Cocoa Bean Fermentation Process Research Articles

PENINGKATAN MUTU BIJI KAKAO MENGGUNAKAN TEKNOLOGI FERMENTASI DI DESA MASARI, PARIGI MOUTONG

Masari village is one of the cocoa production centers in Parigi Moutong regency, but in recent years cocoa production has decreased. The decline in cocoa production was due to the low selling value of cocoa beans, thereby reducing farmers' income. Farmers uncertain income caused the transition of crop commodities into plantation and food crops. The low selling value of cocoa beans was caused by the low quality of cocoa beans. One of the factors that caused the low quality of beans was post-harvest management, which is the cocoa bean fermentation process that is rarely done by the farmers. This community service program aims to provide knowledge about the correct bean fermentation method so they can improve the quality of cocoa beans. Service activities are carried out using counseling and training methods followed by demonstrations on how to ferment cocoa beans. In general, the counseling activity about the selection of cocoa pods that are ready to be harvested and fermentation procedures went well. The seed demonstration activity was also successful which was indicated by brown cocoa beans and had a fragrant smell. The target community already can carry out a good cocoa bean fermentation process. Cocoa bean fermentation technology can be used to improve the physical and chemical quality of cocoa beans so it can increase the value and sales of cocoa beans. The increase in sales will encourage farmers to develop a cocoa-based farming system.

ANALYSIS OF COCOA FERMENTATION WITH THE ADDITION OF YEAST AND LACTIC ACID BACTERIA

Abstract One of the factors that affect the quality of cocoa beans is post-harvest handling such as the fermentation process. Fermentation plays an important role in determining the final quality of dried cocoa beans. Chocolate flavors can be produced from microbial fermentation from high-quality cocoa bean raw materials by utilizing microbes. This study aims to calculate the number of microbes at the beginning and end of the cocoa bean fermentation process, observing the pH value, temperature, and color of cocoa beans. The treatment carried out is a fermentation variation consisting of: (1) natural fermentation or without the addition of microorganisms; (2) controlled fermentation with the addition of microorganisms consisting of Saccharomyces cerevisiae, Lactic Acid Bacteria simultaneously added at the beginning of fermentation. The addition of microorganisms is 106 CFU / 1kg of cocoa beans each. Fermentation is carried out in a fermentation box with a capacity of 1 kg of fresh cocoa, at room temperature (33-35oC), for 4 days. The analysis carried out includes calculating the number of microbes at the beginning and end of fermentation, measuring pH, and organoleptic observations of the color of fermented cocoa beans. The results of the experiment can be concluded that there is an increase in the addition of mixed inoculum (Saccharomyces cerevisiae and Lactobacillus lactis) affecting the influence of chemical changes of the substrate during fermentation; the temperature value in cocoa fermentation ranges from 30 – 32 oC, the pH value in cocoa fermentation does not have a significant increase; and the color of cocoa beans from mixed inoculum fermentation is close to good with a dominant brown color against purple

Chemical and Organoleptic Characteristics of Cocoa Powder from Fermented and Unfermented Cocoa Beans from Masni District and Sidey in Manokwari Regency

beverage industry, such as chocolate drinks and ingredients for cakes, puddings, and ice cream. The fermentation process of cocoa beans is one of the determining factors for the quality of cocoa powder products produced. This is because during the fermentation process, physical, chemical, and organoleptic characteristics are formed, which become the standard for high-quality cocoa powder products. One of the factors that influences the success of the cocoa bean fermentation process is the fermentation time. The right fermentation time is expected to produce high-quality cocoa beans with a distinctive chocolate flavor, according to consumer preferences. The aim of this study was to determine the chemical, and organoleptic characteristics of cocoa powder from cocoa beans originating from Masni District and Sidey District with different fermentation times. The study consisted of six treatments: cocoa beans processed by the community (BKT), cocoa beans without fermentation (BKTF), cocoa beans fermented for 3 days (BK3), 4 days (BK4), 5 days (BK5), and 6 days (BK6). The differences between the treatments of unfermented cocoa beans and the fermentation time affect the physicochemical and organoleptic characteristics of the resulting cocoa powder. The fermentation process tends to improve the color of cocoa powder from dark brown to a lighter shade of brown, increase the moisture content, decrease the fat content, and significantly influence the organoleptic characteristics. Fermenting cocoa beans for 5 days results in cocoa powder with the best physical, chemical, and organoleptic characteristics. The organoleptic test of cocoa powder from cocoa beans fermented 5 days (BK5) show a slightly light brown to brown color, a strong to very strong cocoa powder flavor, a taste from less bitter to moderately bitter, and acidity levels from less acidic to moderately acidic.

Penerapan Fermentasi Biji Kakao (Theobroma cacao L.) di Kabupaten Pidie Jaya, Aceh

Cocoa is one of the primary commodities in Meureudu and Trienggadeng Sub-districts, Pidie Jaya Regency. Many cocoa farmers in the sub-districts still need to understand the process and benefits of fermenting cocoa beans. Bean fermentation is essential because it can increase biochemical changes and the concentration of flavour precursors in cocoa beans so that it has a high selling value. This community service activity was implemented using counselling methods, demonstrations, and the submission of fermenters. It was held for two days assisting and attended by 59 of 70 farmers from two groups. Results showed the participants' positive response to the partaking activities and their interest in fermenting cocoa. Through the activities that have been carried out, cocoa farmers understand and can maintain a good cocoa bean fermentation process. Assistance to farmer groups must be continuously conducted so that cocoa farmers remain consistent in fermenting cocoa beans to produce better quality cocoa.

Studi Meta-analisis: Pengaruh Penambahan Kultur Starter pada Profil Fermentasi, Mikroorganisme, dan Metabolit Hasil Fermentasi Biji Kakao (Theobroma cacao L.)

Fermentation of cocoa beans can produce flavour precursors and colour changes in chocolate. Generally, this process is carried out for 5-7 days without adding starter culture. Adding a starter culture is considered to improve the quality of cocoa beans and shorten the fermentation time. The purpose of this study was to compare the starter cultures used in the cocoa bean fermentation process through a meta-analysis approach. Twenty-four related articles have been screened from the initial number of 110 articles. There are five starter cultures and six parameters that can be processed by Confidence Interval (CI) analysis. Calculating p and I2 values ​​using STATA software was performed to see variations between studies and test the significance of their effects during the fermentation process through p values. The variation test between studies showed that the profiles of lactic acid bacteria (LAB) and acetic acid bacteria (AAB) were heterogeneous. While the yeast profile, levels of lactic acid, acetic acid and pH between studies are homogeneous. The results of the study showed that the addition of starter culture will affect the levels of yeast profile (ES: 0.470; 95% CI: 0.371 to 0.569; p = 0.0); LAB profile (ES: 0.747; 95% CI: 0.600 to 0.894; p = 0,0); AAB profile (ES: 0.808; 95% CI: 0.663 to 0.953; p = 0.0); lactic acid (ES: -0.003; 95% CI: -0.162 to 0.156; p = 0.039); acetic acid (ES: 0.189; 95% CI: 0.01 to 0.368; p = 0.039) and pH (ES: 0.109; 95% CI: 0.001 to 0.218; p = 0.049). Using pure starter cultures can increase the number of microbes of the type added and increase metabolic activity by showing a decrease in acetic acid levels in cocoa beans at the end of the fermentation process. However, no changes were seen in pH or lactic acid levels.

Temperature and pH Monitoring System Design in the Fermentation of Cocoa Beans Based on Android

The fermentation of cocoa beans is a key step in increasing their quality. Temperature and pH have an impact on the fermentation process' performance. Until, temperature and pH measurements in the cocoa bean fermentation process have been done with a thermometer and a pH meter, and have been heavily impacted by experience and knowledge. The goal of this research is to create a temperature and pH monitoring system for the fermentation of cocoa beans using an android platform. The sensor must be able to measure temperature and pH during the fermentation process, and the sensor reading data must be displayed on the android display, according to the system design criteria. A temperature sensor, pH sensor, microcontroller, TP-Link or WiFi modem, ThingSpeak (data storage), and Smartphone make up the system's hardware. Arduino CC software version 1.8.14 was used to create the software. The system hardware was made up of numerous components, including the NodeMCU ESP32 microcontroller, SKU SEN0161 pH sensor, DS18B20 temperature sensor, 4.2k resistor, jumper cable, and 5 volt charger adaptor, according to the results. A plastic container houses the electronic system circuit. The monitoring system can read the temperature and pH during the fermentation process, which lasts 112 hours or 6 days, according to the performance test findings. During fermentation, the temperature of cocoa beans ranged from 27.19 °C to 41.75 °C, with the pH fluctuating between 2.50 and 7.10.

Improving Data Security with Blockchain and Internet of Things in the Gourmet Cocoa Bean Fermentation Process.

Brazil was one of the largest cocoa producers in the world, mainly supported by the South of Bahia region. After the 1980s, the witch-broom disease demolished plantations, and farmers were forced into bankruptcy. The worldwide search for gourmet cocoa has rekindled interest in this production, whose fermentation process is a key step in obtaining fine cocoa, thanks to the fact that many processing properties and sensory attributes are developed in this phase. This article presents a blockchain-IoT-based system for the control and monitoring of these events, aiming to catalog in smart contracts valuable information for improvement of the cocoa fermentation process, and future research. Blockchain is used as a distributed database that implements an application-level security layer. A proof of concept was modeled and the performance of the emulated system was evaluated in the OMNet simulator, where a technique based on the SNMP protocol was applied to reduce the amount of data exchanged and resources served/consumed in this representation. Then, a physical platform was developed and preliminary experiments were performed on a real farm, as a way to verify the improvement of the cocoa fermentation process when using a technological approach.

Oxygen Uptake Rate and Carbon dioxide Evolution Rate on the Fermentation Process of Cocoa Beans (Theobroma Cacao L.)

Fermentation is one of the most critical stages in the postharvest handling process of cocoa beans. Temperature changes during the fermentation process characterize the success of the cocoa bean fermentation process. In general, aeration conditions during the fermentation process effects changes in fermentation air temperature. This study aims to determine cumulative oxygen uptake and cumulative carbon dioxide evolution during cocoa bean fermentation. Fermenter equipped with a controlled system of air temperatures and aeration rates was used to determine changes in oxygen uptake rate and carbon dioxide evolution rate during the fermentation process. The variables observed were including: length of pod storage time before fermentation (X₁; days), aeration rate (Xâ‚‚; l.min-1), and fermenter temperature (X₃; ℃). Response surface methodology was used to determine the relationship between cumulative oxygen uptake as well as cumulative carbon dioxide evolution and the observed variables (X₁, Xâ‚‚, X₃) The results shows that cumulative oxygen uptake and cumulative carbon dioxide evolution obtained in various treatments during the fermentation process are consecutive: 6.66-34.29 g Oâ‚‚ kgvs-¹ and 12.15-50.97 g COâ‚‚ kgvs-¹. Based on the variance analysis of response surface methodology, R² value of the cumulative oxygen uptake is about 88.1%, while R² value of the cumulative carbon dioxide evolution value is about 87.3 %. The highest amount of oxygen consumption and carbon dioxide production was obtained at X₁ = 5 days, Xâ‚‚ = 0.25- 0.4 l.min-1, and X₃ ≤ 50℃.

Enumeration and Identification of Bacterial Spores in Cocoa Powders

The presence of bacterial spores in cocoa powders is inevitable due to the cocoa bean fermentation process, during which members of the genera Bacillus and Geobacillus are typically present. Spores are a concern in heat-treated foods when they survive heat treatments and the finished product supports germination, growth, and potentially toxin production. In this study, available methods for the enumeration of total mesophilic and thermophilic spores (TMS and TTS, respectively) were evaluated, leading to the recommendation of one global method specifically for cocoa powders. The proposed method was validated during a ring test on seven selected cocoa powders and applied during routine analyses on commercial powders. The method includes dilution of cocoa powder using buffered peptone water, heating at 80°C for 10 min for TMS and TTS counts, and heating at 100°C for 30 min for a heat-resistant (HR) spore count. Tryptic soy agar is used as a recovery medium with a maximal concentration of cocoa powder of 2.5 mg/mL (to prevent growth inhibition) and a nonnutrient agar overlay to prevent swarming of bacteria. Plates are incubated for at least 72 h at 30°C for recovery of mesophilic bacteria and 55°C for thermophilic bacteria. Suitable alternatives to specific method parameters are provided. Median values of total spore concentrations are low (<400 CFU/g for TMS and <75 CFU/g for TTS), and concentrations of HR spores are very low (<5 CFU/g). Importantly, the relation between concentrations of HR spores in cocoa powder and incidence of spoilage of heat-treated beverages containing cocoa is currently unclear. In the powders included in this study, Bacillus subtilis and Bacillus licheniformis were the predominant spore-forming species identified (49 and 39%, respectively). Both species are known for high variability in spore heat resistance. The development of reliable and sensitive molecular methods is therefore required to assess the risk of spoilage caused by spores present in cocoa powders.

Genome-Scale Metabolic Reconstruction of Acetobacter pasteurianus 386B, a Candidate Functional Starter Culture for Cocoa Bean Fermentation.

Acetobacter pasteurianus 386B is a candidate functional starter culture for the cocoa bean fermentation process. To allow in silico simulations of its related metabolism in response to different environmental conditions, a genome-scale metabolic model for A. pasteurianus 386B was reconstructed. This is the first genome-scale metabolic model reconstruction for a member of the genus Acetobacter. The metabolic network reconstruction process was based on extensive genome re-annotation and comparative genomics analyses. The information content related to the functional annotation of metabolic enzymes and transporters was placed in a metabolic context by exploring and curating a Pathway/Genome Database of A. pasteurianus 386B using the Pathway Tools software. Metabolic reactions and curated gene-protein-reaction associations were bundled into a genome-scale metabolic model of A. pasteurianus 386B, named iAp386B454, containing 454 genes, 322 reactions, and 296 metabolites embedded in two cellular compartments. The reconstructed model was validated by performing growth experiments in a defined medium, which revealed that lactic acid as the sole carbon source could sustain growth of this strain. Further, the reconstruction of the A. pasteurianus 386B genome-scale metabolic model revealed knowledge gaps concerning the metabolism of this strain, especially related to the biosynthesis of its cell envelope and the presence or absence of metabolite transporters.

Screening of lactic acid bacteria and yeast strains to select adapted anti-fungal co-cultures for cocoa bean fermentation

Contamination with filamentous fungi during cocoa bean fermentation and drying reduces the quality of cocoa beans and poses a health risk for consumers due to the potential accumulation of mycotoxins. The aim of this study was to develop anti-fungal lactic acid bacteria (LAB)-yeast co-cultures by selecting anti-fungal strains best adapted to the cocoa bean fermentation process from 362 LAB and 384 yeast strains isolated from cocoa bean post-harvest processes. The applied multiphasic screening approach included anti-fungal activity tests in vitro and in vivo and assessment of the carbon metabolism and stress tolerance of the anti-fungal strains in a cocoa pulp simulation medium. The anti-fungal strains, Lactobacillus fermentum M017, Lb. fermentum 223, Hanseniaspora opuntiae H17, and Saccharomyces cerevisiae H290, were selected based on their high fungal growth inhibition capacity and their well-adapted metabolism. Up to seven filamentous fungal strains of the genera Aspergillus, Penicillium, and Gibberella were inhibited on average by 63 and 75% of the maximal inhibition zone by M017 and 223, respectively, and by 25 and 31% by the strains H17 and H290, respectively. Both Lb. fermentum strains converted the medium's glucose, fructose, and citric acid into 20.4–23.0 g/l of mannitol, 3.9–6.2 g/l acetic acid, and 8.6–10.3 g/l lactic acid, whereas the two yeast strains metabolized glucose and fructose to produce 7.4–18.4 g/l of ethanol. The Lb. fermentum strains were further characterized as particularly tolerant towards ethanol, acetic acid, and heat stress and both yeast strains tolerated high amounts of ethanol and lactic acid in the medium. Finally, the anti-fungal in vivo assays revealed that the two Lb. fermentum strains completely inhibited growth of the citrinin-producing strain, P. citrinum S005, and the potentially fumonisin-producing strain, G. moniliformis S003, on the surface of cocoa beans. Furthermore, growth of the aflatoxin-producer A. flavus S075 was inhibited after 10-14 days by all four selected anti-fungal strains, i.e. Lb. fermentum M017, Lb. fermentum 223, H. opuntiae H17, and Sacc. cerevisiae H290, at 51–95% when applied as single cultures and at 100% when the strains were combined into four co-cultures, each composed of a Lb. fermentum and one of the two yeast strains. As a conclusion, these four LAB-yeast co-cultures are recommended for future applications to limit the growth of filamentous fungi and the concomitant mycotoxin production during the fermentation of cocoa beans.

A lab-scale model system for cocoa bean fermentation.

Lab-scale systems modelling the spontaneous cocoa bean fermentation process are useful tools to research the influence of process parameters on the fermentation and the final bean quality. In this study in Honduras, a 1-kg lab-scale fermentation (LS-F) was compared to a 300-kg on-farm fermentation (OF-F) in a multiphasic approach, analysing microbial counts, microbial species diversity, physico-chemical parameters, and final dried bean quality. Yeast and total aerobic counts of up to 8logCFU/g during the LS-F were comparable to the OF-F, while counts for lactic acid bacteria and acetic acid bacteria were up to 3logCFU/g lower during the LS-F than during the OF-F. While species of the genera Hansenia, Saccharomyces, and Acetobacter dominated most of the fermentation processes, the genera dominating the drying phases were Pichia, Trichosporon, Pediococcus, and Acetobacter. Dried beans resulting from the LS-F, compared to the OF-F, were similar in contents of acetic acid, 6 times lower in lactic acid, up to 4 times higher in residual sugars, and 3-12 times higher in polyphenols. Dried beans processed at LS showed a similar flavour profile in terms of astringency, bitterness, acidity, and brown, fine, and cocoa flavours, but 2 units higher off-flavours than OF processed beans. With 81%, the share of well-fermented beans from the LS-F complied with industrial standards, whereas 7% over-fermented beans were above the threshold. Conclusively, the 5-day model fermentation and subsequent drying successfully mimicked the on-farm process, providing a high-throughput method to screen microbial strains to be used as starter cultures.

Assessment of the contribution of cocoa-derived strains of Acetobacter ghanensis and Acetobacter senegalensis to the cocoa bean fermentation process through a genomic approach

Acetobacter ghanensis LMG 23848T and Acetobacter senegalensis 108B are acetic acid bacteria that originate from a spontaneous cocoa bean heap fermentation process and that have been characterised as strains with interesting functionalities through metabolic and kinetic studies. As there is currently little genetic information available for these species, whole-genome sequencing of A. ghanensis LMG 23848T and A. senegalensis 108B and subsequent data analysis was performed. This approach not only revealed characteristics such as the metabolic potential and genomic architecture, but also allowed to indicate the genetic adaptations related to the cocoa bean fermentation process. Indeed, evidence was found that both species possessed the genetic ability to be involved in citrate assimilation and displayed adaptations in their respiratory chain that might improve their competitiveness during the cocoa bean fermentation process. In contrast, other properties such as the dependence on glycerol or mannitol and lactate as energy sources or a less efficient acid stress response may explain their low competitiveness. The presence of a gene coding for a proton-translocating transhydrogenase in A. ghanensis LMG 23848T and the genes involved in two aromatic compound degradation pathways in A. senegalensis 108B indicate that these strains have an extended functionality compared to Acetobacter species isolated from other ecosystems.

The cocoa bean fermentation process: from ecosystem analysis to starter culture development.

Cocoa bean fermentation is still a spontaneous curing process to facilitate drying of nongerminating cocoa beans by pulp removal as well as to stimulate colour and flavour development of fermented dry cocoa beans. As it is carried out on farm, cocoa bean fermentation is subjected to various agricultural and operational practices and hence fermented dry cocoa beans of variable quality are obtained. Spontaneous cocoa bean fermentations carried out with care for approximate four days are characterized by a succession of particular microbial activities of three groups of micro-organisms, namely yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB), which results in well-fermented fully brown cocoa beans. This has been shown through a plethora of studies, often using a multiphasic experimental approach. Selected strains of several of the prevailing microbial species have been tested in appropriate cocoa pulp simulation media to unravel their functional roles and interactions as well as in small plastic vessels containing fresh cocoa pulp-bean mass to evaluate their capacity to dominate the cocoa bean fermentation process. Various starter cultures have been proposed for successful fermentation, encompassing both cocoa-derived and cocoa nonspecific strains of (hybrid) yeasts, LAB and AAB, some of which have been implemented on farms successfully.

Comparative genome analysis of the candidate functional starter culture strains Lactobacillus fermentum 222 and Lactobacillus plantarum 80 for controlled cocoa bean fermentation processes.

BackgroundLactobacillus fermentum 222 and Lactobacillus plantarum 80, isolates from a spontaneous Ghanaian cocoa bean fermentation process, proved to be interesting functional starter culture strains for cocoa bean fermentations. Lactobacillus fermentum 222 is a thermotolerant strain, able to dominate the fermentation process, thereby converting citrate and producing mannitol. Lactobacillus plantarum 80 is an acid-tolerant and facultative heterofermentative strain that is competitive during cocoa bean fermentation processes. In this study, whole-genome sequencing and comparative genome analysis was used to investigate the mechanisms of these strains to dominate the cocoa bean fermentation process.ResultsThrough functional annotation and analysis of the high-coverage contigs obtained through 454 pyrosequencing, plantaricin production was predicted for L. plantarum 80. For L. fermentum 222, genes encoding a complete arginine deiminase pathway were attributed. Further, in-depth functional analysis revealed the capacities of these strains associated with carbohydrate and amino acid metabolism, such as the ability to use alternative external electron acceptors, the presence of an extended pyruvate metabolism, and the occurrence of several amino acid conversion pathways. A comparative genome sequence analysis using publicly available genome sequences of strains of the species L. plantarum and L. fermentum revealed unique features of both strains studied. Indeed, L. fermentum 222 possessed genes encoding additional citrate transporters and enzymes involved in amino acid conversions, whereas L. plantarum 80 is the only member of this species that harboured a gene cluster involved in uptake and consumption of fructose and/or sorbose.ConclusionsIn-depth genome sequence analysis of the candidate functional starter culture strains L. fermentum 222 and L. plantarum 80 revealed their metabolic capacities, niche adaptations and functionalities that enable them to dominate the cocoa bean fermentation process. Further, these results offered insights into the cocoa bean fermentation ecosystem as a whole and will facilitate the selection of appropriate starter culture strains for controlled cocoa bean fermentation processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1927-0) contains supplementary material, which is available to authorized users.

Applying meta-pathway analyses through metagenomics to identify the functional properties of the major bacterial communities of a single spontaneous cocoa bean fermentation process sample

A high-resolution functional metagenomic analysis of a representative single sample of a Brazilian spontaneous cocoa bean fermentation process was carried out to gain insight into its bacterial community functioning. By reconstruction of microbial meta-pathways based on metagenomic data, the current knowledge about the metabolic capabilities of bacterial members involved in the cocoa bean fermentation ecosystem was extended. Functional meta-pathway analysis revealed the distribution of the metabolic pathways between the bacterial members involved. The metabolic capabilities of the lactic acid bacteria present were most associated with the heterolactic fermentation and citrate assimilation pathways. The role of Enterobacteriaceae in the conversion of substrates was shown through the use of the mixed-acid fermentation and methylglyoxal detoxification pathways. Furthermore, several other potential functional roles for Enterobacteriaceae were indicated, such as pectinolysis and citrate assimilation. Concerning acetic acid bacteria, metabolic pathways were partially reconstructed, in particular those related to responses toward stress, explaining their metabolic activities during cocoa bean fermentation processes. Further, the in-depth metagenomic analysis unveiled functionalities involved in bacterial competitiveness, such as the occurrence of CRISPRs and potential bacteriocin production. Finally, comparative analysis of the metagenomic data with bacterial genomes of cocoa bean fermentation isolates revealed the applicability of the selected strains as functional starter cultures.

MAPPING THE EFFECTS OF STARTER CULTURE ADDITION ON COCOA BEAN FERMENTATION

Fermentation is an essential step in the formation of aroma and flavour of cocoa beans. During the fermentation, cocoa bean pulp is degraded to form metabolic products, one of which functions as the precursor for the aroma and flavour of cocoa beans. Previous research suggested that the addition of starter culture on the cocoa bean fermentation process can improve the fermentation performance. In this research the effects of various starters culture combinations, including Saccharomyces cerevisiae var. Chevalieri, Lactobacillus plantarum, and Acetobacter aceti were studied. The addition of starter culture was confirmed by analysis of microbial population during the fermentation. Fermentation performances were measured as fermentation index and cocoa bean pH, as well as sugar and metabolic product concentration. The obtained results showed that in general the addition of starter culture accelerated sugar consumption and increased metabolites production. The highest fermentation index was 1.38±0.07, obtained from fermentation added with starter culture combination containing Saccharomyces cerevisiae var. Chevalieri and Lactobacillus plantarum. This fermentation advanced faster and was completed at 4 days, or shortened the overall fermentation time. On the other hand fermentation added with starter culture containing only Acetobacter aceti gave the lowest fermentation index, 0.96±0.04

Enhancement of Cocoa Quality By the Indigenous Yeast Candida Tropicalis KLK4 Through Cocoa Bean Fermentation

An indigenous yeast strain that isolated from fermented cocoa bean has a role in cocoa bean fermentation process, was characterized and identified to be member of the Candida tropicalis  species  based on phenotypic characteristics and the D1/D2 region of the large subunit rRNA gene. Properties of importance for cocoa bean fermentation, namely sucrose, glucose, and citrate assimilation capacity, pH-, ethanol-, and heat-tolerance, were examined for isolate. The Quality of fermented cocoa bean was analyzed by cut test. Candida tropicalis KLK4 was tolerance to low pH value, high temperature, ethanol and could assimilate citrate, reflecting it is able to adapt to cocoa fermentation environment. The utilization of Candida tropicalis KLK4 strain as a starter culture for cocoa fermentation can enhance cocoa  quality.

Complete genome sequence and comparative analysis of Acetobacter pasteurianus 386B, a strain well-adapted to the cocoa bean fermentation ecosystem

BackgroundAcetobacter pasteurianus 386B, an acetic acid bacterium originating from a spontaneous cocoa bean heap fermentation, proved to be an ideal functional starter culture for coca bean fermentations. It is able to dominate the fermentation process, thereby resisting high acetic acid concentrations and temperatures. However, the molecular mechanisms underlying its metabolic capabilities and niche adaptations are unknown. In this study, whole-genome sequencing and comparative genome analysis was used to investigate this strain’s mechanisms to dominate the cocoa bean fermentation process.ResultsThe genome sequence of A. pasteurianus 386B is composed of a 2.8-Mb chromosome and seven plasmids. The annotation of 2875 protein-coding sequences revealed important characteristics, including several metabolic pathways, the occurrence of strain-specific genes such as an endopolygalacturonase, and the presence of mechanisms involved in tolerance towards various stress conditions. Furthermore, the low number of transposases in the genome and the absence of complete phage genomes indicate that this strain might be more genetically stable compared with other A. pasteurianus strains, which is an important advantage for the use of this strain as a functional starter culture. Comparative genome analysis with other members of the Acetobacteraceae confirmed the functional properties of A. pasteurianus 386B, such as its thermotolerant nature and unique genetic composition.ConclusionsGenome analysis of A. pasteurianus 386B provided detailed insights into the underlying mechanisms of its metabolic features, niche adaptations, and tolerance towards stress conditions. Combination of these data with previous experimental knowledge enabled an integrated, global overview of the functional characteristics of this strain. This knowledge will enable improved fermentation strategies and selection of appropriate acetic acid bacteria strains as functional starter culture for cocoa bean fermentation processes.

Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, and Acetobacter pasteurianus predominate during well-performed Malaysian cocoa bean box fermentations, underlining the importance of these microbial species for a successful cocoa bean fermentation process

Two spontaneous Malaysian cocoa bean box fermentations (one farm, two plantation plots) were investigated. Physical parameters, microbial community dynamics, yeast and bacterial species diversity [mainly lactic acid bacteria (LAB) and acetic acid bacteria (AAB)], and metabolite kinetics were monitored, and chocolates were produced from the respective fermented dry cocoa beans. Similar microbial growth and metabolite profiles were obtained for the two fermentations. Low concentrations of citric acid were found in the fresh pulp, revealing low acidity of the raw material. The main end-products of the catabolism of the pulp substrates glucose, fructose, and citric acid by yeasts, LAB, and AAB were ethanol, lactic acid, acetic acid, and/or mannitol. Hanseniaspora opuntiae, Lactobacillus fermentum, and Acetobacter pasteurianus were the prevalent species of the two fermentations. Saccharomyces cerevisiae, Lactobacillus plantarum, Lactobacillus pentosus, and Acetobacter ghanensis were also found during the mid-phase of the fermentation processes. Leuconostoc pseudomesenteroides and Acetobacter senegalensis were among the prevailing species during the initial phase of the fermentations. Tatumella saanichensis and Enterobacter sp. were present in the beginning of the fermentations and they could be responsible for the degradation of citric acid and/or the production of gluconic acid and lactic acid, respectively. The presence of facultative heterofermentative LAB during the fermentations caused a high production of lactic acid. Finally, as these fermentations were carried out with high-quality raw material and were characterised by a restricted microbial species diversity, resulting in successfully fermented dry cocoa beans and good chocolates produced thereof, it is likely that the prevailing species H. opuntiae, S. cerevisiae, Lb. fermentum, and A. pasteurianus were responsible for it.