The Functional Role of Lactic Acid Bacteria in Cocoa Bean Fermentation

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Among various lactic acid bacterial strains tested, cocoa-specific strains of Lactobacillus fermentum were 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.Fermented dry cocoa beans are the basic raw material for chocolate production. Cocoa beans are the seeds of the cocoa tree, Theobroma cacao L. The key microorganisms for successful cocoa bean fermentation processes are yeasts, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) (1, 3-5, 14, 18-21). Although the LAB species diversity involved in the onset of any spontaneous cocoa bean fermentation is wide, not much is known about how cocoa-specific LAB species, such as Lactobacillus fermentum, adapt physiologically and what their targeted functional roles are during fermentation (1,3,4,14,(19)(20)(21). The present study aimed at the kinetic investigation of carbohydrate fermentation and citric acid conversion by various LAB strains to unravel this.The LAB strains used throughout this study are listed in Table 1. Monoculture fermentations were performed in 1.5 liters of a cocoa pulp simulation medium (CPSM) for LAB (16) in Biostat B-DCU fermentors (Sartorius AG/B. Braun Biotech International, Melsungen, Germany) anaerobically for 48 h. Inoculum build-up, fermentor setup, online control of temperature (Table 2), pH profile, agitation, and sampling were as described previously (13, 16). All fermentations were performed in duplicate. The results and figures presented hereinafter are representative for both fermentations.During fermentation, bacterial growth (CFU per ml) was quantified through plating of 10-fold serial dilutions of the samples in saline (0.85% [wt/vol] NaCl solution) on CPSM agar (CPSM containing 1.5% [wt/vol] agar, pH 5.5) that was incubated at the appropriate fermentation temperature for 24 h. Metabolite concentrations were determined through high-performance anion-exchange chromatography using a standard addition protocol (glucose, fructose, mannitol, and citric acid) (15, 23) and high-performance liquid chromatography using external standards (lactic acid, acetic acid, and ethanol) (16). Cell count and metabolite (with external standards) measurements were performed on three independent samples. The errors on the measurements are represented as standard deviations. Gas chromatography (GC) was used for the qualitative determination of 2,3-butanediol and acetoin (15). Concentrations of carbon dioxide in the fermentor gas effluents were determined online through GC (13). The carbon recovery (CR, expressed as percentage) was calculated by dividing the total amount of carbon recovered in the metabolites by the total amount of carbon present in the carbon sources.All LAB strains tested were able to grow in CPSM ( Fig. 1 and T fermented fructose but not glucose and converted citric acid. Due to the initial low pH (3.5), Enterococcus casseliflavus M484 and Lactobacillus amylovorus DCE 471 were not able to...

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confidence: 99%

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confidence: 99%

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

Lefeber

Janssens

Moens

et al. 2011

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“…The main LAB metabolites are various organic acids (mainly lactic and acetic acid), carbon dioxide, ethanol, mannitol, and glycerol (8). LAB also produce volatiles, such as diacetyl, acetoin, and acetaldehyde, but their impact on the flavor of cocoa has not yet been investigated (9). However, recent data suggest that these microbes might have only a slight influence on cocoa flavor and quality, since cocoa fermentations lacking LAB had similar shell weights and gave acceptable chocolates with no differences in sensory rankings (10).…”

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confidence: 99%

Tuning Chocolate Flavor through Development of Thermotolerant Saccharomyces cerevisiae Starter Cultures with Increased Acetate Ester Production

Meersman¹,

Steensels²,

Struyf³

et al. 2016

Microbial starter cultures have extensively been used to enhance the consistency and efficiency of industrial fermentations. Despite the advantages of such controlled fermentations, the fermentation involved in the production of chocolate is still a spontaneous process that relies on the natural microbiota at cocoa farms. However, recent studies indicate that certain thermotolerant Saccharomyces cerevisiae cultures can be used as starter cultures for cocoa pulp fermentation. In this study, we investigate the potential of specifically developed starter cultures to modulate chocolate aroma. Specifically, we developed several new S. cerevisiae hybrids that combine thermotolerance and efficient cocoa pulp fermentation with a high production of volatile flavor-active esters. In addition, we investigated the potential of two strains of two non-Saccharomyces species that produce very large amounts of fruity esters (Pichia kluyveri and Cyberlindnera fabianii) to modulate chocolate aroma. Gas chromatography-mass spectrometry (GC-MS) analysis of the cocoa liquor revealed an increased concentration of various flavor-active esters and a decrease in spoilage-related off-flavors in batches inoculated with S. cerevisiae starter cultures and, to a lesser extent, in batches inoculated with P. kluyveri and Cyb. fabianii. Additionally, GC-MS analysis of chocolate samples revealed that while most shortchain esters evaporated during conching, longer and more-fat-soluble ethyl and acetate esters, such as ethyl octanoate, phenylethyl acetate, ethyl phenylacetate, ethyl decanoate, and ethyl dodecanoate, remained almost unaffected. Sensory analysis by an expert panel confirmed significant differences in the aromas of chocolates produced with different starter cultures. Together, these results show that the selection of different yeast cultures opens novel avenues for modulating chocolate flavor.

“…Thus, the result of the fermentation process strongly depends on the microbial population of the pulp and postharvest practices on the farm (4). It was shown recently that lactic acid bacteria (LAB) play a prominent role in the microbial community of cocoa pulp fermentation, as their metabolic contribution is a key to the success of the fermentation process (5,6). The primary routes of carbon metabolism by lactobacilli are summarized in Fig.…”

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confidence: 99%

Core Fluxome and Metafluxome of Lactic Acid Bacteria under Simulated Cocoa Pulp Fermentation Conditions

Adler

Bolten

Dohnt

et al. 2013

bIn the present work, simulated cocoa fermentation was investigated at the level of metabolic pathway fluxes (fluxome) of lactic acid bacteria (LAB), which are typically found in the microbial consortium known to convert nutrients from the cocoa pulp into organic acids. A comprehensive 13 C labeling approach allowed to quantify carbon fluxes during simulated cocoa fermentation by (i) parallel 13 C studies with [ 13 C 6 ]glucose, [1,2-13 C 2 ]glucose, and [ 13 C 6 ]fructose, respectively, (ii) gas chromatography-mass spectrometry (GC/MS) analysis of secreted acetate and lactate, (iii) stoichiometric profiling, and (iv) isotopomer modeling for flux calculation. The study of several strains of L. fermentum and L. plantarum revealed major differences in their fluxes. The L. fermentum strains channeled only a small amount (4 to 6%) of fructose into central metabolism, i.e., the phosphoketolase pathway, whereas only L. fermentum NCC 575 used fructose to form mannitol. In contrast, L. plantarum strains exhibited a high glycolytic flux. All strains differed in acetate flux, which originated from fractions of citrate (25 to 80%) and corresponding amounts of glucose and fructose. Subsequent, metafluxome studies with consortia of different L. fermentum and L. plantarum strains indicated a dominant (96%) contribution of L. fermentum NCC 575 to the overall flux in the microbial community, a scenario that was not observed for the other strains. This highlights the idea that individual LAB strains vary in their metabolic contribution to the overall fermentation process and opens up new routes toward streamlined starter cultures. L. fermentum NCC 575 might be one candidate due to its superior performance in flux activity.T he worldwide annual production of cocoa beans has reached 4.4 million metric tons (1 [http://faostat3.fao.org/home/index .html#DOWNLOAD; selection: production Ͼ crops Ͼ regions: world {total} Ͼ elements: production {tonnes} Ͼ items: cocoa beans Ͼ years: 2011]). The process of chocolate preprocessing involves pod opening, bean (pulp) fermentation, and bean drying, followed by roasting of the cocoa beans (2). A critical step determining the cocoa bean quality is the fermentation of the fresh cocoa pulp, whose main functions are acetic acid-and heat-induced death of the cocoa bean, reduction in bitterness, and formation of valuable aroma precursors (3). Cocoa bean fermentation is a spontaneous process that is carried out under rather uncontrolled conditions. Thus, the result of the fermentation process strongly depends on the microbial population of the pulp and postharvest practices on the farm (4). It was shown recently that lactic acid bacteria (LAB) play a prominent role in the microbial community of cocoa pulp fermentation, as their metabolic contribution is a key to the success of the fermentation process (5, 6). The primary routes of carbon metabolism by lactobacilli are summarized in Fig. 1. The LAB convert carbohydrates, i.e., fructose and glucose, and citrate, contained in the pulp, into lactate, aceta...