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

AbstractCocoa 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.

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Microbiological and Physicochemical Characterization of Small-Scale Cocoa Fermentations and Screening of Yeast and Bacterial Strains To Develop a Defined Starter Culture

Applied and Environmental Microbiology ◽

10.1128/aem.01144-12 ◽

2012 ◽

Vol 78 (15) ◽

pp. 5395-5405 ◽

Cited By ~ 86

Author(s):

Gilberto Vinícius de Melo Pereira ◽

Maria Gabriela da Cruz Pedrozo Miguel ◽

Cíntia Lacerda Ramos ◽

Rosane Freitas Schwan

Keyword(s):

Acetic Acid ◽

Lactic Acid ◽

Citric Acid ◽

Dominant Species ◽

Starter Culture ◽

Physicochemical Characterization ◽

Cocoa Bean ◽

Small Scale ◽

Content Type ◽

Cocoa Bean Fermentation

ABSTRACTSpontaneous cocoa bean fermentations performed under bench- and pilot-scale conditions were studied using an integrated microbiological approach with culture-dependent and culture-independent techniques, as well as analyses of target metabolites from both cocoa pulp and cotyledons. Both fermentation ecosystems reached equilibrium through a two-phase process, starting with the simultaneous growth of the yeasts (withSaccharomyces cerevisiaeas the dominant species) and lactic acid bacteria (LAB) (Lactobacillus fermentumandLactobacillus plantarumwere the dominant species), which were gradually replaced by the acetic acid bacteria (AAB) (Acetobacter tropicaliswas the dominant species). In both processes, a sequence of substrate consumption (sucrose, glucose, fructose, and citric acid) and metabolite production kinetics (ethanol, lactic acid, and acetic acid) similar to that of previous, larger-scale fermentation experiments was observed. The technological potential of yeast, LAB, and AAB isolates was evaluated using a polyphasic study that included the measurement of stress-tolerant growth and fermentation kinetic parameters in cocoa pulp media. Overall, strainsL. fermentumUFLA CHBE8.12 (citric acid fermenting, lactic acid producing, and tolerant to heat, acid, lactic acid, and ethanol),S. cerevisiaeUFLA CHYC7.04 (ethanol producing and tolerant to acid, heat, and ethanol), andAcetobacter tropicalisUFLA CHBE16.01 (ethanol and lactic acid oxidizing, acetic acid producing, and tolerant to acid, heat, acetic acid, and ethanol) were selected to form a cocktail starter culture that should lead to better-controlled and more-reliable cocoa bean fermentation processes.

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Dynamics and Biodiversity of Populations of Lactic Acid Bacteria and Acetic Acid Bacteria Involved in Spontaneous Heap Fermentation of Cocoa Beans in Ghana

Applied and Environmental Microbiology ◽

10.1128/aem.02189-06 ◽

2007 ◽

Vol 73 (6) ◽

pp. 1809-1824 ◽

Cited By ~ 187

Author(s):

Nicholas Camu ◽

Tom De Winter ◽

Kristof Verbrugghe ◽

Ilse Cleenwerck ◽

Peter Vandamme ◽

...

Keyword(s):

Acetic Acid ◽

Lactic Acid ◽

Lactic Acid Bacteria ◽

Fermentation Process ◽

Denaturing Gradient Gel Electrophoresis ◽

Acetic Acid Bacteria ◽

Cocoa Bean ◽

Rrna Gene ◽

Cocoa Bean Fermentation ◽

Leuconostoc Pseudomesenteroides

ABSTRACT The Ghanaian cocoa bean heap fermentation process was studied through a multiphasic approach, encompassing both microbiological and metabolite target analyses. A culture-dependent (plating and incubation, followed by repetitive-sequence-based PCR analyses of picked-up colonies) and culture-independent (denaturing gradient gel electrophoresis [DGGE] of 16S rRNA gene amplicons, PCR-DGGE) approach revealed a limited biodiversity and targeted population dynamics of both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during fermentation. Four main clusters were identified among the LAB isolated: Lactobacillus plantarum, Lactobacillus fermentum, Leuconostoc pseudomesenteroides, and Enterococcus casseliflavus. Other taxa encompassed, for instance, Weissella. Only four clusters were found among the AAB identified: Acetobacter pasteurianus, Acetobacter syzygii-like bacteria, and two small clusters of Acetobacter tropicalis-like bacteria. Particular strains of L. plantarum, L. fermentum, and A. pasteurianus, originating from the environment, were well adapted to the environmental conditions prevailing during Ghanaian cocoa bean heap fermentation and apparently played a significant role in the cocoa bean fermentation process. Yeasts produced ethanol from sugars, and LAB produced lactic acid, acetic acid, ethanol, and mannitol from sugars and/or citrate. Whereas L. plantarum strains were abundant in the beginning of the fermentation, L. fermentum strains converted fructose into mannitol upon prolonged fermentation. A. pasteurianus grew on ethanol, mannitol, and lactate and converted ethanol into acetic acid. A newly proposed Weissella sp., referred to as “Weissella ghanaensis,” was detected through PCR-DGGE analysis in some of the fermentations and was only occasionally picked up through culture-based isolation. Two new species of Acetobacter were found as well, namely, the species tentatively named“ Acetobacter senegalensis” (A. tropicalis-like) and “Acetobacter ghanaensis” (A. syzygii-like).

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

Pelita Perkebunan (a Coffee and Cocoa Research Journal) ◽

10.22302/iccri.jur.pelitaperkebunan.v36i3.443 ◽

2020 ◽

Vol 36 (3) ◽

pp. 236-248

Author(s):

Maria Denis Lozano Tovar ◽

Geraldine Tibasosa ◽

Carlos Mario González ◽

Karen Ballestas Alvarez ◽

Martha Del Pilar Lopez Hernandez ◽

...

Keyword(s):

Acetic Acid ◽

Starter Culture ◽

Cocoa Bean ◽

Rrna Gene ◽

Cocoa Beans ◽

Isolation And Identification ◽

Pichia Kudriavzevii ◽

Cocoa Bean Fermentation ◽

Cocoa Fermentation

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.

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Metagenome-assembled genomes contributes to unravel the microbiome of cocoa fermentation

Applied and Environmental Microbiology ◽

10.1128/aem.00584-21 ◽

2021 ◽

Author(s):

O.G.G. Almeida ◽

E.C.P De Martinis

Keyword(s):

Acetic Acid ◽

Lactic Acid ◽

Lactic Acid Bacteria ◽

Acetic Acid Bacteria ◽

Raw Material ◽

Microbial Succession ◽

Metabolic Potential ◽

Short Reads ◽

Leuconostoc Pseudomesenteroides ◽

Cocoa Fermentation

Metagenomic studies about cocoa fermentation have mainly reported on the analysis of short reads for determination of Operational Taxonomic Units. However, it is also important to determine MAGs, which are genomes deriving from the assembly of metagenomics. For this research, all the cocoa metagenomes from public databases were downloaded, resulting in five datasets: one from Ghana and four from Brazil. Besides, in silico approaches were used to describe putative phenotypes and metabolic potential of MAGs. A total of 17 high-quality MAGs were recovered from these microbiomes, as follows: (i) fungi - Yamadazyma tenuis (n=1); (ii) lactic acid bacteria - Limosilactobacillus fermentum (n=5), Liquorilactobacillus cacaonum (n=1) , Liquorilactobacillus nagelli (n=1), Leuconostoc pseudomesenteroides (n=1) and Lactiplantibacillus plantarum subsp. plantarum (n=1); (iii) acetic acid bacteria - Acetobacter senegalensis (n=2) and Kozakia baliensis (n=1) and (iv) Bacillus subtilis (n=1) Brevundimonas sp. (n=2) and Pseudomonas sp. (n=1). Medium-quality MAGs were also recovered from cocoa microbiomes, including some detected for the first time in this environment ( Liquorilactobacillus vini , Komagataeibacter saccharivorans and Komagataeibacter maltaceti ) and other previously described ( Fructobacillus pseudoficulneus and Acetobacter pasteurianus ). Taken all together, the MAGs were useful to provide an additional description of the microbiome of cocoa fermentation, revealing previously overlooked microorganisms, with prediction of key phenotypes and biochemical pathways. Importance The production of chocolate starts with the harvesting of cocoa fruits and the spontaneous fermentation of the seeds, in a microbial succession that depends on yeasts, lactic acid bacteria and acetic acid bacteria in order to eliminate bitter and astringent compounds present in the raw material, which will be further roasted and grinded to originate the cocoa powder that will enter the food processing industry. The microbiota of cocoa fermentation is not completely know, and yet it advanced from culture-based studies to the advent of Next Generation DNA sequencing, with the generation of a myriad of data, that need bioinformatic approaches to be properly analysed. Although the majority metagenomic of studies have been based on short reads (OTUs), it is also important to analyse entire genomes to determine more precisely possible ecological roles of different species. Metagenome-assembled genomes (MAGs) are very useful for this purpose, and in this paper, MAGs from cocoa fermentation microbiomes were described, as well the possible implications of their phenotypic and metabolic potentials are discussed.

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Oxidation of Metabolites Highlights the Microbial Interactions and Role ofAcetobacter pasteurianusduring Cocoa Bean Fermentation

Applied and Environmental Microbiology ◽

10.1128/aem.03344-13 ◽

2014 ◽

Vol 80 (6) ◽

pp. 1848-1857 ◽

Cited By ~ 64

Author(s):

Frédéric Moens ◽

Timothy Lefeber ◽

Luc De Vuyst

Keyword(s):

Acetic Acid ◽

Lactic Acid ◽

Starter Culture ◽

Acetic Acid Bacterium ◽

Microbial Interactions ◽

Cocoa Bean ◽

Acid Oxidation ◽

Functional Roles ◽

Cocoa Bean Fermentation ◽

Oxidation Of Ethanol

ABSTRACTFour cocoa-specific acetic acid bacterium (AAB) strains, namely,Acetobacter pasteurianus386B,Acetobacter ghanensisLMG 23848T,Acetobacter fabarumLMG 24244T, andAcetobacter senegalensis108B, were analyzed kinetically and metabolically during monoculture laboratory fermentations. A cocoa pulp simulation medium (CPSM) for AAB, containing ethanol, lactic acid, and mannitol, was used. All AAB strains differed in their ethanol and lactic acid oxidation kinetics, whereby onlyA. pasteurianus386B performed a fast oxidation of ethanol and lactic acid into acetic acid and acetoin, respectively. OnlyA. pasteurianus386B andA. ghanensisLMG 23848Toxidized mannitol into fructose. Coculture fermentations withA. pasteurianus386B orA. ghanensisLMG 23848TandLactobacillus fermentum222 in CPSM for lactic acid bacteria (LAB) containing glucose, fructose, and citric acid revealed oxidation of lactic acid produced by the LAB strain into acetic acid and acetoin that was faster in the case ofA. pasteurianus386B. A triculture fermentation withSaccharomyces cerevisiaeH5S5K23,L. fermentum222, andA. pasteurianus386B, using CPSM for LAB, showed oxidation of ethanol and lactic acid produced by the yeast and LAB strain, respectively, into acetic acid and acetoin. Hence, acetic acid and acetoin are the major end metabolites of cocoa bean fermentation. All data highlight thatA. pasteurianus386B displayed beneficial functional roles to be used as a starter culture, namely, a fast oxidation of ethanol and lactic acid, and that these metabolites play a key role as substrates forA. pasteurianusin its indispensable cross-feeding interactions with yeast and LAB during cocoa bean fermentation.

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Interesting Starter Culture Strains for Controlled Cocoa Bean Fermentation Revealed by Simulated Cocoa Pulp Fermentations of Cocoa-Specific Lactic Acid Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.00594-11 ◽

2011 ◽

Vol 77 (18) ◽

pp. 6694-6698 ◽

Cited By ~ 33

Author(s):

Timothy Lefeber ◽

Maarten Janssens ◽

Frédéric Moens ◽

William Gobert ◽

Luc De Vuyst

Keyword(s):

Acetic Acid ◽

Lactic Acid ◽

Citric Acid ◽

Lactic Acid Bacteria ◽

Starter Culture ◽

Lactobacillus Fermentum ◽

Cocoa Bean ◽

Bacterial Strains ◽

Content Type ◽

Cocoa Bean Fermentation

ABSTRACTAmong various lactic acid bacterial strains tested, cocoa-specific strains ofLactobacillus fermentumwere best adapted to the cocoa pulp ecosystem. They fermented glucose to lactic acid and acetic acid, reduced fructose to mannitol, and converted citric acid into lactic acid and 2,3-butanediol.

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The Key to Acetate: Metabolic Fluxes of Acetic Acid Bacteria under Cocoa Pulp Fermentation-Simulating Conditions

Applied and Environmental Microbiology ◽

10.1128/aem.01048-14 ◽

2014 ◽

Vol 80 (15) ◽

pp. 4702-4716 ◽

Cited By ~ 50

Author(s):

Philipp Adler ◽

Lasse Jannis Frey ◽

Antje Berger ◽

Christoph Josef Bolten ◽

Carl Erik Hansen ◽

...

Keyword(s):

Acetic Acid ◽

Lactic Acid ◽

Lactic Acid Bacteria ◽

Phosphoenolpyruvate Carboxykinase ◽

Isotope Labeling ◽

Acetic Acid Bacteria ◽

Building Blocks ◽

Conversion Rates ◽

Cellular Energetics ◽

Cocoa Fermentation

ABSTRACTAcetic acid bacteria (AAB) play an important role during cocoa fermentation, as their main product, acetate, is a major driver for the development of the desired cocoa flavors. Here, we investigated the specialized metabolism of these bacteria under cocoa pulp fermentation-simulating conditions. A carefully designed combination of parallel13C isotope labeling experiments allowed the elucidation of intracellular fluxes in the complex environment of cocoa pulp, when lactate and ethanol were included as primary substrates among undefined ingredients. We demonstrate that AAB exhibit a functionally separated metabolism during coconsumption of two-carbon and three-carbon substrates. Acetate is almost exclusively derived from ethanol, while lactate serves for the formation of acetoin and biomass building blocks. Although this is suboptimal for cellular energetics, this allows maximized growth and conversion rates. The functional separation results from a lack of phosphoenolpyruvate carboxykinase and malic enzymes, typically present in bacteria to interconnect metabolism. In fact, gluconeogenesis is driven by pyruvate phosphate dikinase. Consequently, a balanced ratio of lactate and ethanol is important for the optimum performance of AAB. As lactate and ethanol are individually supplied by lactic acid bacteria and yeasts during the initial phase of cocoa fermentation, respectively, this underlines the importance of a well-balanced microbial consortium for a successful fermentation process. Indeed, AAB performed the best and produced the largest amounts of acetate in mixed culture experiments when lactic acid bacteria and yeasts were both present.

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Fermentation of Cocoa Beans

10.5772/intechopen.98756 ◽

2021 ◽

Author(s):

Romel E. Guzmán-Alvarez ◽

José G. Márquez-Ramos

Keyword(s):

Acetic Acid ◽

Scientific Information ◽

Acetic Acid Bacteria ◽

Cocoa Bean ◽

Microbial Succession ◽

Cocoa Bean Fermentation ◽

Wide Range ◽

The World ◽

Flavor Precursors ◽

Cocoa Fermentation

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.

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

Fermentation ◽

10.3390/fermentation7010042 ◽

2021 ◽

Vol 7 (1) ◽

pp. 42

Author(s):

Lucie Farrera ◽

Alexandre Colas de la Noue ◽

Caroline Strub ◽

Benjamin Guibert ◽

Christelle Kouame ◽

...

Keyword(s):

Acetic Acid ◽

Lactic Acid ◽

Acid Production ◽

Acetic Acid Bacteria ◽

Intraspecific Diversity ◽

Cocoa Bean ◽

Acetic Acid Production ◽

Cocoa Bean Fermentation ◽

The Impact ◽

Selection Of

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.

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

Fermentation ◽

10.3390/fermentation7030192 ◽

2021 ◽

Vol 7 (3) ◽

pp. 192

Author(s):

Endang Sutriswati Rahayu ◽

Rokhmat Triyadi ◽

Rosyida N. B. Khusna ◽

Titiek Farianti Djaafar ◽

Tyas Utami ◽

...

Keyword(s):

Acetic Acid ◽

Lactic Acid ◽

Lactic Acid Bacteria ◽

Ochratoxin A ◽

Lactobacillus Plantarum ◽

Acetic Acid Bacteria ◽

Starter Cultures ◽

Cocoa Bean ◽

Cocoa Bean Fermentation ◽

Candida Famata

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.

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