Cocoa Fermentation Process Research Articles

Exploring volatile compounds and microbial dynamics: Kluyveromyces marxianus and Hanseniaspora opuntiae reduce Forastero cocoa fermentation time

Traditional cocoa bean fermentation is a spontaneous process and can result in heterogeneous sensory quality. For this reason, yeast-integrated starter cultures may be an option for creating consistent organoleptic profiles. This study proposes the mixture of Hanseniaspora opuntiae and Kluyveromyces marxianus (from non-cocoa fermentation) as starter culture candidates. The microorganisms and volatile compounds were analyzed during the cocoa fermentation process, and the most abundant were correlated with predominant microorganisms. Results showed that Kluyveromyces marxianus, isolated from mezcal fermentation, was identified as the dominant yeast by high-throughput DNA sequencing. A total of 63 volatile compounds identified by HS-SPME-GC–MS were correlated with the more abundant bacteria and yeast using Principal Component Analysis and Agglomerative Hierarchical Clustering. This study demonstrates that yeasts from other fermentative processes can be used as starter cultures in cocoa fermentation and lead to the formation of more aromatic esters, decrease the acetic acid content.

Computational mathematical modeling in the cocoa fermentation process in four isolated systems

In the southern region of Bahia, the cocoa culture has been a source of income and moved the economy for a long time, but the emergence of the “witch's broom” (Moniliophthora Perniciosa) caused the decline of this productive sector. Given the importance of cocoa for the region, makes sense to use it as an object of study for the development of academic research that can foster the reheating of this branch of the economy. In this context, for the processing of cocoa to occur to be able to use in the manufacture of chocolate and derivatives it needs to go through 5 distinct stages, being: harvesting, breaking, fermentation, drying, and storage. The stages in question are responsible for more than 45% of the organoleptic properties of cocoa, i.e. affect the final quality of the product. Fermentation is one of the most important stages since it is through fermentation that cocoa develops most of its characteristics, such as color, flavor, and aroma. The step by step of cocoa fermentation is to let the mass ferment, without stirring, until the temperature reaches 32ºC or if it does not reach the ideal temperature, wait to complete 48h. There are cases in which temperatures can be reached between 40ºC to 60ºC. Thus, the idea arose to create a cocoa fermenter model that could increase the efficiency of the fermentation cycle, and boost its scale and quality. Nowadays the process is done manually, requiring the worker to perform the laborious task of monitoring and stirring the cocoa mass at short intervals, over hours. This work compares four fermenter models through the analysis of heat diffusion. The method is theoretical with the help of computer modeling, where the propagation of heat from a source throughout the system filled with a mass of cocoa is evaluated.

Antagonistic Activity of Cellulase-Producing Yeasts Isolated from Cocoa Fermentation Against Pathogenic Molds Collected from Damaged Cocoa Fruits

The presence of pathogenic molds in post-harvested cocoa beans poses a significant problem to most Indonesian farmers. Pathogenic molds secrete phytopathogenic toxins which cause rotting, seriously damaging the cocoa fruits. The common pathogenic molds found in damaged cocoa beans are Phytophthora palmivora and Phytophthora megakarya. These molds are so far controlled by using chemical pesticides, whose long-term use is potentially harmful to human health and the environment. Thus, a healthier and safer alternative is needed, especially using biological agents such as yeasts. Some yeasts produce cellulose-degrading enzymes that can possibly break down the fungal cell wall which is composed mostly of the polysaccharide’s chitin and glucan. In this study, yeasts were isolated during the cocoa fermentation process, followed by screening for cellulolytic activity using direct planting and paste method, and subsequent antagonistic testing by double culture technique. Out of 128 yeast isolates, 77 possessed cellulase activity, with 6 of them having the highest activity index. Antagonistic activity test of these 6 isolates (C1.0.4, C1.1.3, C2.3.10, C2.3.14, C3.5.11, and C3.3.1) against the pathogenic molds resulted in isolate C3.3.1 showing the highest inhibition percentage from the 2

Isolation of Lactic Acid Bacteria from Cocoa Bean Fermentation as Potential Antibacterial Agent against ESKAPE Pathogens

This study aimed to evaluate the antibacterial activity of microorganisms isolated before and during cocoa fermentation against ESKAPE pathogens. Microorganisms from cocoa fermentation process were isolated on the selective media and were tested against ESKAPE pathogens. Total titratable acidity, minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of cell-free supernatant from lactic acid bacteria (LAB) were determined. The presence of bacteriocin and antibacterial activity were determined. LAB was identified through molecular and biochemical tests. Ninety-five and 134 isolates were obtained from the fermentation of Sungai Balung 25 (BR25) clone and mixed clones, respectively. Screening of antibacterial activity showed that 26 isolates of LAB from the cocoa fermentation process had antibacterial activity against ESKAPE pathogens (zone of inhibition ≥11 mm). Cell-free supernatant from some LAB demonstrated potent antibacterial activity against some ESKAPE pathogens through MIC and MBC and evaluation. Furthermore, one isolate (mix48M01) exhibited antibacterial activity against Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter spp., methicillin-resistant Staphylococcus aureus, and Enterococcus raffinousus. An increase in the diameter of inhibition zone was observed as the lactic acid production increased. Gram-positive bacteria isolates were more resistant to LAB than Gram-negative bacteria. Bacteriocin with good antibacterial activity was also detected from the LAB examined. Molecular analysis showed that 13 LAB shared >99% sequence similarity to Lactobacillus fermentum CECT 562 while one isolate shared sequence similarity <98%, indicating that it might be a novel Lactobacillus species. Isolated LAB from this study exhibited high antibacterial activity against ESKAPE pathogens and could be investigated further as potential probiotics and antibacterial agents in the future.

Exploration of Lactiplantibacillus fabifermentans and Furfurilactobacillus rossiae as potential cocoa fermentation starters.

AimsTo investigate the characteristics of two minority autochthonous LAB species, with particular regard to those properties that could be exploited in an improved cocoa fermentation process from a quality and safety point of view.Methods and ResultsBacterial, yeast and mould strains characteristic of spontaneously fermented Dominican cocoa beans were isolated and identified by 16S or 26S rRNA gene sequencing. The potential of two autochthonous strains of LAB belonging to the species Lactiplantibacillus fabifermentans and Furfurilactibacillus rossiae were investigated. The two selected LAB strains were able to utilize glucose and fructose, produced mainly D‐L lactic acid and had a good ability to resist to cocoa‐related stress conditions such as low pH, high temperature and high osmotic pressure, as well as to grow in sterile cocoa pulp. The strains did not inhibit the growth of yeasts and acetic acid bacteria, that are essential to the cocoa fermentation process, and possessed a complex pool of peptidases especially active on hydrophobic amino acids. The strains also showed antifungal activity against mould species that can be found at the final stages of cocoa fermentation, as Aspergillus tamarii, A. nidulans, Lichtheimia ornata and Rhizomucor pusillus.ConclusionsThe tested strains are good candidates for the design of starter cultures for a controlled cocoa fermentation process.Significance and Impact of the StudyThis research showcases the potential of two alternative LAB species to the dominating Lactiplantibacillus plantarum and Limosilactibacillus fermentum as cocoa fermentation starters, with an interesting activity in improving the safety and quality of the process.

Functional yeast starter cultures for cocoa fermentation.

SummaryThe quest to develop a performant starter culture mixture to be applied in cocoa fermentation processes started in the 20th century, aiming at achieving high‐quality, reproducible chocolates with improved organoleptic properties. Since then, different yeasts have been proposed as candidate starter cultures, as this microbial group plays a key role during fermentation of the cocoa pulp‐bean mass. Yeast starter culture‐initiated fermentation trials have been performed worldwide through the equatorial zone and the effects of yeast inoculation have been analysed as a function of the cocoa variety (Forastero, Trinitario and hybrids) and fermentation method (farm‐, small‐ and micro‐scale) through the application of physicochemical, microbiological and chemical techniques. A thorough screening of candidate yeast starter culture strains is sometimes done to obtain the best performing strains to steer the cocoa fermentation process and/or to enhance specific features, such as pectinolysis, ethanol production, citrate assimilation and flavour production. Besides their effects during cocoa fermentation, a significant influence of the starter culture mixture applied is often found on the cocoa liquors and/or chocolates produced thereof. Thus, starter culture‐initiated cocoa fermentation processes constitute a suitable strategy to elaborate improved flavourful chocolate products.

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.

Evaluation of Indigenous Pichia kudriavzevii from cocoa fermentation for a probiotic candidate

Abstract. Wulan R, Astuti RI, Rukayadi Y, Meryandini A. 2021. Evaluation of Indigenous Pichia kudriavzevii from cocoa fermentation for a probiotic candidate. Biodiversitas 22: 1317-1325. Currently, probiotics are becoming a concern along with a healthy lifestyle awareness. Besides bacteria, yeast can be used as a probiotic candidate with specific functional properties. Research on yeast as a probiotic is still limited (except for Saccharomyces boulardii). Previous research has isolated yeasts from the cocoa fermentation process. This study aimed to evaluate the probiotics properties and antioxidant activity of yeast strains isolated from spontaneous cocoa fermentation in Sukabumi, Indonesia. Previous research has isolated 23 yeast strains from spontaneous cocoa (Theobroma cacao L.) fermentation. Of the 23 yeast strains isolated from cocoa fermentation, 22 strains showed negative hemolysis as an indicator of non-pathogenic properties. Ten yeast strains were able to grow at 37 °C and 41 °C, pH 3, 0.5% bile salts, had autoaggregation ability (63.99-95.33%), and co-aggregation with S. typhimurium ATCC14028 (>80%), as character requirement for probiotic candidates. The genetic identification of the ten yeast strains showed that they were 99% identical to Pichia kudriavzevii. Based on its antioxidant activity, the P. kudriavzevii 2P10 metabolites had the highest percentage of inhibition (68.51%) against DPPH free radicals and resistance to H2O2 oxidative stress up to 3 mM. It can be concluded that P. kudriavzevii 2P10 is a promising probiotic candidate for functional foods and health purposes.

A Combined Metagenomics and Metatranscriptomics Approach to Unravel Costa Rican Cocoa Box Fermentation Processes Reveals Yet Unreported Microbial Species and Functionalities.

Cocoa fermentation is the first step in the post-harvest processing chain of cocoa and is important for the removal of the cocoa pulp surrounding the beans and the development of flavor and color precursors. In the present study, metagenomic and metatranscriptomic sequencing were applied to Costa Rican cocoa fermentation processes to unravel the microbial diversity and assess the function and transcription of their genes, thereby increasing the knowledge of this spontaneous fermentation process. Among 97 genera found in these fermentation processes, the major ones were Acetobacter, Komagataeibacter, Limosilactobacillus, Liquorilactobacillus, Lactiplantibacillus, Leuconostoc, Paucilactobacillus, Hanseniaspora, and Saccharomyces. The most prominent species were Limosilactobacillus fermentum, Liquorilactobacillus cacaonum, and Lactiplantibacillus plantarum among the LAB, Acetobacter pasteurianus and Acetobacter ghanensis among the AAB, and Hanseniaspora opuntiae and Saccharomyces cerevisiae among the yeasts. Consumption of glucose, fructose, and citric acid, and the production of ethanol, lactic acid, acetic acid, and mannitol were linked to the major species through metagenomic binning and the application of metatranscriptomic sequencing. By using this approach, it was also found that Lacp. plantarum consumed mannitol and oxidized lactic acid, that A. pasteurianus degraded oxalate, and that species such as Cellvibrio sp., Pectobacterium spp., and Paucilactobacillus vaccinostercus could contribute to pectin degradation. The data generated and results presented in this study could enhance the ability to select and develop appropriate starter cultures to steer the cocoa fermentation process toward a desired course.

Influence of S. cerevisiae and P. kluyveri as starters on chocolate flavour.

Fermented cocoa beans (Theobroma cacao L.) are a pivotal raw material for chocolate production. A cocktail yeast applied in the cocoa fermentation process can promote the formation of pleasant metabolites. Saccharomyces, Pichia and Hanseniaspora have been widely used in fermentation to improve the final product organoleptic profile, highlighting that fermentation is a critical point for chocolate flavour precursor production. This study aims to evaluate the impact of Pichia kluyveri and Saccharomyces cerevisiae strains as starter cultures on the fermentation for two cocoa hybrids, FA13 and CEPEC2002. During fermentation processes, volatile organic compounds (VOCs) and protein profiles were assessed. Chocolates produced were also assessed regarding the presence of VOCs. Eighty VOCs were identified using gas chromatography coupled to mass spectrometry analysis. Mass spectrometry provided the protein profile evolution during fermentation and showed that the profiles changed with inoculation type (spontaneous versus inoculated fermentation). Chocolate obtained from FA13 inoculated with S. cerevisiae strain contained a greater amount of organics acids, being categorised as sourer than chocolate produced by spontaneous fermentation of FA13. CEPEC2002 inoculated with S. cerevisiae strain in co-culture with P. kluyveri strain generated less sour and sweeter chocolate than spontaneous fermentation only. Chocolates from inoculated assays with starter cultures were more accepted by evaluators, highlighting that P. kluyveri and S. cerevisiae influence the composition of VOCs. Besides, protein profiles also changed throughout fermentation. Further investigation should be conducted to clarify protein degradation dynamics during inoculated fermentations to define which of the microbial cultures positively affect the chocolate sensory characteristics. © 2021 Society of Chemical Industry.

Yeast isolation and identification during on-farm cocoa natural fermentation in a highly producer region in northern Brazil

The cocoa seeds from Brazilian Amazon are recognized for the high international market value in addition to the desirable aroma and taste. We aimed to identify yeast cultures in a natural cocoa fermentation process in one of the Amazonian regions of great importance in the cocoa beans market. Natural fermentation was carried out for seven days according to the methodologies of the producer and at 24 h intervals, seed samples were collected and physical-chemical and microbiological analyzes were performed. The contents of lipids, proteins, ash and moisture did not differ (p ≥ 0.05) differently from temperature, pH and total titratable acidity (p ≤ 0.05). We identified three species in the fermentation: Pichia kudriavzevii, Torulaspora delbrueckii and Saccharomyces cerevisiae, the most common being during the process. We can verify the importance of knowing the microbiota active in cocoa fermentation to propose improvements during this process of great economic importance for the Amazon region.

Co-culturing fructophilic lactic acid bacteria and yeast enhanced sugar metabolism and aroma formation during cocoa beans fermentation

Glucose and fructose are the main fermentable sugars in cocoa pulp. During fermentation, glucose is consumed within 48–72 h and fructose only after 120 h, mainly associated with the preferential use of glucose by microorganisms. In the first stage of this study, the complete genome sequence of a lactic acid bacterium with high fructose consumption capacity (Lactobacillus plantarum LPBF35) was reported. The notable genomic features of L. plantarum LPBF35 were the presence of alcohol/acetaldehyde dehydrogenase gene and improved PTS system, confirming its classification as a “facultatively” fructophilic bacterium. Subsequently, this bacterium was introduced into cocoa fermentation process in single and mixed cultures with Pediococcus acidilactici LPBF66 or Pichia fermentans YC5.2. Community composition by Illumina-based amplicon sequencing and viable counts indicated suppression of wild microflora in all treatments. At the beginning of the fermentation processes, cocoa pulp consisted of approximately 73.09 mg/g glucose and 73.64 mg/g fructose. The L. plantarum LPBF35 + P. fermentans YC5.2 process showed the lowest levels of residual sugars after 72 h of fermentation (7.89 and 4.23 mg/g, for fructose and glucose, respectively), followed by L. plantarum LPBF35 + Ped. acidilactici LPBF66 (8.85 and 6.42 mg/g, for fructose and glucose, respectively), single L. plantarum LPBF35 treatment (4.15 and 10.15 mg/g, for fructose and glucose, respectively), and spontaneous process (22.25 and 14.60 mg/g, for fructose and glucose, respectively). The positive interaction between L. plantarum LPBF35 and P. fermentans YC5.2 resulted in an improved formation of primary (ethanol, lactic acid, and acetic acid) and secondary (2-methyl-1-butanol, isoamyl acetate, and ethyl acetate) metabolites during fermentation. The primary metabolites accumulated significantly in cocoa beans fermented by P. fermentans YC5.2 + L. plantarum LPBF35, causing important reactions of color development and key flavor molecules formation. The results of this study suggest that fructophilic lactic acid bacteria and yeast is a microbial consortium that could improve sugar metabolism and aroma formation during cocoa beans fermentation.

The Contribution of Fermentation Technology to the Value Added of Certification Cocoa in the Subak Abian Amerta Nadi Tuwed Village Melaya District Jembrana Regency

The development of cocoa as an export commodity has become an important issue in the global trade sector, where cocoa is one of the export commodities. The implementation of a sustainable certification program with fermentation processing is very important, this is because consumers' demands are increasingly critical, especially in the aspects of quality, health and product safety to how the product can be socially beneficial and not cause negative impacts on the environment.
 The study was conducted on sustainable certification cocoa farmers in Jembrana Regency. Determination of respondents carried out intentionally (purposive sampling) as many as 40 respondents did cocoa fermentation and as many as 30 respondents who did not ferment cocoa taken by survey methods. Data analysis techniques using different test analysis and value added analysis using the Hayami method. 
 The results showed that the income of cocoa farmers experienced a significant increase in the fermentation and non-fermentation processing. The fermentation production process with an output of 118 kg gets a profit of Rp. 8.911,- with a percentage or level of profit of 39,27 percent of the output value of Rp. 20.911, so from the calculation of profit margins in the cocoa fermentation process the net profit received by farmers is Rp. 8.065,- per production process. Based on the analysis of farmer's income and analysis of added value of cocoa fermentation shows that there is an increase in farmer's income and there is added value obtained by farmers who carry out cocoa fermentation, it provides benefits for cocoa farmers in Jembrana District in addition to increasing income, cocoa added value, and can maintain quality cocoa. It is expected that farmers who participate in certification will conduct fermentation processing to maintain the quality of cocoa beans and increase income, it is necessary to support from local and provincial governments as well as other parties as a steakholder providing technical guidance to cocoa farmers.

Effect of Addition of a Specific Mixture of Yeast, Lactic and Acetic Bacteria in the Fermentation Process to Improve the Quality and Flavor of Cocoa Beans in Colombia

Cocoa fermentation process is fundamental to generate flavors and aromas that are characteristics of chocolate. In Colombia, this process is carried out by microbiota that spontaneously colonizes cocoa beans, therefore the quality of the fermentation is inconsistent. Taking into account that the fermentation of cocoa beans is carried out by a consortium of microorganisms, the aim of thisresearch was to describe the effect of the addition of a specific mixture of yeasts, acetic acid bacteria, and lactic acid bacteria on the physicochemical and organolepticcharacteristics of cocoa beans (clone CCN 51). Isolates of two yeasts (Wickerhamomyces anomalus and Debaryomyces hansenii), three acetic acid bacteria (AAB), (Gluconobacter japonicus, Acetobacter tropicalis, and Acetobacter pasteurianus) and three lactic acid bacteria (LAB) (Pediococcus acidilactici, Lactobacillus brevis, and Lactobacillus plantarum) obtained fromprevious cocoa fermentations selected for their pectinases and acid production capacities in a specific mixture were used. Using the micro-fermentation technique, the effect of a biological starter was evaluated under different viable microorganismsratios (Yeasts: LAB: AAB as follows, 1: 1: 1, 1: 2: 2, 1: 2: 1, 1: 1: 2, 2: 1: 1, 2: 2: 1, 2: 1: 2, and 2: 2: 2). The concentration of each microorganism was standardized at 1x107 cfu/mL, then the biomass of 4 mL for ratio 1 and 8 mL for ratio 2 of each suspension of microorganisms was added at time zero. Different doses of inoculum were 0%, 1%, 2%, 3%, 4%, and 5% v/w mL inoculum/g cocoa beans. A beneficial effecton the sensory quality of cocoa beans was evidenced by the addition of microorganisms; the best proportion of microorganisms was 2:1:2 (yeasts:LAB:AAB) and the best inoculum dose was 3% (v/w) showing lower acidity, astringency, and bitterness, and emphasizing the cocoa flavors, fruity, nutty, and panela malt.

Genome-scale metabolic modeling of Acetobacter pasteurianus 386B reveals its metabolic adaptation to cocoa fermentation conditions

Acetobacter pasteurianus 386B has been selected as a candidate functional starter culture to better control the cocoa fermentation process. Previously, its genome has been sequenced and a genome-scale metabolic model (GEM) has been reconstructed. To understand its metabolic adaptation to cocoa fermentation conditions, different flux balance analysis (FBA) simulations were performed and compared with experimental data. In particular, metabolic flux distributions were simulated for two phases that characterize the growth of A. pasteurianus 386B under cocoa fermentation conditions, predicting a switch in respiratory chain usage in between these phases. The possible influence on the resulting energy production was shown using a reduced version of the GEM. FBA simulations revealed the importance of the compartmentalization of the ethanol oxidation reactions, namely in the periplasm or in the cytoplasm, and highlighted the potential role of ethanol as a source of carbon, energy, and NADPH. Regarding the latter, the physiological function of a proton-translocating NAD(P)+ transhydrogenase was further investigated in silico. This study revealed the potential of using a GEM to simulate the metabolism of A. pasteurianus 386B, and may provide a general framework toward a better physiological understanding of functional starter cultures in food fermentation processes.

Functional Biodiversity of Yeasts Isolated from Colombian Fermented and Dry Cocoa Beans.

Yeasts play an important role in the cocoa fermentation process. Although the most relevant function is the degradation of sugars and the production of ethanol, there is little understanding of the enzyme activities and attributes that allow them to survive even after drying. The present study explored the functional biodiversity of yeasts associated with Criollo Colombian cocoa fermented beans, able to survive after drying. Twelve species belonging to 10 genera of osmo-, acid-, thermo-, and desiccation-tolerant yeasts were isolated and identified from fermented and dry cocoa beans, with Pichia kudriavzevii and Saccharomyces cerevisiae standing out as the most frequent. For the first time, we reported the presence of Zygosaccharomyces bisporus in cocoa fermented beans. It was found that resistance to desiccation is related to the different degradation capacities of fermentation substrates, which suggests that associative relationships may exist between the different yeast species and their degradation products. Besides, the increased thermotolerance of some species was related to the presence of polyphenols in the medium, which might play a fundamental role in shaping the microbial community composition.

Exploring the contribution of fructophilic lactic acid bacteria to cocoa beans fermentation: Isolation, selection and evaluation

Fructophilic lactic acid bacteria (FLAB) are a recently discovered group whose main characteristic is to prefer D-fructose over D-glucose. In this study, laboratory cocoa beans fermentation was analyzed by Illumina-based amplicon sequencing, indicating the presence of potential FLAB of the genera Fructobacillus and Lactobacillus. Eighty efficient fructose-fermenting isolates, obtained from fermenting cocoa pulp beans mass, were identified by 16S rRNA gene sequencing as Pediococcus acidilactici (n = 52), Lactobacillus plantarum (n = 10), Pediococcus pentosaceus (n = 10), Bacillus subtilis (n = 4), and Leuconostoc pseudomesenteroides (n = 4). The growth characteristics of all the 10 L. plantarum strains classified them as “facultatively” fructophilic bacteria, i.e., they grew on glucose without an external electron acceptor but the growth on fructose was faster. Among them, L. plantarum LPBF 35 was characterized by producing a range of aroma-impacting compounds (acetaldehyde, ethyl acetate, nonanal, and octanoic acid), being introduced into a cocoa fermentation process. Although the process started with approximately equal amounts of glucose and fructose, a concomitant, but faster utilization of fructose, was observed in cocoa fermentation conducted with L. plantarum LPBF 35 (with no residual fructose observed) when compared to control fermentation using a glucophilic strain (8.77 mg/g residual fructose) and a spontaneous process (8.38 mg/g residual fructose). L. plantarum LPBF 35 also showed an ideal profile of organic acid metabolism (citric acid consumption and lactic acid production) associated with cocoa fermentation. These results proved new insights on cocoa microbial activity and brings new perspectives on the use of lactic acid bacteria as starter culture.

Microorganisms during cocoa fermentation: systematic review

Introduction. Cocoa (Theobroma cacao L.) originates from Ecuador. It is one of the oldest foods in the world. The fact that cocoa is the main component in chocolate industry makes it one of the most quoted raw materials today. The chemical, physical, microbiological, and sensory properties of cocoa determine its quality and, as a result, economic and nutritional value. The research objective was to conduct a detailed analysis of cocoa fermentation process and to study the transformations this raw material is subjected to during processing.
 Study objects and methods. The present article introduces a substantial bibliographic review based on three databases: Science Direct, Scopus, and Medline. The scientific publications were selected according to several factors. First, they had to be relevant in terms of cocoa fermentation. Second, they were written in English or Spanish. Third, the papers were indexed in high-impact journals. The initial selection included 350 articles, while the final list of relevant publications featured only 50 works that met all the requirements specified above.
 Results and discussion. The main characteristics of yeasts, lactic bacteria, and acetic bacteria were analyzed together with their main parameters to describe their activities during different stages of alcoholic, lactic, and acetic fermentation. A thorough analysis of the main enzyme-related processes that occur during fermentation makes it possible to optimize the use of substrates, temperature, time, pH, acidity, and nutrients. As a result, the finished product contains an optimal concentration of volatile compounds that are formed in the beans during fermentation. The study featured the main strains of fermentation-related microorganisms, their activities, main reactions, and products.
 Conclusion. This study makes it possible to improve the process of fermentation to obtain beans with a better chemical composition.

Effect of aroma potential of Saccharomyces cerevisiae fermentation on the volatile profile of raw cocoa and sensory attributes of chocolate produced thereof

This research describes on-farm-inoculated cocoa bean fermentation with two selected strains of high aroma-producing Saccharomyces cerevisiae isolated from wine fermentation and their effects on volatile profile of resulted raw cocoa material and sensory characteristics of chocolates produced. Both strains produced suitably flavour-active esters, aldehydes, ketones and pyrazines in raw cocoa. Inoculation of S. cerevisiae at 0.5 g kg−1 significantly improved the volatile profile of raw cocoa beans and enabled production of chocolate with enhanced intense fresh fruits and floral notes. Chocolate produced thereof presented better overall quality attributed whatever the fermentation time in comparison to the chocolate issued from spontaneous fermentation. Our results suggested the ways of using selected enhanced yeasts as S. cerevisiae for on-farm implementation of bean fermentation as promising alternative possibilities to improve the cocoa fermentation process, desirable flavourful raw cocoa material and high-aroma-quality chocolates produced.

Antifungal activity of yeasts and lactic acid bacteria isolated from cocoa bean fermentations

The aim of this study was to select yeast and LAB strains to be used as a mean of biological control against fungal growth during cocoa fermentation process. Their antifungal activity was assessed against six spoilage fungi isolated from both fermented and dried cocoa beans and belonging to Aspergillus and Penicillium genera. An initial screening was carried out by using the overlay method where the plates were examined for fungi inhibition zone around LAB and yeast streaks. Then, the most active strains were studied in inhibition test in 96-well microplates where mould growth was measured by microplate reader at 490 nm. The nature of their antifungal strenght (organic acid and/or proteins) was also evaluated. The most promising candidates as biological agents belonged to the species Lactobacillus fermentum, Lactobacillus plantarum, Saccharomyces cerevisiae and Candida ethanolica and their antifungal strength was attributed mainly to organic acid production (for LAB) and proteinaceous compounds (for yeasts) or to their synergic effect.