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

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Water kefir is a sour, alcoholic, and fruity fermented beverage of which the fermentation is started with water kefir grains. These water kefir grains consist of polysaccharide and contain the microorganisms responsible for the water kefir fermentation. In this work, a water kefir fermentation process was followed as a function of time during 192 h to unravel the community dynamics, the species diversity, and the kinetics of substrate consumption and metabolite production. The majority of the water kefir ecosystem was found to be present on the water kefir grains. The most important microbial species present were Lactobacillus casei/ paracasei, Lactobacillus harbinensis, Lactobacillus hilgardii, Bifidobacterium psychraerophilum/crudilactis, Saccharomyces cerevisiae, and Dekkera bruxellensis. The microbial species diversities in the water kefir liquor and on the water kefir grains were similar and remained stable during the whole fermentation process. The major substrate, sucrose, was completely converted after 24 h of fermentation, which coincided with the production of the major part of the water kefir grain polysaccharide. The main metabolites of the fermentation were ethanol and lactic acid. Glycerol, acetic acid, and mannitol were produced in low concentrations. The major part of these metabolites was produced during the first 72 h of fermentation, during which the pH decreased from 4.26 to 3.45. The most prevalent volatile aroma compounds were ethyl acetate, isoamyl acetate, ethyl hexanoate, ethyl octanoate, and ethyl decanoate, which might be of significance with respect to the aroma of the end product.

Section: Methodsmentioning

confidence: 99%

Microbial Species Diversity, Community Dynamics, and Metabolite Kinetics of Water Kefir Fermentation

Laureys

Vuyst

2014

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178

205

“…Their succession during cocoa bean fermentation is important for the required metabolic activities in the fermenting cocoa pulp-bean mass, although the occurrence of these microbial communities may overlap (2,3,(5)(6)(7). Yeast species involved in cocoa bean fermentation mainly encompass Saccharomyces cerevisiae and Hanseniaspora guilliermondii, H. opuntiae, and H. uvarum, which are responsible for pectinolysis of the cocoa pulp and the production of ethanol from carbohydrates (mainly glucose) (8)(9)(10)(11)(12)(13)(14)(15). LAB species involved in cocoa bean fermentation, in particular Lactobacillus fermentum, consume carbohydrates (glucose and fructose) and citric acid and produce lactic acid, acetic acid, and mannitol (6,7,12,(16)(17)(18)(19).…”

mentioning

confidence: 99%

“…LAB species involved in cocoa bean fermentation, in particular Lactobacillus fermentum, consume carbohydrates (glucose and fructose) and citric acid and produce lactic acid, acetic acid, and mannitol (6,7,12,(16)(17)(18)(19). Concerning AAB, mainly Acetobacter species are found, which oxidize ethanol (produced by yeast) and lactic acid (produced by LAB) into acetic acid (6,7,12,15,17,18,20,21).…”

mentioning

confidence: 99%

Oxidation of Metabolites Highlights the Microbial Interactions and Role of Acetobacter pasteurianus during Cocoa Bean Fermentation

Moens

Lefeber

Vuyst

2014

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102

Four cocoa-specific acetic acid bacterium (AAB) strains, namely, Acetobacter pasteurianus 386B, Acetobacter ghanensis LMG 23848 T , Acetobacter fabarum LMG 24244 T , and Acetobacter senegalensis 108B, 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 only A. pasteurianus 386B performed a fast oxidation of ethanol and lactic acid into acetic acid and acetoin, respectively. Only A. pasteurianus 386B and A. ghanensis LMG 23848 T oxidized mannitol into fructose. Coculture fermentations with A. pasteurianus 386B or A. ghanensis LMG 23848T and Lactobacillus fermentum 222 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 of A. pasteurianus 386B. A triculture fermentation with Saccharomyces cerevisiae H5S5K23, L. fermentum 222, and A. pasteurianus 386B, 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 that A. pasteurianus 386B 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 for A. pasteurianus in its indispensable crossfeeding interactions with yeast and LAB during cocoa bean fermentation.

“…Acetate then diffuses into the beans (2)(3)(4), where it initiates a cascade of chemical and biochemical reactions leading to precursor molecules for cocoa flavor (2,5,6). Potential substrates for AAB are lactate and ethanol, which are individually produced by lactic acid bacteria (LAB; mainly Lactobacillus fermentum) and yeasts (diverse yeasts such as Saccharomyces cerevisiae, Hanseniaspora opuntiae, and Candida krusei), respectively, during the fermentation process (6)(7)(8)(9)(10)(11)(12). Hereby, the degradation of lactate by AAB is desired, since the remaining lactate may provide an off flavor in the final cocoa product (11,13,14).…”

mentioning

confidence: 99%

“…Obviously, the most prevalent AAB species is Acetobacter pasteurianus (13,(15)(16)(17). In addition, Acetobacter ghanensis and Acetobacter senegalensis are found during spontaneous cocoa bean fermentation (9,13,17,18). Further studies provided first insights into the basic microbiological properties of such strains and macroscopic dynamics during cocoa pulp fermentation (12,15,(18)(19)(20).…”

mentioning

confidence: 99%

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

Adler

Frey

Berger

et al. 2014

c Acetic 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 parallel 13 C 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.A cetic acid bacteria (AAB) play an important role in cocoa fermentation (1). During fermentation of the pulp that surrounds the cocoa beans, they form acetate. Acetate then diffuses into the beans (2-4), where it initiates a cascade of chemical and biochemical reactions leading to precursor molecules for cocoa flavor (2, 5, 6). Potential substrates for AAB are lactate and ethanol, which are individually produced by lactic acid bacteria (LAB; mainly Lactobacillus fermentum) and yeasts (diverse yeasts such as Saccharomyces cerevisiae, Hanseniaspora opuntiae, and Candida krusei), respectively, during the fermentation process (6-12). Hereby, the degradation of lactate by AAB is desired, since the remaining lactate may provide an off flavor in the final cocoa product (11,13,14). In recent years, AAB have been extensively analyzed for their contribution to cocoa fermentation. Obviously, the most prevalent AAB species is Acetobacter pasteurianus (13,(15)(16)(17). In addition, Acetobacter ghanensis and Acetobacter senegalensis are found during spontaneous cocoa bean fermentation (9,13,17,18). Further studies provided first insights into the basic microbiological properties of such strains and macroscopic dynamics during cocoa pulp fermentation (12,15,(18)(19)(20). At this point, it appears to be relevant to resolve the metabolic contribution of AAB in greater detail. The basic know...