Microbiological and physicochemical characterization of small-scale cocoa fermentations and screening of yeast and bacterial strains to develop a defined starter culture.
ABSTRACT: Spontaneous 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 (with Saccharomyces cerevisiae as the dominant species) and lactic acid bacteria (LAB) (Lactobacillus fermentum and Lactobacillus plantarum were the dominant species), which were gradually replaced by the acetic acid bacteria (AAB) (Acetobacter tropicalis was 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, strains L. fermentum UFLA CHBE8.12 (citric acid fermenting, lactic acid producing, and tolerant to heat, acid, lactic acid, and ethanol), S. cerevisiae UFLA CHYC7.04 (ethanol producing and tolerant to acid, heat, and ethanol), and Acetobacter tropicalis UFLA 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.
Project description:Traditional fermentations of the local Ecuadorian cocoa type Nacional, with its fine flavor, are carried out in boxes and on platforms for a short time. A multiphasic approach, encompassing culture-dependent and -independent microbiological analyses of fermenting cocoa pulp-bean samples, metabolite target analyses of both cocoa pulp and beans, and sensory analysis of chocolates produced from the respective fermented dry beans, was applied for the investigation of the influence of these fermentation practices on the yeast and bacterial species diversity and community dynamics during cocoa bean fermentation. A wide microbial species diversity was found during the first 3 days of all fermentations carried out. The prevailing ethanol-producing yeast species were Pichia kudriavzevii and Pichia manshurica, followed by Saccharomyces cerevisiae. Leuconostoc pseudomesenteroides (glucose and fructose fermenting), Fructobacillus tropaeoli-like (fructose fermenting), and Lactobacillus fermentum (citrate converting, mannitol producing) represented the main lactic acid bacterial species in the fermentations studied, resulting in intensive heterolactate metabolism of the pulp substrates. Tatumella saanichensis and Tatumella punctata were among the members of the family Enterobacteriaceae present during the initial phase of the cocoa bean fermentations and could be responsible for the production of gluconic acid in some cases. Also, a potential new yeast species was isolated, namely, Candida sorbosivorans-like. Acetic acid bacteria, whose main representative was Acetobacter pasteurianus, generally appeared later during fermentation and oxidized ethanol to acetic acid. However, acetic acid bacteria were not always present during the main course of the platform fermentations. All of the data taken together indicated that short box and platform fermentation methods caused incomplete fermentation, which had a serious impact on the quality of the fermented dry cocoa beans.
Project description: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 13C 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.
Project description:The growth of filamentous fungi during the spontaneous cocoa bean fermentation leads to inferior cocoa bean quality and poses a health risk for consumers due to the potential accumulation of mycotoxins. We recently developed anti-fungal cultures with the capacity to inhibit the growth of mycotoxigenic filamentous fungi on cocoa beans. However, it is not clear how these anti-fungal cultures affect the fermentation process and cocoa bean quality. For that, the anti-fungal co-cultures, Lactobacillus fermentum M017-Saccharomyces cerevisiae H290 (A) and Lb. fermentum 223-S. cerevisiae H290 (B), were applied to 180-kg box fermentations in Honduras in three time-independent replications each including a spontaneous control fermentation. The comparison of inoculated and spontaneous fermentation processes revealed that the co-cultures only marginally affected the fermentation process and cocoa bean quality. Microorganisms reached maximal levels of 6.2-7.6 log CFU/g of yeasts and acetic acid bacteria and 7.9-9.5 log CFU/g of lactic acid bacteria during all fermentations and led to maximal metabolite concentrations in bean cotyledons of 4-12 mg/g ethanol, 2-6 mg/g lactic acid and 6-14 mg/g acetic acid. The fermentation and drying processes resulted in 38-90 mg epicatechin equivalents/g in the cotyledons of dried beans. However, the co-cultures led to up to ten times higher mannitol levels in cotyledons of inoculated beans compared to beans during spontaneous fermentation, and caused a slower fermentation process, detectable as up to 8-12 °C lower temperatures in the centre of the fermenting pulp-bean mass and up to 22% lower proportions of well-fermented beans after drying. Co-culture B-with Lb. fermentum 223 -led to improved cocoa bean quality compared to co-culture A-with Lb. fermentum M017 -, i.e. cocoa beans with 0.5-1.9 mg/g less acetic acid, 4-17% higher shares of well-fermented beans and, on a scale from 0 to 10, to 0.2-0.6 units lower astringency, up to 1.1 units lower off-flavours, and 0.2-0.9 units higher cocoa notes. Therefore, the anti-fungal co-culture B is recommended for future applications and its capacity to limit fungal growth and mycotoxin production during industrial-scale cocoa bean fermentation should be investigated in further studies.
Project description:Microbial strains for starter culture-initiated sourdough productions are commonly isolated from a fermenting flour-water mixture. Yet, starter culture strains isolated from matrices other than sourdoughs could provide the dough with interesting metabolic properties and hence change the organoleptic properties of the concomitant breads. Furthermore, the selection of sourdough starter cultures does not need to be limited to lactic acid bacteria (LAB), as other food-grade microorganisms are sometimes found in sourdoughs. Therefore, different strains belonging to LAB, acetic acid bacteria (AAB), and coagulase-negative staphylococci (CNS) that originated from different fermented food matrices (fermenting cocoa pulp-bean mass, fermented sausage, and water kefir), were examined as to their prevalence in a wheat sourdough ecosystem during 72-h fermentations. Limosilactobacillus fermentum IMDO 222 (fermented cocoa pulp-bean mass isolate) and Latilactobacillus sakei CTC 494 (fermented sausage isolate) seemed to be promising candidates as sourdough starter culture strains, as were the AAB strains Acetobacter pasteurianus IMDO 386B and Gluconobacter oxydans IMDO A845 (both isolated from fermented cocoa pulp-bean mass), due to their competitiveness in the wheat flour-water mixtures. Wheat breads made with G. oxydans IMDO A845 sourdoughs were significantly darker than reference wheat breads.
Project description:Few data have been published on the occurrence and functional role of acetic acid bacteria (AAB) in lambic beer production processes, mainly due to their difficult recovery and possibly unknown role. Therefore, a novel aseptic sampling method, spanning both the spatial and temporal distributions of the AAB and their substrates and metabolites, was combined with a highly selective medium and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as a high-throughput dereplication method followed by comparative gene sequencing for their isolation and identification, respectively. The AAB (Acetobacter species more than Gluconobacter species) proliferated during two phases of the lambic beer production process, represented by Acetobacter orientalis during a few days in the beginning of the fermentation and Acetobacter pasteurianus from 7 weeks until 24 months of maturation. Competitive exclusion tests combined with comparative genomic analysis of all genomes of strains of both species available disclosed possible reasons for this successive dominance. The spatial analysis revealed that significantly higher concentrations of acetic acid (from ethanol) and acetoin (from lactic acid) were produced at the tops of the casks, due to higher AAB counts and a higher metabolic activity of the AAB species at the air/liquid interface during the first 6 months of lambic beer production. In contrast, no differences in AAB species diversity occurred throughout the casks.IMPORTANCE Lambic beer is an acidic beer that is the result of a spontaneous fermentation and maturation process. Acidic beers are currently attracting attention worldwide. Part of the acidity of these beers is caused by acetic acid bacteria (AAB). However, due to their difficult recovery, they were never investigated extensively regarding their occurrence, species diversity, and functional role in lambic beer production. In the present study, a framework was developed for their isolation and identification using a novel aseptic sampling method in combination with matrix-assisted laser desorption ionization-time of flight mass spectrometry as a high-throughput dereplication technique followed by accurate molecular identification. The sampling method applied enabled us to take spatial differences into account regarding both enumerations and metabolite production. In this way, it was shown that more AAB were present and more acetic acid was produced at the air/liquid interface during a major part of the lambic beer production process. Also, two different AAB species were encountered, namely, Acetobacter orientalis at the beginning and Acetobacter pasteurianus in a later stage of the production process. This developed framework could also be applied for other fermentation processes.
Project description:Pyrroquinoline quinone-dependent alcohol dehydrogenase (PQQ-ADH) is a key enzyme in the ethanol oxidase respiratory chain of acetic acid bacteria (AAB). To investigate the effect of PQQ-ADH on acetic acid production by Acetobacter pasteurianus JST-S, subunits I (adhA) and II (adhB) of PQQ-ADH were over-expressed, the fermentation parameters and the metabolic flux analysis were compared in the engineered strain and the original one. The acetic acid production was improved by the engineered strain (61.42 g L-1) while the residual ethanol content (4.18 g L-1) was decreased. Analysis of 2D maps indicated that 19 proteins were differently expressed between the two strains; of these, 17 were identified and analyzed by mass spectrometry and two-dimensional gel electrophoresis. With further investigation of metabolic flux analysis (MFA) of the pathway from ethanol and glucose, the results reveal that over-expression of PQQ-ADH is an effective way to improve the ethanol oxidation respiratory chain pathway and these can offer theoretical references for potential mechanism of metabolic regulation in AAB and researches with its acetic acid resistance.
Project description:Under extreme acidic environments, bacteria exploit several acid resistance (AR) mechanisms for enhancing their survival, which is concerned with several aspects, such as issues in human health and fermentation for acidic products. Currently, knowledge of bacterial AR mainly comes from the strong acid (such as hydrochloric acid) stresses, whereas AR mechanisms against organic weak acids (such as acetic acid), which are indeed encountered by bacteria, are less understood. Acetic acid bacteria (AAB), with the ability to produce acetic acid up to 20 g/100 mL, possess outstanding acetic acid tolerance, which is conferred by their unique AR mechanisms, including pyrroloquinoline quinine-dependent alcohol dehydrogenase, acetic acid assimilation and molecular chaperons. The distinguished AR of AAB toward acetic acid may provide a paradigm for research in bacterial AR against weak organic acids. In order to understand AAB's AR mechanism more holistically, omics approaches have been employed in the corresponding field. However, the currently reported transcriptomic study was processed under a low-acidity (1 g/100 mL) environment, which could not reflect the general conditions that AAB are usually faced with. This study performed RNA-Seq transcriptomic analysis investigating AR mechanisms in Acetobacter pasteurianus CGMCC 1.41, a widely used vinegar-brewing AAB strain, at different stages of fermentation, namely, under different acetic acid concentrations (from 0.6 to 6.03 g/100 mL). The results demonstrated the even and clustered genomic distribution of up- and down-regulated genes, respectively. Difference in AR between AAB and other microorganisms was supported by the down-regulation of urea degradation and trehalose synthesis-related genes in response to acetic acid. Detailed analysis reflected the role of ethanol respiration as the main energy source and the limited effect of acetic acid assimilation on AR during fermentation as well as the competition between ethanol respiratory chain and NADH, succinate dehydrogenase-based common respiratory chain. Molecular chaperons contribute to AR, too, but their regulatory mechanisms require further investigation. Moreover, pathways of glucose catabolism and fatty acid biosynthesis are also related to AR. Finally, 2-methylcitrate cycle was proposed as an AR mechanism in AAB for the first time. This study provides new insight into AR mechanisms of AAB, and it also indicates the existence of numerous undiscovered AR mechanisms.
Project description:Forastero hybrid cocoa bean fermentations have been carried out in a box (B) and in a heap (H), with or without the inoculation of Saccharomyces cerevisiae and Torulaspora delbrueckii as starter cultures. The bacteria, yeasts, and microbial metabolites (volatile and nonvolatile organic compounds) were monitored during fermentation to assess the connection between microbiota and the release of metabolites during this process. The presence of starter cultures was detected, by means of culture-dependent analysis, during the first 2 days of both fermentations. However, no statistical difference was observed in any of the physicochemical or microbiological analyses. Plate counts revealed the dominance of yeasts at the beginning of both fermentations, and these were followed by acetic acid bacteria (AAB) and lactic acid bacteria (LAB). Hanseniaspora opuntiae, S. cerevisiae, Pichia pijperi, Acetobacter pasteurianus, and Lactobacillus fermentum were the most abundant operational taxonomic units (OTUs) during both fermentation processes (B and H), although different relative abundances were observed. Only the diversity of the fungal species indicated a higher level of complexity in the B fermentations than in the H fermentations (P < 0.05), as well as a statistically significant difference between the initially inoculated starter cultures (P < 0.01). However, the microbial metabolite analysis indicated different distributions of the volatile and nonvolatile compounds between the two procedures, that is, B and H (P < 0.05), rather than between the inoculated and noninoculated fermentations. The box fermentations showed faster carbohydrate metabolism and greater production of organic acid compounds, which boosted the formation of alcohols and esters, than did the heap fermentations. Overall, the microbial dynamics and associations between the bacteria, yeasts, and metabolites were found to depend on the type of fermentation.IMPORTANCE In spite of the limited effectiveness of the considered inoculated starter strains, this study provides new information on the microbial development of box and heap cocoa fermentations, under inoculated and noninoculated conditions, as we coupled yeast/bacterial amplicon-based sequencing data with microbial metabolite detection. The information so far available suggests that microbial communities have played an important role in the evolution of aroma compounds. Understanding the pathways that microorganisms follow during the formation of aromas could be used to improve the fermentation processes and to enhance chocolate quality.
Project description:Kefir is a functional beverage that contains lactic and acetic acid bacteria (LAB, AAB) and yeasts. This work's aim was to study the chemical, microbial, and functional characteristics of kefir produced from cow's milk and soy milk. After fermentation, free amino acids were 20.92?mg 100?mL-1 and 36.20?mg 100?mL-1 for cow's milk and soy milk kefir, respectively. Glutamic acid was majority in both, suggesting that microbial proteolysis leads to an increase in free amino acids including glutamic acid. 108-109?CFU?mL-1 LAB, 106-107?CFU?mL-1 AAB, and 106-107?CFU?mL-1 yeasts were counted in cow's milk kefir, whereas soy milk kefir contained greatly lower yeasts and AAB. Lactococcus lactis, Kazachstania unispora, and Saccharomyces cerevisiae were isolated as major microorganisms in both kefirs. Acetobacter orientalis only existed in cow's milk kefir. Cow's milk and soy milk showed ACE inhibitory activity, which significantly increased after fermentation. Both kefirs also exhibited antioxidant activity and bactericidal activity against Escherichia coli, Salmonella Typhimurium, and Staphylococcus aureus.
Project description:Cocoa bean fermentation relies on the sequential activation of several microbial populations, triggering a temporal pattern of biochemical transformations. Understanding this complex process is of tremendous importance as it is known to form the precursors of the resulting chocolate's flavour and taste. At the same time, cocoa bean fermentation is one of the least controlled processes in the food industry. Here, a quantitative model of cocoa bean fermentation is constructed based on available microbiological and biochemical knowledge. The model is formulated as a system of coupled ordinary differential equations with two distinct types of state variables: (i) metabolite concentrations of glucose, fructose, ethanol, lactic acid and acetic acid and (ii) population sizes of yeast, lactic acid bacteria and acetic acid bacteria. We demonstrate that the model can quantitatively describe existing fermentation time series and that the estimated parameters, obtained by a Bayesian framework, can be used to extract and interpret differences in environmental conditions. The proposed model is a valuable tool towards a mechanistic understanding of this complex biochemical process, and can serve as a starting point for hypothesis testing of new systemic adjustments. In addition to providing the first quantitative mathematical model of cocoa bean fermentation, the purpose of our investigation is to show how differences in estimated parameter values for two experiments allow us to deduce differences in experimental conditions.