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

Abstract

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...

Metabolic cross-feeding allows a gut microbial community to overcome detrimental diets and alter host behaviour

“…Furthermore the AA labelling in the 24h and 48h samples showed very similar patterns as the one detected in Ap and Lp co-cultures using labelled glucose. Our results show that Ap synthesizes multiple AAs from lactate and are in agreement with earlier reports that lactate can serve as an important precursor for AA synthesis in other Acetobacteraceae (Adler et al, 2014). Interestingly, the average amount of 13 C-Ile synthesized from 13 C 3 -lactate was similar to that measured in the co-cultures of Ap and Lp synthesized from 13 C-glucose ( Figure 3 and 5 and Figure S5, S7 and S8), enforcing the similarity between the co-culture condition and the lactate Ap condition.…”

“…Concomitantly, this interaction also spurs the growth of Ap as this bacterium grows better in the fly in the presence of Lp. This positive effect of Lp on AA synthesis by Ap is best explained by the preferential use of lactate by Ap for the production of AAs (Adler et al, 2014) which coincidentally is one of the main metabolic byproducts produced and secreted by Lp. These metabolic interactions within the Ap/Lp community allow these two bacteria to create a "circular economy" in which they both optimally use the available nutritional resources provided by the host diet, allowing them both to overcome detrimental host diets and boosting their metabolic output.…”

Microbial acidification, alcoholization, and aroma production during spontaneous lambic beer production

“…The AAB metabolism irreversibly switches to overoxidation only when ethanol is depleted, as this metabolic pathway is repressed in the presence of ethanol . During cocoa bean fermentation, it has been shown that AAB can convert lactic acid into acetoin, which may be of benefit in lambic beer production too, as acetoin may contribute to the lambic beer aroma . The AAB also possess intracellular esterases, catalyzing the condensation of acetic acid and ethanol into ethyl acetate, a common ester of lambic beers …”

“…105,106 During cocoa bean fermentation, it has been shown that AAB can convert lactic acid into acetoin, which may be of benefit in lambic beer production too, as acetoin may contribute to the lambic beer aroma. 69,107,108 The AAB also possess intracellular esterases, catalyzing the condensation of acetic acid and ethanol into ethyl acetate, a common ester of lambic beers. 109 Brettanomyces species Brettanomyces yeasts characterize the fourth phase of the lambic beer production process (after eight months of fermentation/maturation; Fig.…”

State‐of‐the‐Art Chocolate Manufacture: A Review

“…For example, AAB can generate acetic acid through the direct metabolism of sugars (Swiegers and others ). According to Adler and others (), the formation of acetate is key in the development of desirable flavors in chocolate.…”