid bacteria. The presence of mold in cocoa bean fermentation is undesired, as it reduces the quality and may produce mycotoxins, which can cause poisoning and death. Aspergillus niger is a fungus that produces ochratoxin A, which is often found in dried agricultural products such as seeds and cereals. In this study, we applied indigenous Candida famata HY-37, Lactobacillus plantarum HL-15, and Acetobacter spp. HA-37 as starter cultures for cocoa bean fermentation. We found that the use of L. plantarum HL-15 individually or in combination Candida famata HY-37, Lactobacillus plantarum HL-15, and Acetobacter spp. HA-37 as a starter for cocoa bean fermentation can inhibit the growth of A. niger YAC-9 and the synthesis of ochratoxin A during fermentation and drying. With biological methods that use indigenous Lactobacillus plantarum HL-15 individually or in combination with Candida famata HY-37 and Acetobacter spp. HA-37, we successfully inhibited contamination by ochratoxin-A-producing fungi. Thus, the three indigenous microbes should be used in cocoa bean fermentation to inhibit the growth of fungi that produce mycotoxins and thus improve the quality.

Fermentation of Cocoa Beans

Cocoa bean fermentation is a spontaneous process driven by an ordered microbial succession of a wide range of yeasts, lactic acid and acetic acid bacteria, some aerobic sporeforming bacteria and various species of filamentous fungi. The process of cocoa fermentation is a very important step for developing chocolate flavor precursors which are attributable to the metabolism of succession microbial. The microbial ecology of cocoa has been studied in much of the world. In Venezuela, studies have been carried out with Criollo, Forastero, and Trinitario cocoa, fermented under various conditions, the results obtained coinciding with the reported scientific information. Fermentation must be associated with the type of cocoa available, carried out knowing the final processing and derivative (paste, butter, powder). The results shown in this chapter correspond to investigations carried out with cocoa from three locations in Venezuela. The quantification, identification, isolation, functionality of the most representative microbiota involved in the fermentation of these grains was sought. This to give possible answers to the fermentation times and improvement of the commercial quality. Likewise, generate greater interest on the part of the producers in carrying out the fermentation.

Changes in Physico-Chemical and Microbiological Properties in Thai Cocoa Bean Fermentation

The purpose of this research is to investigate the quality-related physical, chemical, and microbiological changes in Thai cocoa beans during fermentation in 2 types of wooden containers. The results will compose a book of guidelines for good Thai cocoa fermentation in order to educate Thai farmers. Fresh Thai cocoa beans have a low pH value (5.0 - 5.5) compared to those from other countries in general (6.0 - 7.0). However, fermented temperature is able to reach 40 - 45 °C in 6 days, which is a main criteria for finishing cocoa fermentation. The color of fresh cocoa beans changes from white to brown within 2 days; after that, cocoa beans are mixed from the top to the bottom of the containers. Three groups of microorganism are evaluated with 3 different sampling points in wooden containers. The results reveal that yeast is grown quickly in 2 days on the top of containers, and then acetic acid bacteria and lactic acid bacteria are grown by the utilized yeast’s metabolites. These behaviors were found in both of the 2 wooden containers; however, a heap of cocoa beans (200 - 250 kgs) in a wooden box showed better quality of cocoa fermentation than a small volume (40 - 50 kgs) in a wooden tray. HIGHLIGHTS Understanding changes in Thai cocoa beans fermentation based on traditional method Two type of wooden containers applied for comparing all quality-related cocoa fermentation Proper cocoa beans fermentation process composed to a book of guideline for Thai Farmers GRAPHICAL ABSTRACT

Small-scale Fermentation of Cocoa Beans and on-Process Monitoring

Cocoa bean fermentation is a critical step in the formation of chocolate aroma precursors. Small-scale fermentation is needed to accommodate fermentation practice during low season and fruit scarcity. The study was conducted to address the minimum amount of wet cocoa beans required for a successful fermentation. In the trials, wet cocoa beans at different quantities, i.e. 1, 2.5, 5, 7.5, 10, 15, and 40 kg, were incubated in separate wooden boxes for 4 days (96 hours) and turned once after 48 hours. Temperature and pH of the pulp and beans were measured every 6 hours. Dry beans were visually inspected to determine the number of grayish and purple cotyledons indicating unfermented beans. In general, smaller scale fermentations, i.e. 1 to 15 kg, resulted in similar profiles with that of standard mini-box fermentation (40 kg) in terms of temperature, pulp pH and bean pH. The 40 kg fermentation resulted in the lowest percentage of purple beans with no grayish beans, meanwhile smaller scale fermentations showed higher number of unfermented beans. The logarithmic regression indicates that as much as 1.4 kg beans producing unfermented beans of less than 20% which meets the criteria of grade 3 based on the Indonesian National Standard for cocoa bean (SNI 2323:2008/Amd1:2010). Data analysis shows that temperature below 35°C for 42 hours was associated with higher percentage of grayish and purple beans. It is concluded that cocoa bean fermentation can be carried out at least at 5 kg scale, and temperature at 42 hours could be a parameter for process monitoring.

Towards a Starter Culture for Cocoa Fermentation by the Selection of Acetic Acid Bacteria

Acetic acid bacteria are involved in many food and beverage fermentation processes. They play an important role in cocoa bean fermentation through their acetic acid production. They initiate the development of some of the flavor precursors that are necessary for the organoleptic quality of cocoa, and for the beans’ color. The development of starter cultures with local strains would enable the preservation of the microbial biodiversity of each country in cocoa-producing areas, and would also control the fermentation. This approach could avoid the standardization of cocoa bean fermentation in the producing countries. One hundred and thirty acetic acid bacteria were isolated from three different cocoa-producing countries, and were identified based on their 16S rRNA gene sequence. The predominate strains were grown in a cocoa pulp simulation medium (CPSM-AAB) in order to compare their physiological traits regarding their specific growth rate, ethanol and lactic acid consumption, acetic acid production, and relative preferences of carbon sources. Finally, the intraspecific diversity of the strains was then assessed through the analysis of their genomic polymorphism by (GTG)5-PCR fingerprinting. Our results showed that Acetobacter pasteurianus was the most recovered species in all of the origins, with 86 isolates out of 130 cultures. A great similarity was observed between the strains according to their physiological characterization and genomic polymorphisms. However, the multi-parametric clustering results in the different groups highlighted some differences in their basic metabolism, such as their efficiency in converting carbon substrates to acetate, and their relative affinity to lactic acid and ethanol. The A. pasteurianus strains showed different behaviors regarding their ability to oxidize ethanol and lactic acid into acetic acid, and in their relative preference for each substrate. The impact of these behaviors on the cocoa quality should be investigated, and should be considered as a criterion for the selection of acetic acid bacteria starters.

Isolation and identification of microbial species found in cocoa fermentation as microbial starter culture candidates for cocoa bean fermentation in Colombia.

Microbial activity involved in the cocoa beans fermentation process is essential to maintain and improve the organoleptic and nutritional qualities of chocolate; therefore, the aim of this investigation was to search and select microbial isolates with the potential to improve the quality of cocoa beans. Fermentation experimentswere conducted on farms located in Maceo (Antioquia), San Vicente de Chucurí (Santander), and Rivera and Algeciras (Huila), Colombia. Yeast, lactic acid bacteria (LAB), acetic acid bacteria (AAB), and mesophilic aerobic microorganisms were obtained from different fermentation batches. The growth of these microorganismswas tested in six treatments as follows: 50% cocoa pulp agar (CPA), high concentrations of glucose (10%), ethanol (5%), and acetic acid (7%), an acidic pH of 3.0, and a high temperature of 50oC for 24 h. The isolates with the highest growth were identified by 18S and 16S rRNA gene analysis, revealing a high diversity ofspecies associated with cocoa fermentation, including eight species of yeasts (Debaryomyces hansenii, Meyerozyma guillermondii, Wickerhanomyces anomalus, Pichia guillermondii, Pichia kudriavzevii, Trichosporon asahii, Candida parapsilosis, and Pichia manshurica), six species of LAB (Pediococcus acidilactici, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus farraginis, Lactobacillus rhamnosus, and Leuconostoc mesenteroides), four species of AAB (Gluconobacter japonicus, Acetobacter tropicalis, Acetobacter pasteurianus, and Acetobacter malorum/tropicalis), and three species of Bacillus spp. (Bacillusaryabhattai /megaterium, Bacillus subtilis, and Bacillus coagulans). In general, microbial populations increased in cocoa batches after 12 h of fermentation and decreased after 84-96 h. All the yeast isolates grew in 10% glucose and CPA, 85.7% in 5% ethanol, and 95% at a pH of 3.0. All the yeast isolates were affectedby 7% acetic acid and incubation at 50oC for 24 h. Eighty-five percent of the LAB grew in 10% glucose, 100% in 5% ethanol, 42.8% in CPA, 64% at a pH of 3.0, and 35.7% grew after being exposed to 50oC for 24 h; all were affected by 7% acetic acid. As for the AAB, 100% grew in 10% glucose, 71% in 7% ethanol, 100% grew in CPA, in 7% acetic acid, and at a pH of 3.0, while 100% were affected by incubation at 50oC. Three yeast isolates, W. anomalus, D. hansenii and M. guillermondii, three LAB isolates, P. acidilactici, L. brevis, and L. plantarum, and three AAB isolates, A. tropicalis, A. pasteurianus and G. japonicus, were selected as promising strains to be used in a microbial starter culture for cocoa bean fermentation to improve the organoleptic quality of cocoa.