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Functional role of yeasts, lactic acid bacteria and acetic acid bacteria in cocoa fermentationprocessesDe Vuyst, Luc; Leroy, Frederic

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DOI:10.1093/femsre/fuaa014

Publication date:2020

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Citation for published version (APA):De Vuyst, L., & Leroy, F. (2020). Functional role of yeasts, lactic acid bacteria and acetic acid bacteria in cocoafermentation processes. FEMS Microbiology Reviews, 44(4), 432-453. https://doi.org/10.1093/femsre/fuaa014

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FEMS Microbiology Reviews, fuaa014, 44, 2020, 432–453

doi: 10.1093/femsre/fuaa014Advance Access Publication Date: 18 May 2020Review Article

REVIEW ARTICLE

Functional role of yeasts, lactic acid bacteria andacetic acid bacteria in cocoa fermentation processesLuc De Vuyst* and Frederic Leroy

Research Group of Industrial Microbiology and Food Biotechnology, Faculty of Sciences and BioengineeringSciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium∗Corresponding author: Research Group of Industrial Microbiology and Food Biotechnology, Faculty of Sciences and Bioengineering Sciences, VrijeUniversiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium. Tel: +32-2-6293245; Fax: +32-2-6292720; E-mail: [email protected]

One sentence summary: Yeasts, lactic acid bacteria and acetic acid bacteria enable pulp removal and cocoa bean curing during cocoa fermentation anddrying processes, which precede roasting of the cured cocoa beans, the starting material for the production of chocolate.

Editor: Bas Teusink

ABSTRACT

Cured cocoa beans are obtained through a post-harvest, batchwise process of fermentation and drying carried out on farmsin the equatorial zone. Fermentation of cocoa pulp-bean mass is performed mainly in heaps or boxes. It is made possible bya succession of yeast, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) activities. Yeasts ferment the glucose of thecocoa pulp into ethanol, perform pectinolysis and produce flavour compounds, such as (higher) alcohols, aldehydes,organic acids and esters. LAB ferment the glucose, fructose and citric acid of the cocoa pulp into lactic acid, acetic acid,mannitol and pyruvate, generate a microbiologically stable fermentation environment, provide lactate as carbon source forthe indispensable growth of AAB, and contribute to the cocoa and chocolate flavours by the production of sugar alcohols,organic acids, (higher) alcohols and aldehydes. AAB oxidize the ethanol into acetic acid, which penetrates into the beancotyledons to prevent seed germination. Destruction of the subcellular seed structure in turn initiates enzymatic andnon-enzymatic conversions inside the cocoa beans, which provides the necessary colour and flavour precursor molecules(hydrophilic peptides, hydrophobic amino acids and reducing sugars) for later roasting of the cured cocoa beans, the firststep of the chocolate-making.

Keywords: acetic acid bacteria; chocolate; cocoa fermentation; flavour; lactic acid bacteria; yeasts

INTRODUCTION

Fermentation of perishable raw materials originating from plantagriculture and animal husbandry is an ancient biotechnologicalprocess that is practiced to produce stable and safe fermentedfood products of high microbiological, organoleptic, nutritionaland health-beneficial quality (Leroy and De Vuyst 2004; Bourdi-chon et al. 2012; Wolfe and Dutton 2015; Tamang, Watanabe andHolzapfel 2016; Marco et al. 2017; Rezac et al. 2018). This pro-cess is based on environmental inoculation of the raw materi-als (often referred to as natural or spontaneous fermentation),intentional inoculation through backslopping (use of a part ofa finished fermented food product to inoculate a fresh batchof raw material), or after addition of a microbial starter culture

(Leroy and De Vuyst 2004; Hutkins 2019). Whereas starter cul-tures are in use for the production of fermented dairy prod-ucts (e.g. yoghurt and cheese), fermented meat products (e.g. fer-mented sausage), bread production (by means of bakers’ yeast)and fermented alcoholic beverages (e.g. beer and wine) sinceseveral decades, several processes for the fermentation of cere-als (e.g. artisan sourdoughs), vegetables (e.g. sauerkraut), fruits(e.g. vinegar), fish (e.g. fish sauce), etc. are still mostly of abackslopping or spontaneous nature, but research on the useof starter cultures for these fermentation processes is ongoing(Hutkins 2019). Some of these spontaneous fermentation pro-cesses are of great economic importance, as in the case of thecash tree crop cocoa (Schwan, Fleet and Afoakwa 2015; Pereira,Soccol and Soccol 2016). They hence would likely take profit of

Received: 14 December 2019; Accepted: 16 May 2020

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the introduction of starter cultures to carry out accelerated andenhanced fermentation processes that lead to end-products ofimproved and standardized quality.

Cocoa originated in the Amazon basin of South America(Zarrillo et al. 2018; Coe and Coe 2019). It was cultivated andconsumed as a beverage constituent in Latin America since pre-historic times. Its consumption, first as cocoa drink and lateron as chocolate, was first introduced into Europe in the 16thcentury. Nowadays, cocoa trees are cultivated on plantationsin the equatorial zone globally. Their fruits have to undergo arather complex and often uncontrolled, on-farm, post-harvest,batchwise process of fermentation and drying to be able touse the fermented dry cocoa beans for further manufacturing(Fig. 1; Schwan and Wheals 2004; Beckett 2009; Badrie et al.2015; Afoakwa 2016; De Vuyst and Weckx 2016a,b; Figueroa-Hernandez et al. 2019; Munoz et al. 2019). Ivory Coast, Ghana,Indonesia, Ecuador, Nigeria, Cameroon and Brazil are the maincocoa-producing countries in the cocoa belt, some 20◦ north andsouth of the equator.

Fermented dry cocoa beans are the principal raw materialfor the production of chocolates and other cocoa-based (food)products (Schwan and Wheals 2004; Afoakwa et al. 2008; Limaet al. 2011; Saltini, Akkerman and Frosch 2013; Badrie et al.2015; Schwan, Fleet and Afoakwa 2015; Pereira, Soccol andSoccol 2016; De Vuyst and Weckx 2016a,b; Ozturk and Young2017; Munoz et al. 2019). Whilst the demand for fermenteddry cocoa beans is increasing, chocolate manufacturers as wellas consumers are concerned about the economical, ethical,environmental and social sustainability of cocoa manufactur-ing throughout its whole production chain. High demands mayresult in an expansion of the cocoa plantations at the cost of for-est area, affect global warming, create potential irregular worksituations, intensify the use of agrochemicals (fertilizers andpesticides), increase residual biomass if not dealt with properly(cocoa pod husks, cocoa bean shells and cocoa pulp sweatings),and parallel cocoa bean price fluctuations and speculations, etc.(Wessel and Quist-Wessel 2015; Kongor et al. 2016; Konstantaset al. 2018; Vasquez et al. 2019). Besides expansion of the cocoaproduction area and improvement of the cocoa yields, alterna-tive fermentation practices, such as novel fermentation meth-ods or the use of functional starter cultures, can be applied toimprove the yield of well-cured cocoa beans and the farmers’income.

Besides the early review of Schwan and Wheals (2004),some recent reviews partially dealt with the potential of startercultures to improve the cocoa fermentation process (Saltini,Akkerman and Frosch 2013; Pereira, Soccol and Soccol 2016;De Vuyst and Weckx 2016a,b; Ozturk and Young 2017; Castro-Alayo et al. 2019; Figueroa-Hernandez et al. 2019; Munoz et al.2019). The present review provides a state-of-the-art overviewof the microbiology and biochemistry of cocoa fermentationprocesses and the importance of controlled, starter culture-initiated fermentation processes to produce fermented drycocoa beans. It will focus in particular on the functional roleof yeasts, lactic acid bacteria (LAB) and acetic acid bacteria(AAB), thereby attempting to cover most of the studies publishedsince 2000.

Role of fermentation in cocoa curing

Fermentation is an essential step in the post-harvest process-ing of cocoa pods, the big oval fibrous fruits of the cocoa tree(Theobroma cacao L.) (Bartley 2005; Motamayor et al. 2008; Badrie

et al. 2015). These fruits differ in size, shape and surface colour.After processing, their seeds, the cured cocoa beans, are used forchocolate production (Fig. 1; Schwan and Wheals 2004; De Vuystand Weckx 2016a,b; Figueroa-Hernandez et al. 2019; Munoz et al.2019). Morpho-geographically, distinction can be made betweenbulk (basic or ordinary) cocoa (Forastero variety; 95% of theworld cocoa production) and fine (flavour) cocoa (Criollo andTrinitario varieties; 5%). However, most cocoa crops in use arehybrids of several varieties. The white to dark purple, freshcocoa beans (the colour depends on their anthocyanin contents)are embedded in a white-grey, sticky, sour-sweet and viscouspulp, the cocoa pulp-bean mass, which is manually or mechani-cally scooped out of the cocoa pods after their harvest (Fig. 1).When applying good agricultural and farming practices, onlymature, healthy and undamaged cocoa pods should be used.The fresh cocoa pulp-bean mass, ideally depleted from placen-tas and residual cocoa pod particles, is either piled into heaps onbanana or plantain leaves on the ground, tightened to removeair and covered with extra leaves to keep the heat producedupon fermentation inside, to activate the fermentation process(the heap fermentation method), or brought into wooden boxes,tightened and covered with leaves before the actual fermenta-tion (the box fermentation method) (Fig. 1). These heap and boxfermentation methods represent the two main cocoa fermenta-tion methods used worldwide. Other methods include baskets,trays and platforms, besides a variety of local farm practices.Once open to the air and during its transfer, the cocoa pulp-bean mass—which is nearly sterile, depending on the intact-ness of the cocoa pods, in turn depending on crop cultivationmanagement—is inoculated by microorganisms from the envi-ronment. Such natural or spontaneous fermentation process isnot only driven by the microorganisms that originate from thecocoa pod surfaces, of which the microbial load depends on thepod ripeness, age and healthy status, but also those from thebanana and plantain leaves, the tools and utensils used on thefarms, the left-overs from previous fermentation runs in thetransport and collecting baskets and in the fermentation boxes,insects or larger animals, as well as the surrounding soil, dustand air (Jespersen et al. 2005; Camu et al. 2007, 2008a; Lefeberet al. 2011a; Papalexandratou et al. 2011b,c, 2013; Maura et al.2016; Ho, Fleet and Zhao 2018; John et al. 2019). The main pur-pose of the fermentation of the cocoa pulp-bean mass is theremoval of the pulp surrounding the beans through endogenousand yeast-mediated pectinolysis to improve the subsequent dry-ing of the non-germinating beans as well as killing of the seedembryo, which is made possible through the curing steps. Thisis necessary for dry storage of the cocoa beans and their fur-ther use as raw material for chocolate production. Indeed, thedegradation and conversion of the pulp substrates, in partic-ular carbohydrates and organic acids, supports the avoidanceof seed germination (and even causes seed death). After enzy-matic reactions inside the seeds, they also support the produc-tion of flavour precursor and flavour-active molecules as wellas colour compounds in the cured beans, which are necessaryfor their further processing into cocoa and chocolate products.Some flavour compounds originate from the cocoa bean vari-ety, which is determined by cultivar, genotype, environmen-tal growth conditions (growth location, weather and soil con-ditions) and agricultural practices, such as agroforestry man-agement, fertilization, pruning and phytosanitary management(Counet et al. 2004; Aculey et al. 2010; Cruz et al. 2013; Kadowet al. 2013; Moreira et al. 2013, 2016, 2018; Trognitz et al. 2013;Ali et al. 2014; Ramos et al. 2014; Sukha et al. 2014; Diomandeet al. 2015; Tran et al. 2015; Vazquez-Ovando et al. 2015; Acierno

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434 FEMS Microbiology Reviews, 2020, Vol. 44, No. 4

Figure 1. From cocoa fruit to chocolate. The first steps of the cocoa processing chain take place at farms in the cocoa-producing countries around the equator. Fermented

dry cocoa beans are further processed into chocolate and other cocoa products in factories worldwide. ∗Grinded cocoa nibs form cocoa mass or cocoa liquor, dependingon the temperature. Cocoa mass/liquor is used for the production of both dark and milk chocolates, together with cocoa butter and sugar. ∗∗Milk powder is used forthe production of milk and white chocolates, the latter further involving only cocoa butter and sugar.

et al. 2016, 2019; Kongor et al. 2016; Menezes et al. 2016; Chetschiket al. 2018; Castro-Alayo et al. 2019; Utrilla-Vazquez et al. 2020).However, possible pulp-preconditioning through pod storage,depulping, or bean spreading/pre-drying (to reduce pulp volume,fermentable carbohydrate content, moisture content, or acidityand thus increase cocoa flavour) is of influence (Nazaruddin et al.2006; Afoakwa et al. 2011, 2012, 2013a,b,c, 2014; Kongor et al. 2016;D’Souza et al. 2017; Qin et al. 2017; Sukha, Umaharan and Butler2017; Hinneh et al. 2018, 2019a,b; Megıas-Perez et al. 2018, 2020;Hamdouche et al. 2019). Overall, fermentation and drying are

determining steps for the expression of the flavour potential ofcured cocoa beans (Rodriguez-Campos et al. 2011, 2012; Tran et al.2015; Kongor et al. 2016; Moreira et al. 2016; Ascrizzi et al. 2017;D’Souza et al. 2017; Castro-Alayo et al. 2019; Hamdouche et al.2019; Munoz et al. 2019; Utrilla-Vazquez et al. 2020). This flavourpotential is further expressed under the appropriate conditionsfor the roasting of the cured cocoa beans and the conching ofthe chocolate batter for texturization and final flavour devel-opment during chocolate-making (Counet et al. 2004; Tran et al.2015; Acierno et al. 2016; Kongor et al. 2016; Menezes et al. 2016;

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Moreira et al. 2016, 2018; Ascrizzi et al. 2017; Garcıa-Alamilla et al.2017; Braga et al. 2018; Hinneh et al. 2018, 2019a,b; Castro-Alayoet al. 2019; Munoz et al. 2019).

During fermentation, continuous physicochemical changesoccur in the cocoa bean cotyledons, driven in particular by tem-perature and pH courses as well as ethanol and organic acid(mainly acetic acid) ingress, together with changes in the natureand concentrations of the pulp nutrients, some of which actas fermentation substrates, and of the microbial metabolitesformed. This causes a temporal succession of the growth andmetabolic activities of the different microbial groups that par-ticipate in the cocoa fermentation process, namely yeasts, LABand AAB, which occur in the aqueous phase of the cocoa pulp,therefore often referred to as cocoa pulp-bean mass fermenta-tion process. In other words, this process is situated outside thecocoa beans, which are cured as a result of the microbial fer-mentation of the pulp followed by drying (Fig. 2; Schwan andWheals 2004; De Vuyst and Weckx 2016a,b; Figueroa-Hernandezet al. 2019; Munoz et al. 2019). The ethanol, acetic acid and hightemperature that are produced during microbial fermentation,along with a variable yet increasing oxygen availability due topulp drainage, cause physicochemical changes in the seeds, inparticular a pH decrease and a temperature increase. These leadto the death of the seed embryo and a damaged and reorga-nized subcellular seed cotyledon structure, originally consist-ing of separated lipid-protein-starch storage cells and polyphe-nol storage or pigment cells. This allows enzymatic and non-enzymatic conversions in the cotyledons to produce flavour pre-cursor molecules, because endogenous substrates and enzymescan now migrate and mix. In addition, leakage or preservationof several seed compounds occurs. The indirect role of aceticacid (and ethanol) in flavour precursor formation and polyphe-nol dynamics has been shown during fermentation-like incuba-tions of cocoa beans (Andersson, Koch and Lieberei 2006; Ali et al.2014; Kadow et al. 2015; De Taeye et al. 2016; Evina et al. 2016; Johnet al. 2016, 2019, 2020; Munoz et al. 2019). Acetic acid migratesinto the cotyledon tissue with the water stream upon initia-tion of seed germination during the initial phase of fermenta-tion of the cocoa pulp-bean mass through a pressure-inducedopening of the shell (testa or seed coat) at the micropyle andhilum of the swelling seeds. The colour and flavour of the curedbeans are determined to a large extent by the leakage of pur-ple anthocyanins, causing bleaching of the cocoa beans, and bythe oxidation of polyphenols and their polymerization into high-molecular-mass compounds as well as condensation and com-plex formation with proteins, oligopeptides, free amino acidsand monosaccharides (forming complex tannins), depending onthe fermentation time (and pod storage/pulp-preconditioning)(Wollgast and Anklam 2000; Nazaruddin et al. 2006; Camu et al.2008b; Suazo, Davidov-Pardo and Arozarena 2014; Albertini et al.2015; De Taeye et al. 2016; Evina et al. 2016; Hue et al. 2016;Mayorga-Gross et al. 2016; Racine et al. 2019). The bitternessand astringency of the raw cocoa beans are reduced due to theleakage of flavonoid polyphenols [flavanols (flavan-3-ols or cat-echins) and proanthocyanidins (oligomers of flavan-3,4-diols)]and methylxanthine alkaloids (1,3,7-trimethylxanthine or caf-feine and 3,7-dimethylxanthine or theobromine), besides effectsoriginating from the formation of insoluble tannins. From ahealth point of view, the leakage of anti-oxidative polyphe-nols, (psycho)stimulatory methylxanthines and most probablyother possibly health-related cocoa bean compounds, such asamines (e.g. mood-elevating phenylethylamine), other alkaloids

(e.g. neuroprotective and psychoactive salsolinol), hydroxycin-namic acid amides (e.g. anti-inflammatory and neuroprotec-tive clovamide) and organic acids (e.g. anti-oxidative caffeicacid), whether or not modified during the curing process, seemsto be disadvantageous (Paoletti et al. 2012; Badrie et al. 2015;Tuenter, Foubert and Pieters 2018; Rawel et al. 2019). Strategiesfor their recovery can nevertheless be envisaged, either fromthe pulp drainage or the cocoa beans. To this end, laboratory-scale (2 L) and pilot-scale (15 L) model fermentation processesin polypropylene boxes, placed in an incubator with controlledtemperature and aeration, have been proposed, using a cocoapulp simulation medium (CPSM; Lefeber et al. 2010) and rehy-drated unfermented dry cocoa beans, to assess the impactof controlled fermentation conditions on bioactive compoundspresent in cocoa beans (Lee et al. 2019; Racine et al. 2019). Finally,it is unclear if the fat content of the cocoa beans varies dur-ing fermentation, but its content should be as high as possible,as the cocoa bean fat (cocoa butter) is a basic ingredient of thefinal chocolate (Lima et al. 2011; Ndife et al. 2013; Trognitz et al.2013; Krahmer et al. 2015; Servent et al. 2018; Sirbu et al. 2018).Further processing of the cured cocoa beans involves, amongstother steps, roasting to start up the chocolate manufacturingprocess, conching to give chocolate its appropriate final flavourand texture, and tempering to give the chocolate bar or pralineits appropriate appearance and durability (Fig. 1; Beckett 2009;Afoakwa 2016; Engeseth and Pangan 2018).

Microbial species diversity and community dynamicsduring cocoa fermentation

The microbial ecology and succession of cocoa fermentationprocesses is complex (Schwan and Wheals 2004; De Vuyst andWeckx 2016a,b; Figueroa-Hernandez et al. 2019; Munoz et al.2019). Cocoa fermentation processes differ in microbial speciesand counts as well as in substrate and metabolite compoundsand concentrations over time, hence leading to variable fermen-tation quality and cocoa flavour. Moreover, cocoa fermentationprocesses are characterized by changing interactions with theenvironment and among the indigenous microorganisms (Adleret al. 2013, 2014; Moens, Lefeber and De Vuyst 2014; Illeghems,Weckx and De Vuyst 2015b; Moreno-Zambrano et al. 2018; Mota-Gutierrez et al. 2018). This variability not only depends on thecocoa variety, agricultural and farm practices, fermentationmethod and duration, etc., but even occurs for fermentation pro-cesses carried out on the same farm, under the same conditionsand with the same cocoa variety (Afoakwa et al. 2008; Cevallos-Cevallos et al. 2018; Rottiers et al. 2019). Nonetheless, as furtheroutlined below, these complex successive microbial activitiesand diversity of the cocoa fermentation process also account forits robustness.

The actual fermentation of the cocoa pulp-bean mass is car-ried out by a temporal succession of activities of yeasts, LABand AAB, whose interactions are determined mainly by theirethanol, lactic acid and acetic acid concentrations produced aswell as the temperature increase upon fermentation. A well-performed cocoa fermentation process, implying good agricul-tural and farm practices, is finished within 4–6 days (Schwanand Wheals 2004; Lima et al. 2011; Saltini, Akkerman and Frosch2013; De Vuyst and Weckx 2016b; Munoz et al. 2019). Although4 days seem to be optimal, the fermentation may last from 2to 10 days or it may even be that no intentional fermentationset-up is foreseen (Schwan and Wheals 2004; Rodriguez-Campos

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Figure 2. Overview of the fermentation of the cocoa pulp-bean mass through the succession of an anaerobic phase, involving yeasts and lactic acid bacteria (LAB),and an aerobic phase, involving acetic acid bacteria (AAB), and the concomitant bioconversion reactions taking place inside the cocoa beans. Uptake of substratesand secretion of metabolites by the microbial cells are not represented. EMP, Embden-Meyerhoff-Parnas pathway. HeFe, heterofermentation. HoFe, homofermentation.PKP, phosphoketolase pathway. TCA, tricarboxylic acid cycle.

et al. 2011, 2012; Lefeber et al. 2012; Papalexandratou et al. 2013;Maura et al. 2016; Mayorga-Gross et al. 2016; De Vuyst and Weckx2016b; Munoz et al. 2019). Overall, the fermentation durationdepends on the cocoa variety and the local farming practicesapplied. If the fermentation lasts longer than 4 days, bacillican also grow under the aerobic conditions, but they are ratherundesirable because of the production of off-flavours, such asshort-chain fatty acids and ammonia (Ardhana and Fleet 2003;Schwan and Wheals 2004; Nielsen et al. 2007a; Lima et al. 2011;Pereira et al. 2012, 2013a; Pereira, Magalhaes-Guedes and Schwan2013b; Ho, Zhao and Fleet 2014; Kouame et al. 2015a,b; De Vuystand Weckx 2016b; Miguel et al. 2017; Agyirifo et al. 2019; Ham-douche et al. 2019; Papalexandratou et al. 2019). Similarly, thegrowth of filamentous fungi can cause off-flavour and myco-toxin formation (Ardhana and Fleet 2003; Schwan and Wheals2004; Guehi et al. 2007, 2008; Sanchez-Hervas et al. 2008; Copettiet al. 2010, 2011, 2014; Mounjouenpou et al. 2010; Lima et al.2011; Kedjebo et al. 2015; De Vuyst and Weckx 2016b; Agyir-ifo et al. 2019). These bacilli and filamentous fungi may thuscause spoilage, although they are sometimes considered as ben-eficial contributors to pectinolysis; certain Bacillus species areeven linked with pyrazine production (Ardhana and Fleet 2003;Schwan and Wheals 2004; Ouattara et al. 2008, 2011; Lima et al.2011; Kouame et al. 2015a,b; De Vuyst and Weckx 2016b). Enter-obacterales (e.g. soil- or plant-associated Erwinia, Pantoea andTatumella species) occur transiently in the beginning of the fer-mentation of the cocoa pulp-bean mass and may participate inthe degradation of pulp pectin and citric acid (Garcia-Armisenet al. 2010; Lefeber et al. 2011a; Papalexandratou et al. 2011c, 2013,

2019; Illeghems et al. 2012; Pereira et al. 2012; Pereira, Magalhaes-Guedes and Schwan 2013b; Ho, Zhao and Fleet 2014; Hamdoucheet al. 2015, 2019; Illeghems, Weckx and De Vuyst 2015b; De Vuystand Weckx 2016b). They can be responsible for the production ofgluconic acid from glucose as well, which is considered undesir-able and impacts the glucose-dependent growth of yeasts andLAB, although gluconic acid may provide the cocoa beans witha mild herbal flavour and mask bitterness caused by caffeine(Lefeber et al. 2011a; Papalexandratou et al. 2011c, 2019).

Whereas the microbiology of the cocoa fermentation processwas initially studied culture-dependently only [since the 1950s;reviewed in Schwan and Wheals (2004), Thompson et al. (2013)and De Vuyst and Weckx (2016a)], cocoa fermentation studiesfrom 2000 on started with phenotypic identification of pickedcolonies [e.g. Ardhana and Fleet (2003) and Kostinek et al. (2008)].This was later followed by molecular identification and ampli-con sequencing of the isolates [since 2005; e.g. Jespersen et al.(2005) and Camu et al. (2007)], culture-independent communityprofiling methods such as DNA-based fingerprinting [since 2005;e.g. Nielsen et al. (2005) and Camu et al. (2007)] and (shotgun)metagenomics (since 2012) that can both unravel the microbialspecies diversity [e.g. Garcia-Armisen et al. (2010), Illeghems et al.(2012) and Agyirifo et al. (2019)] and the metabolic potential ofthe microbial communities detected, including meta-pathwayreconstruction [e.g. Illeghems, Weckx and De Vuyst (2015b) andAgyirifo et al. (2019)]. All these studies have brought deeperinsights into the microbial community dynamics and speciesdiversity of the complex cocoa fermentation process. Some ofthese studies have been accompanied with a metabolite target

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analysis of pulp and/or beans, unravelling the dynamics of thepulp substrate consumption and conversions as well as those ofthe microbial metabolite production and conversions in the pulpand/or beans, now also including volatile organic compounds ofseed and microbial origin [e.g. Ardhana and Fleet (2003), Camuet al. (2007), Aculey et al. (2010) and Moreira et al. (2016)]. Recentmulti-omic approaches and multivariate analyses have linkedthe microbiology and biochemistry of cocoa pulp-bean mass fer-mentation processes with the flavour profiles of the cured cocoabeans and chocolates produced thereof [e.g. Ho, Fleet and Zhao(2018) and Mota-Gutierrez et al. (2018)].

The actual cocoa fermentation process can be split into twoprogressive phases that differ in environmental conditions, inparticular oxygen availability, which are further referred to as ananaerobic phase and an aerobic phase. Besides microbial activ-ities in the pulp, different enzymatic activities take place insidethe beans during these two cocoa fermentation process phases.

Anaerobic yeast and lactic acid bacteria phase

Yeasts and LABYeasts and LAB grow simultaneously for about 24–36 h of fer-mentation during the anaerobic phase of the cocoa fermenta-tion process, which takes off at an environmental temperatureof around 25–30◦C under carbohydrate-rich and acidic condi-tions (Schwan and Wheals 2004; De Vuyst and Weckx 2016a,b;Figueroa-Hernandez et al. 2019; Munoz et al. 2019). These con-ditions are characterized by high glucose and fructose con-centrations in the mature pulp (formed from sucrose throughmainly pulp invertase activity and—to a lesser extent—yeastinvertase activity) and a low pH of approximately 3.0 (becauseof the presence of citric acid), respectively. This simultane-ous growth of yeasts and LAB may be ascribed to their non-competitive interactions. The yeasts preferentially grow on theavailable glucose, fermenting it to pyruvate through glycolysisfor ATP and reducing equivalent production and further pro-ducing ethanol and carbon dioxide (which maintains the anaer-obic conditions) for redox balancing (Fig. 2). Alternatively, theLAB species that first grow in the cocoa pulp-bean mass and donot require oxygen for growth prefer to consume fructose andperform citric acid conversion (Fig. 2). Fructose is metabolizedeither homofermentatively (glycolysis) or heterofermentatively(phosphoketolase pathway) to pyruvate, while citric acid is con-verted into acetic acid and oxaloacetic acid. The latter is furtherconverted into pyruvate as well, which will finally yield eitherlactic acid, acetic acid, or pyruvate metabolites, in particular2,3-butanedione (diacetyl; buttery notes), 3-hydroxy-2-butanone(acetoin; buttery notes) and 2,3-butanediol (Ganzle 2015; vonWright and Axelsson 2019). The conversion of citric acid into lac-tic acid and acetic acid causes an increase of the pH from 3.0 to4.0, because of the higher pKa values of the latter organic acidscompared to citric acid. This low pH course contributes to themaintenance of a microbiologically stable environment, whichis dominated by dedicated acid-tolerant yeast and LAB species.The growth of LAB may even be favoured by the carbon dioxideproduction by the yeasts and, later on, by the release of vita-mins and other nutrients from yeast cells that autolyze uponfermentation (Agyirifo et al. 2019). It is assumed that yeasts donot contribute to citric acid conversion during cocoa fermenta-tion, as citric acid metabolism by yeasts is aerobic (De Vuyst andWeckx 2016b). Nonetheless, citric acid assimilation can some-times be shown in vitro for yeast isolates from fermenting cocoapulp-bean mass (Jespersen et al. 2005; Daniel et al. 2009; Ho, Zhao

and Fleet 2014; Samagaci et al. 2016). However, the yeasts areresponsible for the depectinization of the pulp through pecti-nase (pectin methylesterase and polygalacturonase) activities(Schwan and Wheals 2004; Meersman et al. 2015, 2017; Sama-gaci et al. 2016). This causes the so-called sweatings of the cocoafermentation process, consisting of a drainage of the depoly-merized pulp as white-yellow to reddish liquid, that either pen-etrate into the ground or are collected for further valorization(Schwan and Wheals 2004; Vasquez et al. 2019). Yet, studiesfocusing on the (bio)chemical analysis of sweatings are veryscarce (Schwan and Wheals 2004; Papalexandratou et al. 2011c;Vasquez et al. 2019). Also, the protease activity of the yeasts maycontribute to a reduction of the shell material of the cocoa beans,which may make them weaker and hence more accessible to aninflux of pulp and/or microbial metabolites upon fermentation(Ho, Zhao and Fleet 2014; Visintin et al. 2016). Furthermore, theyeasts produce glycerol through glycolysis, which contributesto a sweet taste and enhanced mouthfeel (Fig. 2; Rodrigues,Ludovico and Leao 2006; Dzialo et al. 2017). Organic acids arealso formed by yeasts, including acetic acid and succinic acidthrough pyruvate metabolism and tricarboxylic acid (TCA) cycleactivity, respectively (Fig. 2). They contribute to the acidity ofthe beans. Further, the yeasts produce acetoin from acetalde-hyde (green apple notes), which can be further reduced to 2,3-butanediol; similarly, diacetyl can be reduced to acetoin and 2,3-butanediol (Fig. 2). Moreover, the yeasts generate both flavourprecursor molecules and flavour-active compounds, such ashigher alcohols (e.g. 3-methylbutanol and 2-phenylethanol) andesters (e.g. ethyl acetate, ethylphenyl acetate and phenylethylacetate), contributing to the floral and fruity notes of the cocoabeans (see below). Yeasts produce higher alcohols either catabol-ically, via the Ehrlich pathway involving consecutive transam-ination, decarboxylation and dehydrogenation of amino acids,or anabolically, as side-products of amino acid biosynthesisstarting from pyruvate (Fig. 2; Rodrigues, Ludovico and Leao2006; Dzialo et al. 2017). Esters are the condensation productsof ethanol and medium-chain fatty acids or higher alcoholsand acetic acid, yielding ethyl esters and acetate esters, respec-tively. The production of these flavour-active compounds makesthe yeasts indispensable for a successful cocoa fermentationprocess, as these compounds can influence the final chocolatearoma profile (Lefeber et al. 2012; Ho, Zhao and Fleet 2014; Meers-man et al. 2015, 2016; Papalexandratou and Nielsen 2016; Ho,Fleet and Zhao 2018).

During the anaerobic phase, the temperature of the ferment-ing cocoa pulp-bean mass increases from around 25–30◦C to 35–40◦C, due to the fermentation activities. Depectinization anddrainage of the cocoa pulp, which enhances with the increas-ing temperature (either by irreversible degradation of struc-tural components of the pulp pectin or by increased activ-ity of yeast endopolygalacturonase), reduces the pulp viscosityand, together with regular mixing of the cocoa pulp-bean mass,allows air ingress, which favours the growth of the residing bac-teria (Schwan and Wheals 2004; De Vuyst and Weckx 2016b;Meersman et al. 2017). The ethanol produced by the yeasts partlypenetrates into the seed cotyledons, is oxidized into acetic acidby AAB later in the fermentation process, flows away with thesweatings, or evaporates (Fig. 2; Schwan and Wheals 2004; DeVuyst and Weckx 2016b).

A rather wide yeast species diversity has been recovered fromfermenting cocoa pulp-bean mass, belonging to mainly the gen-era Candida, Hanseniaspora, Pichia and Saccharomyces (Ardhanaand Fleet 2003; Jespersen et al. 2005; Nielsen et al. 2005, 2007a;

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Galvez et al. 2007; Daniel et al. 2009; Nielsen, Jakobsen and Jes-persen 2010; Papalexandratou and De Vuyst 2011; Papalexandra-tou et al. 2011c, 2013, 2019; Illeghems et al. 2012; Pereira et al. 2012,2013a, 2017; Crafack et al. 2013; Meersman et al. 2013; Moreiraet al. 2013, 2016, 2017; Pereira, Magalhaes-Guedes and Schwan2013b; Ho, Zhao and Fleet 2014, 2015; Ramos et al. 2014; Arana-Sanchez et al. 2015; Batista et al. 2015, 2016; Hamdouche et al.2015, 2019; Kone et al. 2016; Maura et al. 2016; Menezes et al. 2016;Miescher Schwenninger, Freimuller Leischtfeld and Gantenbein-Demarchi 2016; Samagaci et al. 2016; Visintin et al. 2016; Koffiet al. 2017; Miguel et al. 2017; Bastos et al. 2018; Ho, Fleet andZhao 2018; Mota-Gutierrez et al. 2018, 2019a,b; Romanens et al.2018; Agyirifo et al. 2019; Almeida et al. 2019; Lee et al. 2019; Ooi,Ting and Siow 2020). The most frequently isolated yeast speciesare Hanseniaspora opuntiae in the beginning to the middle of thecocoa fermentation process, and Saccharomyces cerevisiae—andin some cases Pichia kudriavzevii—from the middle and later timepoints of the fermentation process. However, other species ofHanseniaspora and Pichia, and even diverse Candida species (andspecies of other genera) may occur, reflecting a great speciesdiversity among the predominant yeast genera occurring dur-ing cocoa fermentation processes; this also reflects their vari-ous and independent origins (Jespersen et al. 2005; Meersmanet al. 2013; Ludlow et al. 2016; Maura et al. 2016; Koffi et al. 2017;Pereira et al. 2017). All these yeast species mainly differ in acidic,ethanol and heat tolerances, explaining their successive growthand prevalence, although substrate competitiveness may notbe excluded (Daniel et al. 2009; Pereira et al. 2012; Crafack et al.2013; Maura et al. 2016; Samagaci et al. 2016; Visintin et al. 2016).Another factor that may determine the abundance of certainyeast species is killer toxin production, i.e. the production ofoligopeptides that inhibit the growth of other yeasts that do notshow killer activity, albeit in a temperature-dependent manner(Samagaci et al. 2014, 2016; Meersman et al. 2015; Romanens et al.2019; Ruggirello et al. 2019). From the above it can be concludedthat yeasts, being represented by several species, have a deter-mining impact on the efficiency and outcome of the cocoa fer-mentation process, either directly or indirectly.

LABOnce the yeast counts start to decline, because of less favourableenvironmental conditions (less glucose, less anaerobic condi-tions, a slightly higher pH and an increasing temperature) andan increasing competitiveness for growth with the LAB, the lat-ter microbial group further proliferates thanks to the appro-priate growth conditions arising after 24–72 h of fermenta-tion (Schwan and Wheals 2004; De Vuyst and Weckx 2016a,b;Figueroa-Hernandez et al. 2019; Munoz et al. 2019). At the startof the cocoa fermentation process, the prevailing fructophilicLAB species use fructose as energy source, with or without citricacid conversion, thereby avoiding too much competition fromthe yeasts. After the prevailing yeast phase, mainly heterofer-mentative LAB species grow, which ferment glucose to lacticacid, acetic acid, ethanol, carbon dioxide and/or mannitol (Fig. 2;Ardhana and Fleet 2003; Camu et al. 2007, 2008a; Galvez et al.2007; Nielsen et al. 2007a; Kostinek et al. 2008; Lefeber et al.2010, 2011a, 2012; Papalexandratou et al. 2011a,b,c, 2013, 2019;Pereira et al. 2012, 2013a; Adler et al. 2013; Crafack et al. 2013;Meersman et al. 2013; Moreira et al. 2013, 2016; Pereira, Mag-alhaes-Guedes and Schwan 2013b; Ouattara et al. 2014, 2017;Ramos et al. 2014; Ho, Zhao and Fleet 2015; Ho, Fleet and Zhao2018; Mota-Gutierrez et al. 2018; Romanens et al. 2018, 2019; Leeet al. 2019). Mannitol production is caused by the use of fruc-tose as alternative external electron acceptor, which results in a

higher competitiveness due to extra ATP production by convert-ing acetyl phosphate into acetic acid instead of ethanol forma-tion (Zaunmuller et al. 2006; Ganzle 2015; von Wright and Axels-son 2019). This lowers the ratio of the concentrations of lacticacid to acetic acid, which contributes to a lower acidity of thecured beans (Lefeber et al. 2010, 2012). As lactic acid, such asthe volatile acetic acid, may penetrate into the beans during thefermentation process, it may yield too acidic beans for choco-late production. Moreover, because of its non-volatility, lacticacid cannot be removed easily during bean drying and the roast-ing and conching steps of chocolate manufacturing. Hence, thegrowth of LAB is sometimes considered as undesirable or evenunnecessary for the fermentation process. Whereas the formerconcern is related to the acidic defects that may occur (lac-tic acid and acetic acid contribute fresh acid and vinegar-likeacid notes, respectively), the latter is mainly based on the factthat LAB do not contribute to desirable ester formation, whichis necessary for the floral and fruity aromas of the beans (Ho,Zhao and Fleet 2015; Meersman et al. 2015; Miguel et al. 2017;Ho, Fleet and Zhao 2018; John et al. 2019). Besides those of theyeasts, the metabolic contributions of the LAB should neverthe-less be considered as key to a successful cocoa fermentationprocess, encompassing their early growth on fructose, their cit-ric acid conversion, as well as their production of lactic acid,acetic acid and mannitol, when growth on both glucose andfructose occurs (Lefeber et al. 2010, 2011b; Adler et al. 2013, 2014;De Vuyst and Weckx 2016a,b). Although lactic acid itself may notplay the same role as acetic acid in modulating bean pH, pep-tides and flavanols, the effects of citric acid conversion in thepulp (avoiding citric acid transfer into the beans) and reductionof fructose to mannitol (possibly diffusing into the beans) canresult into other processes that contribute to the cocoa flavourthan those caused by acetic acid. This refers in particular to theconversion of amino acids in the pulp (contributing to cocoaflavour production as yeasts do) and the provision of a sweetand cool taste due to mannitol accumulation (Lefeber et al. 2010,2011b; De Vuyst and Weckx 2016a,b; John et al. 2020). As is thecase for acetic acid, lactic acid may display a flavanol-protectiveeffect, i.e. reducing polyphenol degradation (Evina et al. 2016;John et al. 2019). Desirable volatile organic compounds contribut-ing to the cocoa flavour produced by LAB encompass pyru-vate metabolites (diacetyl and acetoin; buttery/creamy notes)and amino acid conversion metabolites, such as the aldehy-des 2-methylbutanal and 3-methylbutanal (malty and chocolatenotes) produced from isoleucine and leucine, respectively, aswell as benzaldehyde (almond and bitter notes) produced fromphenylalanine (see below).

The exhaustion of the energy sources glucose and fructose, acontinuous increase of the ethanol, lactic acid and acetic acidconcentrations, and a rise in temperature of the fermentingcocoa pulp-bean mass eventually cause a decline of the LABcounts and a further decline of the yeast counts (Schwan andWheals 2004; De Vuyst and Weckx 2016b).

The LAB species diversity recovered from fermenting cocoapulp-bean mass is rather restricted. Fructophilic species thatoccur in the early stages of the cocoa fermentation processencompass Fructobacillus pseudoficulneus, Fructobacillus tropeaoliand Leuconostoc pseudomesenteroides, as well as Lactobacillus plan-tarum, the latter species being reclassified as Lactiplantibacillusplantarum (Ardhana and Fleet 2003; Camu et al. 2007, 2008a;Galvez et al. 2007; Nielsen et al. 2007a; Kostinek et al. 2008;Lefeber et al. 2011a; Pereira et al. 2012, 2013a; Papalexandratouet al. 2013; Snauwaert et al. 2013; Pereira, Magalhaes-Guedes andSchwan 2013b; Ho, Zhao and Fleet 2014, 2015; Ramos et al. 2014;

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Hamdouche et al. 2015; Bortolini et al. 2016; Miescher Schwen-ninger, Freimuller Leischtfeld and Gantenbein-Demarchi 2016;Moreira et al. 2016, 2017; Visintin et al. 2016; Miguel et al. 2017;Bastos et al. 2018; Romanens et al. 2018; Agyirifo et al. 2019; Leeet al. 2019). Strictly heterofermentative LAB species that prevailduring the middle and late phases of the cocoa fermentationprocess are mostly represented by Lactobacillus fermentum, whichhas been reclassified as Limosilactobacillus fermentum (Camu et al.2007, 2008a; Nielsen et al. 2007a; Kostinek et al. 2008; Lefeberet al. 2011a; Illeghems et al. 2012; Pereira et al. 2012, 2013a; Mor-eira et al. 2013, 2017; Papalexandratou et al. 2013, 2019; Pereira,Magalhaes-Guedes and Schwan 2013b; Ho, Zhao and Fleet 2014;Ramos et al. 2014; Hamdouche et al. 2015, 2019; Illeghems, DeVuyst and Weckx 2015a; Batista et al. 2016; Bortolini et al. 2016;Miescher Schwenninger, Freimuller Leischtfeld and Gantenbein-Demarchi 2016; Visintin et al. 2016; Miguel et al. 2017; Bastos et al.2018; Mota-Gutierrez et al. 2018; Romanens et al. 2018; Agyirifoet al. 2019; Lee et al. 2019). These species differ in their capaci-ties to use glucose, fructose and/or citric acid. The fructophilicLAB species use fructose as energy source and ferment it to lac-tic acid with or without citric acid as co-substrate. The use ofthe glycolysis as main carbon flux by strains of Lacp. plantarummay explain the low competitiveness of this LAB species uponfurther fermentation, despite their tolerance toward acidity andethanol, as opposed to heterofermentation that can lead to extraATP generation as mentioned above (Lefeber et al. 2010; Adleret al. 2013; Illeghems, De Vuyst and Weckx 2015a). The preva-lence of strictly heterofermentative Liml. fermentum strains canbe ascribed to their conversion of citric acid, production of man-nitol and tolerance toward acidity, ethanol and heat (Lefeberet al. 2010, 2011b; Adler et al. 2013; Illeghems, De Vuyst andWeckx 2015a). However, distinction can be made between het-erolactic Liml. fermentum strains that consume glucose as energysource, fructose as alternative external electron acceptor andcitric acid as co-substrate, to maintain their redox balance and toincrease their intracellular pH and ATP generation for compet-itiveness, and strains that consume fructose as energy sourceand citrate as co-substrate, to increase their citrate flux towardlactic acid to produce enough ATP under glucose-limiting con-ditions (Adler et al. 2013). In the beginning of the cocoa fermen-tation process, also species belonging to the genera Enterococcusand Weissella may occur (Camu et al. 2007,2008a; De Bruyne et al.2008, 2010; Snauwaert et al. 2013; Bortolini et al. 2016; Papalexan-dratou et al. 2019), whereas sometimes other species of the Lacto-bacillaceae (encompassing lactobacilli, Lactobacillus-related gen-era and pediococci) are found during the main LAB fermenta-tion phase (Nielsen et al. 2007b; Kostinek et al. 2008; De Bruyneet al. 2009; Illeghems et al. 2012; Moreira et al. 2013; Papalexan-dratou et al. 2013, 2019; Ramos et al. 2014; Ho, Zhao and Fleet2015; Batista et al. 2016; Menezes et al. 2016; Miescher Schwen-ninger, Freimuller Leischtfeld and Gantenbein-Demarchi 2016;Miguel et al. 2017; Agyirifo et al. 2019).

Aerobic acetic acid bacteria phase

The AAB species that are already present from the beginning ofthe cocoa fermentation process and have survived the anaer-obic yeast and LAB growth phases start to grow during theaerobic phase, which occurs after approximately 48 h of fer-mentation (Schwan and Wheals 2004; De Vuyst and Weckx2016a,b; Figueroa-Hernandez et al. 2019; Munoz et al. 2019). Thisis enabled by further air ingress upon sweating and regular mix-ing of the cocoa pulp-bean mass during fermentation. They

reach their maximal counts after 72–96 h of fermentation. Pos-sessing a non-functional glycolysis, AAB, in particular Aceto-bacter species, oxidize the ethanol produced by the yeasts intoacetic acid (partial oxidation for ATP generation) (Fig. 2). Becauseof this exothermic reaction, they increase the temperature toand even beyond 50◦C. Whereas ethanol serves as the mainenergy source, lactate produced by the LAB is the main carbonsource for the Acetobacter species (Camu et al. 2007, 2008a; Adleret al. 2014; Moens, Lefeber and De Vuyst 2014; Pelicaen et al.2019). Simultaneously with the oxidation of ethanol, lactic acidis oxidized mainly into acetoin (via α-acetolactate) and, to a lowextent, into acetic acid because of a low pyruvate decarboxylaseactivity of Acetobacter (Fig. 2; Adler et al. 2014; Moens, Lefeber andDe Vuyst 2014; Pelicaen et al. 2019). Moreover, lactic acid can beused as the sole carbon source by Acetobacter pasteurianus (Peli-caen et al. 2019). Hence, the highest acetic acid concentrationsare produced in the presence of both yeasts and LAB, under-lying the importance of the latter microbial group, as balancedconcentrations of ethanol and lactic acid are necessary for opti-mal growth of Acetobacter (Adler et al. 2014). This produces thenecessary acetic acid for bean curing. Acetoin is an overflowmetabolite of the formation of pyruvate from lactic acid thattakes place instead of pyruvate oxidation through the TCA cyclein the case that ethanol (repressing the TCA cycle and hence pre-venting catabolic breakdown of acetic acid) is oxidized to aceticacid (Fig. 2). As a result, biomass formation from lactic acid accel-erates, for instance through the amino acid supply from pyru-vate (Adler et al. 2014). The oxidation of the non-volatile lacticacid contributes to the acidity (pH-lowering effect) and flavour(less acidic taste, buttery flavour) of the cured cocoa beans. As forthe growth of LAB, the growth of AAB is sometimes consideredunnecessary for cocoa fermentation, as too high acetic acid con-centrations may cause too acidic beans and as they have no sig-nificant direct impact, compared to yeasts, on the production ofdesirable volatile organic compounds in both beans and choco-lates (Ho, Zhao and Fleet 2015; Ho, Fleet and Zhao 2018). How-ever, the acetic acid produced by AAB is indispensable for cocoabean curing (Schwan and Wheals 2004; Misnawi 2008; Kongoret al. 2016; De Vuyst and Weckx 2016a,b; Munoz et al. 2019). Theminimization of the role of LAB and AAB by Ho and collabora-tors has been based on the investigation of controlled cocoa fer-mentation processes, carried out in small plastic boxes, in thepresence and absence of appropriate antibiotics to investigatethe role of individual microbial groups, in particular yeasts (Ho,Zhao and Fleet 2014, 2015; Ho, Fleet and Zhao 2018). However,during these small-scale plastic box fermentations, the growthof LAB and AAB was not prevented completely and neither couldthe effect of increased concentrations of reducing sugars, aminoacids and pyrazines be explained by favoring the growth ofyeasts only.

Although part of the acetic acid evaporates, the highest tem-peratures in the fermenting cocoa pulp-bean mass are reachedif further overoxidation of acetic acid, and also of lactic acid(and mannitol), into carbon dioxide and water takes place bythe prevailing AAB (Fig. 2; Schwan and Wheals 2004; De Vuystand Weckx 2016b; Figueroa-Hernandez et al. 2019; Munoz et al.2019). This overoxidation causes a slight pH increase, whereasthe high temperature finally halts the cocoa fermentation pro-cess, resulting in a decline of the yeast, LAB and AAB communitycounts but possibly favouring the outgrowth of residing Bacillusspores (Schwan and Wheals 2004; De Vuyst and Weckx 2016b;Figueroa-Hernandez et al. 2019; Munoz et al. 2019).

The AAB species diversity recovered from fermenting cocoapulp-bean mass is even more restricted than that of the LAB.

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It encompasses Acetobacter ghanensis and Acetobacter senegalen-sis that mainly occur at the start of the cocoa fermentationprocess, whereas A. pasteurianus—and in some cases Acetobac-ter lovaniensis, Acetobacter syzygii, or Acetobacter tropicalis—prevailduring the last phase of this process (Ardhana and Fleet 2003;Camu et al. 2007, 2008a; Cleenwerck et al. 2007, 2008; Galvez et al.2007; Nielsen et al. 2007a; De Vuyst et al. 2008; Papalexandratouet al. 2009, 2011a,b,c, 2013; Lefeber et al. 2011a; Illeghems et al.2012; Pereira et al. 2012, 2013a; Romero et al. 2012; Romero-Corteset al. 2012; Crafack et al. 2013; Meersman et al. 2013; Pereira,Magalhaes-Guedes and Schwan 2013b; Ho, Zhao and Fleet 2014,2015; Ramos et al. 2014; Yao et al. 2014; Soumahoro et al. 2015;Batista et al. 2016; Bortolini et al. 2016; Miescher Schwenninger,Freimuller Leischtfeld and Gantenbein-Demarchi 2016; Moreiraet al. 2016, 2017; Visintin et al. 2016; Bastos et al. 2018; Ho, Fleetand Zhao 2018; Mota-Gutierrez et al. 2018; Romanens et al. 2018;Agyirifo et al. 2019; Hamdouche et al. 2019; Lee et al. 2019).These species differ in the speed with which they can oxidizeethanol and lactic acid simultaneously (Moens, Lefeber and DeVuyst 2014). The prevalence of A. pasteurianus strains is dueto their ethanol-, lactic acid- and mannitol-oxidizing capaci-ties and their tolerances toward acidity and heat (Lefeber et al.2011b; Illeghems, De Vuyst and Weckx 2013; Moens, Lefeber andDe Vuyst 2014; Illeghems et al. 2016). The growth of glucose-oxidising Gluconobacter and Gluconacetobacter species that can bepresent in the fermenting cocoa pulp-bean mass is not desir-able, as this may lead to the production of gluconic acid, whenethanol and lactic acid are depleted. This mainly occurs whenno appropriate substrate degradation and metabolite produc-tion by the prevailing microorganisms occurs in the first phaseof the cocoa fermentation process, leaving glucose and gluconicacid for AAB and yeast growth late in the fermentation pro-cess and possibly producing off-flavours (Papalexandratou et al.2011c; Moens, Lefeber and De Vuyst 2014).

Drying phase

Drying, either sun drying on platforms or artificial drying, ofthe fermented cocoa beans reduces their moisture content andhence the occurrence of living microorganisms (Schwan andWheals 2004; De Vuyst and Weckx 2016a,b; Figueroa-Hernandezet al. 2019; Munoz et al. 2019). Under conditions of a higher tem-perature, higher pH and higher availability of oxygen during thedrying step compared to the fermentation step, certain yeast,LAB and AAB species can be recovered, besides bacilli and fil-amentous fungi (Schwan and Wheals 2004; Hamdouche et al.2015; Romanens et al. 2018). During the drying phase, severalcocoa bean compounds are degraded or modified and others canbe produced (e.g. tetra- and trimethylpyrazines through Maillardreactions under the appropriate environmental conditions).

Enzymatic and non-enzymatic conversions inside thecocoa beans during cocoa fermentation

Anaerobic and aerobic phasesThe acetic acid produced by the AAB penetrates into the cocoaseeds and decreases the pH of the cotyledons from 6.5 to 4.8;due to this low pH in the cotyledons, the undissociated aceticacid kills the seed embryo, together with the ethanol ingressand the heat effect of the fermentation process (Fig. 2; Schwanand Wheals 2004; Misnawi 2008; Kongor et al. 2016; De Vuystand Weckx 2016a,b; Munoz et al. 2019). This prevents seed ger-mination and destroys the internal seed cotyledon structurecompletely. These physicochemical changes result in desirable

enzymatic and non-enzymatic conversions and the release ofseed compounds, as mentioned above. All this is affected by thelength of the anaerobic and aerobic phases, which is determinedby the fermentation progress that in turn determines the speedof the courses of the pH decrease and temperature increase, theduration and extent of the seed acidification, the final seed pH(poorly fermented beans have a high pH of 5.5–5.8, whereas well-fermented beans have a low pH of 4.7–5.4) and the oxygen ten-sion (Schwan and Wheals 2004; Afoakwa et al. 2008; De Vuystand Weckx 2016a,b; Munoz et al. 2019; John et al. 2020). Hence,the different endogenous enzymes will become gradually acti-vated and inactivated as a function of time during the fermen-tation and drying steps [older literature reviewed in Schwan andWheals (2004), Kongor et al. (2016), De Vuyst and Weckx (2016a,b),Munoz et al. (2019) and Rawel et al. (2019)].

During the aerobic phase, the seed polyphenol oxidasebecomes active (Fig. 2). Its pH optimum is situated at a slightlyacidic pH of 5.4, but the enzyme is gradually inactivated dur-ing fermentation and drying. Because it produces quinones from(epi)catechin through oxidation, it contributes to the desirablebrown pigmentation of the cured cocoa beans. During the pre-ceding anaerobic phase, the seed invertase, glycosidases andproteases are active (Fig. 2). The invertase, optimally active atan acidic pH of 4.5, is active mainly at the beginning of thefermentation process and hydrolyses sucrose into the reduc-ing sugars glucose and fructose that serve as flavour precur-sors. Invertase loses its activity upon fermentation, due to theincreasing temperature. The glycosidases α-arabinosidase andβ-galactosidase, optimally active under acidic conditions (pH4.0–4.5), elaborate sugar moieties from anthocyanins (arabinoseand galactose from cyanidin-3-α-L-arabinoside and cyanidin-3-β-D-galactoside, respectively) and convert them into antho-cyanidins. Moreover, glycosylated terpenoids (e.g. linalyl glyco-side) are hydrolysed enzymatically. Proteases, mainly asparticendoprotease (optimally active at an acidic pH of 3.5) and ser-ine carboxypeptidase (optimally active at a higher but slightlyacidic pH of 5.8), act on a vicilin-class (7S) globulin (composed ofthree subunits of 47, 31 and 15 kDa, respectively) and an albu-min (21 kDa), the main cocoa seed proteins. Major differencesin protein content of non-fermented cocoa beans are attributedto their geographical origin (Kumari et al. 2018). However, a sim-ilarity in protein profiles of cocoa beans from the same regiondoes not necessarily mean that they belong to the same cocoahybrid (Kumari et al. 2018). The cooperative action of both pro-teases is necessary to form the desirable cocoa aroma precursorsto generate volatile organic compounds with cocoa-specific andnutty notes (Voigt, Taube and Wostemeyer 2018). This greatlydepends on the degree and time course of acidification, giventheir different pH optima. In particular, the aspartic endopro-tease releases a limited number of hydrophobic oligopeptides,more from the cocoa seed vicilin than albumin, in a differen-tial and sequential cleavage site-specific way. These hydropho-bic oligopeptides are then converted by the cleavage-specificexo-carboxypeptidase into tens to hundreds of hydrophilic pep-tides. This lowers the ratio of vicilin to albumin seed proteinsand increases the amounts of oligopeptides and free hydropho-bic amino acids, in particular isoleucine, leucine, valine, ala-nine, phenylalanine and tyrosine. Such hydrophilic peptides andhydrophobic amino acids serve as cocoa flavour precursors (deBrito et al. 2000; Bucheli et al. 2001; Buyukpamukcu et al. 2001;Amin et al. 2002, 2003; Laloi et al. 2002; Yusep et al. 2002; Schwanand Wheals 2004; Guilloteau et al. 2005; Rohsius, Matissek andLieberei 2006; Jinap et al. 2008; Kratzer et al. 2009; Adeyeye et al.2010; Bertazzo et al. 2011; Romero-Cortes et al. 2013; Marseglia

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et al. 2014; Kadow et al. 2015; Caligiani et al. 2016; Hue et al. 2016;John et al. 2016, 2019, 2020; Kongor et al. 2016; Kumari et al. 2016,2018; Mayorga-Gross et al. 2016; Sousa et al. 2016; Voigt et al. 2016;Janek et al. 2016a,b; Apriyanto et al. 2017; D’Souza et al. 2018; Mor-eira et al. 2018; Voigt, Taube and Wostemeyer 2018; Rottiers et al.2019; Scalone et al. 2019). The peptide profiles of cured beans interms of number, diversity and relative abundance reflect theirfermentation degree. This is independent of their geographi-cal origin, which is not the case for the protein contents of thebeans (Mayorga-Gross et al. 2016; D’Souza et al. 2018; Kumari et al.2018). A maximum number of peptides is found after 48–96 hof fermentation (Mayorga-Gross et al. 2016; D’Souza et al. 2018;Munoz et al. 2019). However, the size of the peptides decreasesas a function of the fermentation time, although several smallpeptides can originate from degradation of multiple proteins(other than vicilin and albumin). These peptide profiles or thenumber of the diverse peptides can thus serve as a biomarkerfor the degree of fermentation and hence replace the classicalcut test (qualitative colour-based model) or fermentation indexmeasurement (quantitative colour-based model) (Misnawi 2008;Aculey et al. 2010; Romero-Cortes et al. 2013; D’Souza et al. 2018;Hinneh et al. 2018; Kumari et al. 2018). However, only certainpeptides are considered as being of influence on the uniquecocoa aroma, although their number is still a matter of debate(Buyukpamukcu et al. 2001; Marseglia et al. 2014; Kumari et al.2016, 2018; Voigt et al. 2016; D’Souza et al. 2018; Voigt, Taubeand Wostemeyer 2018; Scalone et al. 2019). Moreover, in vitroproteolytic digestions of cocoa vicilin indicate that short pep-tides (formed at acidic pH of 4.4–5.2) might be cocoa-specificaroma compound precursors, whereas slightly longer peptides(formed at slightly acidic but higher pH of 4.8–5.6) might benutty-specific aroma compound precursors (Voigt, Taube andWostemeyer 2018). Hence, cocoa fermentation processes thatresult in a strong acidification inside the beans, the pH of whichdecreases below 5.2, could result in cured cocoa beans with ahigh cocoa aroma. In contrast, those processes that result in aweak acidification inside the beans, leading to a pH above 5.2,could result in cured cocoa beans with a high nutty aroma. Thisagain underlines the importance of the extent and speed of thepH course inside the cocoa beans during fermentation.

Cocoa bean flavour formationChocolate possesses a characteristic aroma and taste,composed of acidity, astringency and bitterness, andcocoa/coffee/nutty/roasted, floral, fruity and spicy notes,besides delivering a melt-in-the-mouth pleasure (Frauendorferand Schieberle 2006, 2008, 2019; Afoakwa et al. 2008; Diabet al. 2014; Aprotosoaie, Luca and Miron 2016; Engeseth andPangan 2018; Barisic et al. 2019; Munoz et al. 2019). The cocoaand chocolate aromas, composed of a mixture of hundreds ofcompounds, are determined by both volatile (mainly aldehydes,esters, organic acids, pyrazines and diketopiperazines) andnon-volatile organic compounds (mainly amino acids, otherorganic acids, saccharides and proanthocyanidins) (Aculey et al.2010; Rodriguez-Campos et al. 2011, 2012; Owusu, Petersen andHeimdal 2012, 2013; Crafack et al. 2014; Ho, Zhao and Fleet2014;Tran et al. 2015; Batista et al. 2016; Meersman et al. 2016;Menezes et al. 2016; Moreira et al. 2016, 2017, 2018; Pereira et al.2017; Visintin et al. 2017; Cevallos-Cevallos et al. 2018; Cuellaret al. 2018; Hinneh et al. 2018; Ho, Fleet and Zhao 2018; Mota-Gutierrez et al. 2018, 2019b; Assi-Clair et al. 2019; Hamdoucheet al. 2019; Lee et al. 2019; Rottiers et al. 2019; Utrilla-Vazquezet al. 2020). These flavour constituents can be of endogenous(pulp and beans) or fermentation origin.

Besides mono- and disaccharides and organic acids (mainlycitric acid next to malic acid, succinic acid and oxalic acid), theaqueous unfermented pulp contains acetophenone (1-phenylethanone; floral/fruity notes), aldehydes (e.g. phenylacetalde-hyde; floral/honey notes), esters (e.g. 2-pentyl acetate or amylacetate; fruity notes), methylketones (e.g. 2-heptanone; cheese-like notes; precursor of 2-heptanol), secondary alcohols (e.g.2-heptanol; fruity/floral/spicy notes) and monoterpenes [con-tribute sweet and flowery notes; e.g. linalool (flowery/leafy/tea-like notes), myrcene (spicy notes) and ocimene (floral/herbalnotes)] (Kadow et al. 2013; Chetschik et al. 2018). Monoterpenesderive from the methyl-erythritol 4-phosphate (MEP) pathwayof the cocoa plant (with carbohydrates as precursors), whereasmethylketones (from secondary alcohols), secondary alcoholsand esters derive from the fatty acid metabolism of the cocoaplant. Moreover, acetate ester formation may take place duringmigration of acetic acid into the cotyledon tissue.

Fermentation of the cocoa pulp-bean mass leads to the pro-duction of (higher) alcohols, aldehydes, ketones, organic acidsand sugar alcohols (LAB and yeasts) and esters (yeasts), asdescribed above (Fig. 2; Schwan and Wheals 2004; De Vuyst andWeckx 2016a,b; Figueroa-Hernandez et al. 2019; Munoz et al. 2019;Mota-Gutierrez et al. 2019b). These compounds are producedin the aqueous phase of the cocoa pulp and contribute to thefermentative flavour of the cured cocoa beans. All these com-pounds can diffuse into the beans, as has been shown duringfermentation-like incubations (Kadow et al. 2013; Ho, Zhao andFleet 2014; Sukha, Umaharan and Butler 2017; Chetschik et al.2018; Ho, Fleet and Zhao 2018; Castro-Alayo et al. 2019). How-ever, some studies on cocoa pulp-bean mass fermentation donot clearly distinguish between flavour (precursor) formation inthe pulp, where the actual fermentation takes place, and thoseprocesses that occur in the beans. In the latter case, volatileorganic compounds can be the result of influx of both micro-bial and pulp metabolites and/or endogenous pathways or enzy-matic conversions taking place in the cocoa beans. Hence, thechocolate flavour is the result of an interplay between the ini-tial composition of the cocoa pulp and beans and the post-harvest treatments of the cocoa pulp-bean mass (methods,duration and mixing frequency of fermentation and drying),which determines the influx and efflux of flavour and health-related cocoa compounds, and the subsequent processing of thecured cocoa beans during chocolate-making (in particular roast-ing, alkalization and conching) (Guehi et al. 2010; Rodriguez-Campos et al. 2011, 2012; Owusu, Petersen and Heimdal 2012,2013; Saltini, Akkerman and Frosch 2013; Suazo, Davidov-Pardoand Arozarena 2014; Tran et al. 2015; Cevallos-Cevallos et al.2018; Castro-Alayo et al. 2019; Hamdouche et al. 2019; Rottierset al. 2019; Hinneh et al. 2019a,b). All these subprocesses havean impact on the dynamics and, therefore, end-concentrationsof the non-volatile and volatile organic flavour compounds thatlead to taste and aroma, respectively.

The volatile fraction of cocoa beans that are ready forchocolate-making is mainly the result of Maillard reactions andStrecker degradation among the flavour precursor molecules[hydrophilic peptides, (hydrophobic) amino acids and reducingsugars elaborated during the fermentation and/or drying steps]that take place during the cocoa bean roasting process, withwhich chocolate manufacturing actually starts (Afoakwa et al.2008; Frauendorfer and Schieberle 2008; Huang and Barringer2011; Owusu, Petersen and Heimdal 2012, 2013; Diab et al. 2014;Engeseth and Pangan 2018; Barisic et al. 2019; Frauendorferand Schieberle 2019; Munoz et al. 2019; Mota-Gutierrez et al.2019b). These reactions produce Strecker amines, carboxylic

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acids [e.g. 2-methyl butanoic acid (or 2-methyl butyric acid)and 3-methylbutanoic acid (isovaleric acid)], and—aboveall—Strecker aldehydes and heterocyclic aroma compounds(furans, furanones, imidazoles, oxazoles, pyrazines, pyridines,pyrroles and thiazoles), which generate different flavournotes in the final chocolate. Additionally, polymerizationreactions produce melanoidin pigments that are responsiblefor the brown colour. 2,5-Diketopiperazines (bitter taste) aresemi-volatile cyclic dipeptides formed from specific pep-tide precursors during roasting (Andruszkiewicz et al. 2019).The most abundant Strecker aldehydes that contribute tochocolate notes are acetaldehyde (originating from alanine;fruity notes), benzaldehyde (phenylalanine; sweet, bitter andalmond notes), 3-methylbutanal (leucine; malty/chocolatenotes), 2-methylbutanal (isoleucine; malty/chocolate notes),2-methylpropanal (valine; malty/nutty/chocolate notes)and phenylacetaldehyde (phenylalanine; floral/honeynotes). The most abundant pyrazines that contribute toburnt/cocoa/coffee/earthy/green/nutty/roasty/toasty notesare 2,3,5,6-tetramethylpyrazine, 2,3,5-trimethylpyrazine, 2,5-dimethylpyrazine and 2,3-dimethylpyrazine. Diacetyl andacetoin are precursors for tetramethylpyrazine formation.Subsequent reactions further produce various compounds,such as the cocoa hexenal (5-methyl-2-phenyl-2-hexenal orbenzeneacetaldehyde; sweet, roasted, rhum- and cocoa-likenotes; bitter taste), produced from phenylalanine, in particularduring roasting and conching. Proline conversion leads to 2-acetyl-1-pyrrole, which contributes to caramel/chocolate/roasty(popcorn-like) notes. Some of the sweet and floral/flowery notescan be obtained from the reduction of 2,3-butanedione andacetoin to 2,3-butanediol upon prolonged conching, as wellas the conversion of benzaldehyde and phenylacetaldehydeinto benzyl alcohol and phenylethyl alcohol, respectively.Degradation of monosaccharides yields furanones (e.g. 4-hydroxy-2,5-dimethyl-3(2H)furanone or furaneol) and pyrones(e.g. 3-hydroxy-2-methyl-4-pyrone), which contribute to thecaramel-like notes of chocolate.

Some of the compounds mentioned above are already ini-tially present in the fermented, dry, unroasted cocoa beans.Indeed, fresh cocoa beans contain secondary alcohols (e.g. 2-heptanol and 2-phenylethanol; floral and fruity notes), alde-hydes (e.g. 2-butanal and 2-hexanal; fruity and grassy notes,respectively), ketones (e.g. methylketones, such as acetophe-none and 2-heptanone; floral and fruity notes) and organicacids (e.g. 2-methylbutanoic acid and 3-methylbutanoic acid;sweaty notes). These compounds may increase (e.g. aldehydesand organic acids) or decrease (e.g. ketones) upon curing of thecocoa beans, being the result of biosynthesis or degradation(seed metabolism), modification (Maillard reactions as describedabove and oxidation of, for instance, linalool to linalool oxide)and influx (transfer of compounds produced in the pulp micro-bially) or efflux (leakage of compounds from the cocoa beansor volatilization from the pulp and beans) (Rodriguez-Camposet al. 2011, 2012; Castro-Alayo et al. 2019; Hamdouche et al. 2019;Rottiers et al. 2019; Mota-Gutierrez et al. 2019b). For instance,the microbial metabolites that are formed during fermentationand the plant metabolites, which are cultivar- and genotype-dependent, may transfer from the pulp through the seed shellthat is likely weakened by fermentation, when the cocoa fer-mentation process proceeds. Here, the role of yeasts and LABis thus of primordial importance to produce (sugar/higher) alco-hols, aldehydes and/or esters during fermentation of the cocoapulp-bean mass. Although pyrazines may also be produced bybacilli during (prolonged) fermentation, they are mainly formed

during drying (in particular tetra- and trimethylpyrazine) androasting (all pyrazines).

However, the production of flavour-active compounds is notalways desirable and can in some instances lead to sensorydefects (Schwan and Wheals 2004; Afoakwa et al. 2008). Althoughit is not always fully clear what is driving this problem ofoff-flavours, over-fermentation and poor drying may be causalfactors, the former leading to an increase in certain bacilliand filamentous fungi and the latter often being ascribed tofailing infrastructure that can both produce these off-flavourcompounds. Such off-flavours are mostly composed of amylacetates, short- and medium-chain fatty acids and phenols(e.g. 2-methoxyphenol or guaiacol), which deliver undesirable,hammy, putrid and smoky notes when formed in excess.

Use of starter cultures

The knowledge on the microbial succession and activities ofthe most prevalent yeast, LAB and AAB species involved incocoa fermentation processes obtained during the last decadeshas prompted the search for appropriate single- or mixed-strain starter cultures for controlled and steered cocoa fermen-tation processes (Schwan and Wheals 2004; Saltini, Akkermanand Frosch 2013; Pereira, Soccol and Soccol 2016; De Vuystand Weckx 2016a,b; Ozturk and Young 2017; Castro-Alayo et al.2019; Figueroa-Hernandez et al. 2019; Munoz et al. 2019; Mota-Gutierrez et al. 2019b). It is now generally accepted that themicrobial activities during fermentation and the subsequenttransfer of volatile organic compounds into the beans play adetermining role in the flavour development of cured cocoabeans and, possibly, chocolates produced thereof. In addition,the cocoa genotype (in particular cocoa bean storage proteins,polysaccharides and polyphenols) and the temperature and pHcourses during fermentation, which determine the enzymaticactivities in the beans, are of crucial importance (Schwan andWheals 2004; Lima et al. 2011; Saltini, Akkerman and Frosch2013; De Vuyst and Weckx 2016a,b; Castro-Alayo et al. 2019;Figueroa-Hernandez et al. 2019; Munoz et al. 2019). It is not clearyet if the fermentation method applied may influence flavourdevelopment, as literature on the comparison of heap, box andtray fermentations is controversial, although there are indica-tions that different fermentation methods may alter the flavourcharacteristics of cocoa liquors and conched chocolates (Owusu,Petersen and Heimdal 2012, 2013; Crafack et al. 2014; Ganeswariet al. 2015; Mota-Gutierrez et al. 2018).

The use of starter cultures may allow faster, time-controlledand efficient cocoa fermentation processes and deliver highyields of cured cocoa beans of consistent and high quality.In turn, this may result in more profit for the farmers, thecocoa traders and the chocolate manufacturers, as further out-lined below. Introducing the practice of starter culture-initiatedcocoa fermentation processes has of course been a challenge,but once proof-of-principle was delivered the research focusshifted from accelerated and time-controlled fermentation pro-cesses (directed by the speed of pulp degradation and carbohy-drate conversion) toward the use of starter cultures with extrafunctional properties, in particular regarding flavour formation.The first starter cultures that have been developed indeed havetargeted an enhanced flow of sweatings by means of pecti-nolytic strains of dedicated yeast species applied as the solestarter cultures, among which S. cerevisiae var. chevalieri andKluyveromyces fragilis (not necessarily fermenting cocoa pulp-bean mass isolates), to improve the pulp breakdown. Although

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this may increase the aeration of the fermenting cocoa pulp-bean mass and decrease the amounts of pulp monosaccharidesavailable for fermentation to organic acids, leading to reducedbean acidity, it has not affected the cocoa bean quality sig-nificantly [Sanchez et al. 1985; Samah, Ptih and Selamat 1992;Buamah, Dzogbefia and Oldham 1997; Dzogbefia, Buamah andOldham 1999; reviewed in Schwan and Wheals (2004) and DeVuyst and Weckx (2016a,b)]. Also, AAB have been used as the soleinoculum, whereby the application of a starter culture strain ofGluconacetobacter xylinus subsp. xylinus has resulted in a fermen-tation process without intermediate LAB fermentation phaseand cured cocoa beans with lower pH and higher acetic acidconcentrations (Samah et al. 1993). However, less cocoa aromahas been obtained than in naturally cured cocoa beans. Yet,greater production of acetic acid by an A. pasteurianus starterculture strain, in combination with a S. cerevisiae starter culturestrain, not only leads to a higher acidic taste but also preservespolyphenols from leakage, resulting in a higher bitterness (Johnet al. 2019).

Concerning functional starter cultures that prevail during thecocoa fermentation process, a direct impact of such a starterculture strain could be the production of flavour-active com-pounds, whereas an indirect impact could be a limitation ofthe production of undesirable off-flavours or compounds. How-ever, the application of flavour-producing starter culture strainsassumes that the flavour precursor molecules or flavour-activecompounds are not only produced in the pulp, where the actualcocoa fermentation process takes place, but also diffuse fromthe pulp into the beans during fermentation, the bean shellbeing the barrier for this transfer as outlined above (Kadow et al.2013; Ho, Zhao and Fleet 2014; Sukha, Umaharan and Butler2017; Chetschik et al. 2018; Ho, Fleet and Zhao 2018; Castro-Alayo et al. 2019). Moreover, once inside the beans, the flavour-active compounds have to be retained during the drying stepand to survive the chocolate-making process to be of impacton the final chocolate flavour. The reason why many studies donot reveal significant improvement of chocolate flavour uponthe use of starter cultures, compared with a spontaneous fer-mentation process, may be ascribed to a production of off-flavours by the background microbiota in the case that theadded starter culture is not competitive enough to allow forits prevalence. Concerning the choice of candidate functionalstarter cultures, distinction should be made between studiesthat have tested culture collection or commercial strains thatdo not necessarily originate from fermenting cocoa pulp-beanmass and studies that have first initiated a thorough screen-ing of strains with desirable functional properties. In almost allstudies the starter cultures were applied as liquid cultures. Allstudies have used different fermentation recipients and batchsizes.

Yeast starter culturesExcept for an early Brazilian study (Schwan 1998), the firststarter culture-initiated cocoa fermentation studies all dealtwith single- or mixed-strain starter cultures involving yeasts.Indeed, Schwan (1998) was the first to evaluate the use of a con-sortium of cocoa pulp-bean mass-derived strains as starter cul-ture for cocoa fermentation processes carried out in woodenboxes (200 kg, Comum hybrid), applying the microbial inocu-lum either at the start of the fermentation process or throughphased addition. The selected strains in this study belong to thepectinolytic, acid-tolerant and ethanol-tolerant yeast species S.cerevisiae var. chevalieri, the lactic acid-producing, acid-tolerant

and heat-tolerant LAB species Lactobacillus lactis (now reclas-sified as Lactobacillus delbrueckii subsp. lactis) and Lacp. plan-tarum and the ethanol-(over)oxidizing, acetic acid-producing,heat-tolerant AAB species Acetobacter aceti and Gluconobacter oxy-dans. This has resulted in a fermentation process comparablewith a natural one, normally cured cocoa beans and accept-able chocolates, but with no enhanced quality. This proof-of-principle study has been followed by tests with pectinase-producing or flavour-producing yeast strains to accelerate thecocoa fermentation process (flow of sweatings) and improvethe flavour of the cured cocoa beans and chocolates producedthereof. First, as follow-up of the study of Sanchez et al. (1985)that has examined strains of several yeast species involved incocoa fermentation processes as to their capacity to ferment anddepectinize cocoa pulp, a yeast starter culture composed of aKluyveromyces marxianus hybrid strain (MMIII-41), not of cocoaorigin but with enhanced pectinolytic activity, has allowed tonot only increase the flow of sweatings but also obtain curedcocoa beans of improved quality and chocolates with enhancedsensory attributes, after fermentation of Brazilian cocoa beans(45 kg) in plastic baskets (Leal et al. 2008). The addition of a strainof S. cerevisiae (UFLA CA11) to cocoa pulp-bean mass of differentcocoa hybrids from Ecuador and Brazil (CCN51, CEPEC2004, FA13,PH16, PS1030, FA13 and/or PS1319) has been tested in woodenboxes (60–100 kg) and has shown an accelerated fermenta-tion process. This has paralleled faster carbohydrate consump-tion and ethanol production, preventing undesirable microbialgrowth. An impact on the volatile organic compounds producedand the resulting chocolate flavour attributes has been found,although this depends on the hybrid used, likely due to differ-ences in pulp composition (Ramos et al. 2014; Menezes et al.2016). An enhanced pulp degradation by inoculating S. cere-visiae var. chevalieri has been confirmed in the case of Indone-sian Forastero cocoa fermentation in plastic bags (Cempakaet al. 2014). An acceleration of the cocoa fermentation processhas also been achieved after simultaneous inoculation of thecocoa pulp-bean mass (100 kg of the PS1319 hybrid in woodenboxes) with selected strains of S. cerevisiae (UFLA CA11), Pichiakluyveri (UFLA YCH194) and H. uvarum (UFLA YCH203), with a 2-log higher cell density of S. cerevisiae, although H. uvarum wassuppressed by the two former yeast species. Eventually, thefinal chocolates possessed a flavour described as bitter, cocoa,coffee, fruity and sour (Batista et al. 2015, 2016). Given its fre-quent isolation from fermenting cocoa pulp-bean mass, a Can-dida sp. starter culture has been tested in Malaysia to fermentcocoa pulp-bean mass (5 kg) in a basket, showing inhibition ofpathogenic bacteria (Mahazar et al. 2015).

Hybrid yeast strains of S. cerevisiae obtained through selec-tive breeding to get an improved heat tolerance and fermen-tation capacity, including high aroma production, have beentested during laboratory- (100 mL of pulp in 250-mL glass bot-tles) and pilot-scale fermentations (50 kg of cocoa pulp-beanmass in baskets) as single-strain starter cultures (thus withoutthe addition of LAB and/or AAB strains) (Meersman et al. 2015,2016). These Malaysian cured cocoa beans have led to the man-ufacturing of high-quality chocolates, as reflected in the flavourprofiles of the cocoa liquors and conched chocolates. This hasbeen related to the production of flavour-active ethyl esters andacetate esters, especially long and more fat-soluble ones, asshort-chain ethyl esters decrease or disappear upon process-ing and a decrease in spoilage-related off-flavours, which washowever strain-dependent. Yet, some of the strains tested havenot prevailed during the whole fermentation process. This studyhas further shown that moderate acidification, in combination

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with a slow and delayed temperature increase, can result in ahigh flavour profile of the cocoa beans and chocolates therefrom(Meersman et al. 2016; Papalexandratou and Nielsen 2016). Alsothe application of strains of P. kluyveri (Indonesian cocoa isolate)and Cyberlindnera fabianii (non-cocoa origin) that produce highconcentrations of fruity esters has enabled the modulation ofthe flavour of the chocolates produced into a sweet taste withfruity, cocoa and roasty notes. An influence on volatile organiccompound production has nonetheless been limited, as thesenon-Saccharomyces yeasts could not prevail during fermentationof the cocoa pulp-bean mass due to their poor competitivenessin a cocoa fermentation ecosystem (Meersman et al. 2016).

Other screening studies have looked at the tolerance towardacidity, ethanol, heat and osmotic stress and/or the enzymaticpotential of candidate yeast strains (Samagaci et al. 2014, 2015;Visintin et al. 2016; Koffi et al. 2018). For instance, pectinolytic P.kudriavzevii and Candida nitrativorans strains isolated from Ivo-rian cocoa bean heap fermentation processes have been pro-posed as candidate yeast starter cultures, solely or in combi-nation (Samagaci et al. 2014, 2015, 2016). It has further beenshown that a great genetic and metabolic intraspecies diversityoccurs among P. kudriavzevii strains regarding the production ofvolatile organic compounds during laboratory-scale fermenta-tions (400 g of cocoa pulp-bean mass in 1-L glass bottles) (Pereiraet al. 2017). Hence, the selection of two highly aromatic strainsof P. kudriavzevii (LPB06 and LPB07) has led to an acceleration ofthe cocoa fermentation process and yielded cured cocoa beanswith a rich aroma composition. The application of monoculturesof a strain of Torulaspora delbrueckii (IC103), possessing severalenzyme activities and co-cultures of this strain with a strain of S.cerevisiae (IC67) has indicated the prevalence of S. cerevisiae uponfermentation of 300 kg of cocoa pulp-bean mass (hybrids PS1319and SJ02) in wooden boxes. A positive impact on the sensory pro-file of the chocolates therefrom could be obtained in the pres-ence of T. delbrueckii too, although this depended on the cocoahybrid (Visintin et al. 2017). In contrast, no differences havebeen found in microbial community dynamics, physicochemi-cal parameters and metabolite production when using two com-mercial strains of S. cerevisiae (ID67) and T. delbrueckii (ID103)as starter cultures during Forastero cocoa hybrid heap (100 kg)and wooden box (200 kg) fermentation processes carried outin Cameroon (Mota-Gutierrez et al. 2018). Although the authorshave indicated different fermentation practices, the cocoa vari-ety and the use of different starter cultures as the main causeof the limited success of the strains inoculated, it is most likelythat the non-cocoa origin and hence non-adaptiveness or non-competitiveness of the strains can explain this. Similarly, theuse of bakers’ yeast (S. cerevisiae) produces unacceptable breadynotes in cured beans and chocolates therefrom (Bittenbenderet al. 2017). Yet, the application of two highly aromatic, com-mercial strains of S. cerevisiae from wine origin have resulted infavourable scores for the volatile organic compound productionprofiles of cocoa beans and concomitant chocolates, leading tointense fruity and floral notes in a strain-dependent way. Theseresults were obtained for cocoa fermentation processes carriedout in 45-L plastic boxes with 30 kg of cocoa pulp-bean mass ofan Ivorian hybrid (Assi-Clair et al. 2019).

Mixed-species and -strain starter culturesThorough screenings and kinetic analyses have been performedto compose a mixed-strain starter culture representing the threemain microbial species involved in cocoa fermentation pro-cesses, namely S. cerevisiae, Liml. fermentum and A. pasteurianus(Lefeber et al. 2010, 2011b; Moens, Lefeber and De Vuyst 2014).

Therefore, fermentations with candidate yeast, LAB and AABstrains have been performed in 10-L laboratory fermentors mak-ing use of cocoa pulp simulation media (CPSM-LAB and CPSM-AAB). This way, the competitiveness of dedicated strains ofheterofermentative, mannitol-producing and citrate-convertingLiml. fermentum, homofermentative and citrate-non-convertingLacp. plantarum and ethanol- and lactic acid-oxidizing, aceticacid-overoxidizing A. pasteurianus has been shown. An acceler-ated cocoa fermentation process has been shown by the addi-tion of a strain of Lacp. plantarum to cocoa pulp-bean mass(16 g) of Indonesian cocoa beans in 50-mL glass bottles too(Kresnowati, Suryani and Affifah 2013). CPSM-LAB and CPSM-AAB has also been used to study the (meta)fluxome of LABand AAB strains, respectively (Adler et al. 2013, 2014). Further-more, the cocoa fermentation process has been simulated inCPSM-LAB during 10-L laboratory fermentations, compensat-ing for the loss of ethanol due to the high fermentation tem-perature and aeration, by means of a carefully selected tri-culture, consisting of a consortium of three strains, namelyS. cerevisiae H5S5K23 (ethanol-producing and -tolerant, citricacid-non-converting, acid- and heat-tolerant), Liml. fermentum222 (formerly known as Lb. fermentum 222; heterofermentative,citric acid-converting, mannitol-producing, moderately acid-tolerant) and A. pasteurianus 386B (ethanol- and lactic acid-oxidizing, acetic acid-producing and -overoxidizing, acid- andheat-tolerant) (Lefeber et al. 2010, 2011b, 2012; Moens, Lefeberand De Vuyst 2014). Whole-genome sequence analysis of theseLAB and AAB strains has confirmed their competitiveness andfunctional properties, such as the presence of relevant andunique carbohydrate and amino acid consumption genes as wellas genes for environmental adaptations and stress responses(Illeghems, De Vuyst and Weckx 2013; Illeghems, Weckx and DeVuyst 2015a; Illeghems et al. 2016; Pelicaen et al. 2019). The preva-lence of facultatively heterofermentative Lacp. plantarum strains,for instance, may be ascribed to possible heterofermentation ofcarbohydrates, several stress responses and the possibility ofbacteriocin production to enhance their competitiveness undercocoa pulp-bean mass fermentation conditions (Illeghems, DeVuyst and Weckx 2015a; Illeghems, Weckx and De Vuyst 2015b).Finally, this mixed-strain starter culture has been implementedon farms in both Ivory Coast and Malaysia, by adding a liquidyeast culture and/or rehydrated lyophilized LAB and AAB pow-ders (Lefeber et al. 2012). Its application has shown that uni-formly fermented cocoa beans can be obtained within four daysof fermentation, due to an early onset and accelerated carbohy-drate fermentation, consistent ethanol production and fast cit-ric acid conversion. This has also indicated the importance ofthe inclusion of a yeast strain to manufacture bulk dark choco-late without unfermented flavours (Lefeber et al. 2012), likelyascribed to its added value concerning the production of flavour-active compounds (Ho, Zhao and Fleet 2014, 2015; Ho, Fleet andZhao 2018).

Another screening study has evaluated the technologicalpotential of yeast, LAB and AAB strains in CPSM supple-mented with fresh cocoa pulp-bean mass and carried out in 2-Lglass bottles; the strains used have been isolated from naturalcocoa fermentation processes performed at bench-scale (plas-tic containers of 0.5 L) and pilot-scale (stainless steel tank of15 L) (Pereira et al. 2012). Several yeast species have been pro-posed as candidate starter cultures, such as Candida humilis,Debaryomyces etchellsii, P. kudriavzevii and P. kluyveri, togetherwith S. cerevisiae. However, their success will depend on theirprevalence, as mentioned above. These researchers have alsosuggested a three-strain inoculum, composed of S. cerevisiae

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(UFLA CYC7.04; ethanol-producing and acid-, ethanol- and heat-tolerant), Liml. fermentum (UFLA CBE8.12; citric acid-fermenting,lactic acid-producing and acid-, ethanol- and heat-tolerant), andA. tropicalis (UFLA CBE16.01; ethanol- and lactic acid-oxidizing,acetic acid-producing and acid-, ethanol- and heat-tolerant).Finally, the impact of yeast strains isolated from a spontaneousMalaysian cocoa fermentation process, belonging to the generaCandida, Hanseniaspora, Saccharomyces and Wickerhamomyces, onthe antioxidant properties of cured cocoa beans has been evalu-ated, making use of a modified CPSM supplemented with freshcocoa pulp-bean mass (500 mL), and has emphasized the poten-tial of strains of Hanseniaspora thailandica and P. kudriavzevii asfunctional yeast starter cultures (Ooi, Sepiah and Khairul Bariah2016; Ooi, Ting and Siow 2020).

Regarding other mixed-strain and -species starter culturestested, it has been shown that a starter culture composed ofstrains of K. marxianus (KM16–6; pectinolytic Ghanaian cocoaisolate), P. kluyveri (highly aromatic commercial wine strain),Liml. fermentum (L18; Ghanaian cocoa isolate) and A. pasteuri-anus (A149; Ghanaian cocoa isolate) results in minor physicaland chemical differences in fermentation characteristics dur-ing small-scale heap and plastic tray (20 kg; mixed Forasterohybrids) fermentation processes carried out in Ghana, com-pared to a spontaneous control (Crafack et al. 2013, 2014). Incontrast, pronounced differences in flavour volatile compoundcompositions have been found in the roasted cocoa liquors andin the flavour profiles of the concomitant non-conched choco-lates. However, the yeast strains added have never dominatedthe cocoa fermentation process, likely because of the low-cell-density inocula used and the poor drainage that occurred. Also,besides yeast strains, several strains of LAB and AAB specieshave been screened as to their tolerance toward acidity, ethanol,heat and osmotic stress (Visintin et al. 2016). A reduction fromseven to three days of fermentation has been achieved with anoptimal inoculum size and microbial succession of a mixed-strain starter culture consisting of S. cerevisiae MTCC 173, Lacp.plantarum MTCC 5422 (formerly known as Lb. plantarum MTCC5422) and A. aceti MTCC 3347, applied during 10-kg box fermen-tations in India, resulting in consistently fermented cocoa beanswith desirable characteristics for the production of acceptablechocolates (Sandhya et al. 2016). Also, the inoculation of strainsof S. cerevisiae UFLA CCMA 0200, Lacp. plantarum CCMA 0238(formerly known as Lb. plantarum CCMA 0238), and A. pasteuri-anus CCMA 0241 during box fermentations (100 kg of cocoapulp-bean mass from hybrid PH15) has resulted in faster glu-cose and fructose consumption and chocolates with distinctflavours (Moreira et al. 2017). The use of a starter culture com-posed of a strain of S. cerevisiae (CCMA 0681) and a strain ofLiml. fermentum (UFLA CHBB 8.12; inoculum of 1.0 log less thanthe yeast strain) and applied during Brazilian cocoa box fer-mentations (100 kg of hybrid PH16) has affected the microbialcommunities involved and has impacted the sensory quality ofthe chocolates produced (astringent, bitter and sour taste; lesscocoa notes) (Miguel et al. 2017). Inocula consisting of strainsof S. cerevisiae, Lb. delbrueckii subsp. lactis (formerly known asLb. lactis), and A. aceti have been assessed as well (Apriyantoet al. 2017). As mentioned above, Ho, Fleet and Zhao (2018) havestudied the impact of yeasts, LAB and AAB on the quality ofcured cocoa beans and chocolates produced thereof under care-fully controlled conditions, making use of an Australian Trini-tario variety and 12.5-L plastic containers containing 3.5 kg ofcocoa pulp-bean mass, thereby testing various inocula based ondifferent mixtures of strains of the yeasts Hanseniaspora guillier-mondii, K. marxianus, P. kudriavzevii and S. cerevisiae, the LAB Liml.

fermentum and Lacp. plantarum and/or the AAB A. pasteurianusand Gluconobacter frateurii. The use of these starter cultures hasresulted in cured cocoa beans with slightly different concentra-tions of lactic acid and acetic acid, being highest in the pres-ence of LAB and AAB, respectively, without significant differ-ences regarding the sensory evaluation of the chocolates pro-duced, underlining the primordial role of yeasts for cocoa fer-mentation.

Still other studies have screened LAB strains, including themas producers of organic acids and/or bacteriocins and yeaststrains, serving as producers of proteinaceous antifungal com-pounds, in a slightly modified CPSM as to their capacity to usethem as co-cultures for the biological control of mycotoxin-producing fungi (Essia Ngang et al. 2015; Ruggirello et al. 2019;Romanens et al. 2019). This set-up has been successful to inhibitan aflatoxin-producing strain of Aspergillus flavus. The combina-tion of strains of Pichia mexicana and Bacillus pumilus has acceler-ated the fermentation (in plastic bags containing 1.5 kg of freshcocoa pulp-bean mass) and drying (aluminium pans) processesand has reduced the growth of mycotoxin-producing fungi dur-ing a cocoa fermentation experiment carried out in the Philip-pines (Gibe and Pangan 2015). Also strains of Bacillus specieshave been screened as to their capacity to produce pectinase,to degrade citrate and to be acid-, ethanol- and heat-tolerantin view of potential starter culture applications for cocoa fer-mentation processes (Yao et al. 2017a,b). Finally, a Lacp. plantarumstarter culture strain has been tested as to its capacity to inhibitthe growth of mycotoxin-producing fungi (Djaafar et al. 2019).

Challenges of starter culture use and opportunities within thedomain of ecological intensificationIf all the experiments and proofs-of-principle mentioned abovewill finally lead to on-farm use of functional starter cultures willdepend on several factors (De Vuyst and Weckx 2016a,b; Ozturkand Young 2017). However, both farmers and chocolate produc-ers will profit from it. First of all, good agricultural and farm-ing practices and the use of high-quality raw material remainprimordial, as starter culture application cannot substitute forthat. Further, not only the introduction of the application ofstarter cultures at farm level or in large estates, but also theirproduction, maintenance and distribution and the accompany-ing farmers’ training and extra costs will be a great challengeand should be covered by the chocolate industry. Hence, practi-cal feasibility (taking into account geography and logistics) andstructural adoption by the cocoa farmers are still to be seen asbottlenecks that lag behind the biotechnological state of affairs.A potential way forward to overcome such important hurdlesis to firmly situate starter culture innovation in an overarch-ing framework of sustainable agriculture. It is indeed the lat-ter that is nowadays creating most of the momentum for foodinnovations, as a comprehensive ecological intensification ofglobal agriculture will be needed to improve productivity andsustainability by mid-century and beyond (Rusere et al. 2019).The political and industrial will to do so is already becomingmore outspoken, which will facilitate the proper biotechnolog-ical means in the Global South to, eventually, benefit resource-poor farmers (Qaim, Krattiger and Von Braun 2000). One of thekey issues, however, will be to obtain farmer support by tailoringsuch plans to the clear benefit of smallholder farming systems.Dedicated analysis of the farming systems and farmer type het-erogeneity will be required to interlink ecosystem characteris-tics with potential ecological intensification (Rusere et al. 2019).There are indeed many different types of farmers, who can becategorized from aspirational innovators, over copy-cats, to the

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change-resistant type (Hammond et al. 2017). Depending on thefarmer profile, the adoption of starter cultures can be either asmooth or difficult process. In any case, smallholder innova-tion is not to be taken as self-evident but offers nonethelesspotential for increased sustainability and improved livelihoods,provided that it is endorsed by the farmer families themselves.For instance, agroecological transition based on agroforestry andcashew intercropping on cocoa plantations is becoming a famil-ial tool of social and ecological transition, ahead of the top-downimplementation by industry and politics (Ruf, Kone and Bebo2019). The local use of starter cultures for cocoa fermentationmay help to achieve similar purposes by improving the yieldsand reducing the losses, as long as its logistic implementationis facilitated and its advantages are made clear to the smallhold-ers that have to adopt them. If that is the case, the latter may aswell end-up catalyzing the transition to more controlled cocoafermentation processes and improved cured cocoa bean yields,hence enhancing the supply of cocoa beans for chocolate pro-duction and allowing uniform bulk as well as tailor-made choco-lates. So, rather than relying on mere financial incentives atfarmer or cooperative level—driven by a consumer demand fornew flavours or a market benefit from higher standardization—areal breakthrough can probably only be achieved if the technol-ogy of starter culture use is understood by both industry andfarmers as a promising path to a more resilient production sys-tem in an ecological and economic system of change.

Fermentation recipients

Except for common heaps, boxes, baskets and platforms, cocoafermentation processes have been carried out in drums andstainless steel tanks (Schwan and Wheals 2004; Pereira et al.2012, 2013a; De Vuyst and Weckx 2016a). The latter recipi-ents have been used to control the environmental contami-nation of the cocoa pulp-bean mass, to facilitate the use ofstarter cultures, to improve the aeration of the cocoa pulp-beanmass and to enhance the mixing of the fermenting cocoa pulp-bean mass and thus have an impact on a homogeneous pHand temperature control, given the importance of these exter-nal factors. However, care has to be taken to keep the heatinside the fermentor to allow a desirable increasing tempera-ture course. Therefore, to take all these factors into account,new fermentor designs and controlled fermentation methodsare needed. Temperature control, applying either a tempera-ture regime or a constant temperature, has been examined onlaboratory-scale through controlled incubation of fermentationrecipients (Pereira et al. 2012, 2017; Ho, Zhao and Fleet 2014, 2015;Mayorga-Gross et al. 2016; Apriyanto et al. 2017; Ho, Fleet andZhao 2018; Romanens et al. 2018; Lee et al. 2019; Racine et al.2019). Experimentally, (micro)fermentation of cocoa pulp-beanmass has been tested in glass bottles (Kresnowati, Suryani andAffifah 2013; Apriyanto et al. 2017; Pereira et al. 2017; John et al.2019), plastic bags (Cempaka et al. 2014; Gibe and Pangan 2015;Bittenbender et al. 2017), plastic baskets, boxes, pots or vessels(Leal et al. 2008; Lefeber et al. 2011a; Pereira et al. 2012; Ho, Zhaoand Fleet 2015; Cevallos-Cevallos et al. 2018; Ho, Fleet and Zhao2018; Romanens et al. 2018; Assi-Clair et al. 2019; Lee et al. 2019;Racine et al. 2019), polystyrene boxes or coolers (Ali et al. 2014;Mayorga-Gross et al. 2016), etc. Some of these laboratory-scalemodel systems provide useful tools to examine the influenceof cocoa genotypes, origins, particular process parameters, orstarter cultures on the cocoa fermentation process and finalcocoa bean quality for chocolate production.

CONCLUSIONS

Our understanding of the microbial ecology of cocoa fermen-tation processes has increased substantially during the lastdecade. Also, more detailed knowledge about the formation offlavour precursors inside the beans upon fermentation is avail-able now. However, as the influence of several factors that mayimpact both the microbial dynamics and flavour compound pro-duction kinetics during fermentation of the cocoa pulp-beanmass is not fully known yet, further studies are necessary tounravel this. Furthermore, appropriate fermentation methodsneed to be applied when introducing new fermentation recip-ients, such as wooden drums or stainless steel tanks. Finally,there is still a long way to go to realize the practical implemen-tation of (lyophilized) starter culture practices for cocoa fermen-tation processes on-farm on a large scale.

ACKNOWLEDGMENTS

The authors acknowledge their financial support from the uni-versity council (SRP7, IRP2 and IOF2442 projects), the Herculesfoundation (grants UABR09004 and UAB13002) and the ResearchFoundation Flanders (FWO-SBO18 project REVICO S004617N).

Conflicts of Interest. None declared.

REFERENCES

Acierno V, Fasciani G, Kiani S et al. PTR-QiToF-MS and HSI forthe characterization of fermented cocoa beans from differ-ent origins. Food Chem 2019;289:591–602.

Acierno V, Yener S, Alewijn M et al. Factors contributing to thevariation in the volatile composition of chocolate: botani-cal and geographical origins of the cocoa beans, and brand-related formulation and processing. Food Res Int 2016;84:86–95.

Aculey PC, Snitkjaer P, Owusu M et al. Ghanaian cocoa beanfermentation characterized by spectroscopic and chromato-graphic methods and chemometrics. J Food Sci 2010;75:S300–7.

Adeyeye EI, Akinyeye RO, Ogunlade I et al. Effect of farm andindustrial processing on the amino acid profile of cocoabeans. Food Chem 2010;118:357–63.

Adler P, Bolten CJ, Dohnt K et al. Core fluxome and metafluxomeof lactic acid bacteria under simulated cocoa pulp fermenta-tion conditions. Appl Environ Microbiol 2013;79:5670–81.

Adler P, Frey LJ, Berger A et al. The key to acetate: metabolicfluxes of acetic acid bacteria under cocoa pulp fermentation-simulating conditions. Appl Environ Microbiol 2014;80:4702–16.

Afoakwa EO, Budu AS, Brown HM et al. Changes in biochemicaland physico-chemical qualities during drying of pulp pre-conditioned and fermented cocoa (Theobroma cacao) beans.Int Food Res J 2014;20:1843–53.

Afoakwa EO, Kongor JE, Takrama J et al. Changes in nib acidifi-cation and biochemical composition during fermentation ofpulp pre-conditioned cocoa (Theobroma cacao) beans. Int FoodRes J 2013b;20:1843–53.

Afoakwa EO, Kongor JE, Takrama JF et al. Effects of pulp pre-conditioning on total polyphenols, o-diphenols and antho-cyanin concentrations during fermentation and drying ofcocoa (Theobroma cacao) beans. J Food Sci Eng 2013a;3:235–45.

Dow


ic.oup.com/fem


ber 2020


Afoakwa EO, Paterson A, Fowler M et al. Flavor formation andcharacter in cocoa and chocolate: a critical review. Crit RevFood Sci Nutr 2008;48:840–57.

Afoakwa EO, Quao J, Takrama J et al. Chemical composition andphysical quality characteristics of Ghanaian cocoa beans asaffected by pulp pre-conditioning and fermentation. J FoodSci Technol 2013c;50:1097–105.

Afoakwa EO, Quao J, Takrama J et al. Effect of pulp precondi-tioning on acidification, proteolysis, sugars and free fattyacids concentration during fermentation of cocoa (Theobromacacao) beans. Int J Food Sci Nutr 2011;62:755–64.

Afoakwa EO, Quao J, Takrama J et al. Influence of pulp-preconditioning and fermentation on fermentative qualityand appearance of Ghanaian cocoa (Theobroma cacao) beans.Int Food Res J 2012;19:127–33.

Afoakwa EO. Chocolate Science and Technology. Ames: Wiley-Blackwell, 2016.

Agyirifo DS, Wamalwa M, Otwe EP et al. Metagenomics anal-ysis of cocoa bean fermentation microbiome identifyingspecies diversity and putative functional capabilities. Heliyon2019;5:e02170.

Albertini B, Schoubben A, Guarnaccia D et al. Effect of fermen-tation and drying on cocoa polyphenols. J Agric Food Chem2015;63:9948–53.

Ali NA, Baccus-Taylor GSH, Sukha DA et al. The effect ofcacao (Theobroma cacao L.) pulp on final flavour. Acta Hortic2014;1047:245–54.

Almeida SFO, Silva LRC, Junior GCAC et al. Diversity of yeastsduring fermentation of cocoa from two sites in the BrazilianAmazon. Acta Amazonica 2019;49:64–70.

Amin I, Jinap S, Jamilah B et al. Analysis of cocoa cotyledons albu-min. Asian J Plant Sci 2003;2:958–62.

Amin I, Jinap S, Jamilah B et al. Oligopeptide patterns producedfrom Theobroma cacao L of various genetic origins. J Sci FoodAgric 2002;82:733–7.

Andersson M, Koch G, Lieberei R. Structure and function of theseed coat of Theobroma cacao L. and its possible impact onflavour precursor development during fermentation. J ApplBot Food Qual 2006;80, 48–62.

Andruszkiewicz PJ, D’Souza RN, Altun I et al. Thermally-inducedformation of taste-active 2,5-diketopiperazines from short-chain peptide precursors in cocoa. Food Res Int 2019;121:217–28.

Apriyanto M, Sutardi S, Supriyanto S et al. Amino acid analysisof cocoa fermented by high performance liquid chromatog-raphy (HPLC). Asian J Dairy & Food Res 2017;36:156–60.

Aprotosoaie AC, Luca SV, Miron A. Flavor chemistry of cocoa andcocoa products - An overview. Compr Rev Food Sci Food Saf2016;15:73–91.

Arana-Sanchez A, Segura-Garcıa LE, Kirchmayr M et al. Iden-tification of predominant yeasts associated with artisanMexican cocoa fermentations using culture-dependent andculture-independent approaches. World J Microbiol Biotechnol2015;31:359–69.

Ardhana MM, Fleet GH. The microbial ecology of cocoa bean fer-mentations in Indonesia. Int J Food Microbiol 2003;86:87–99.

Ascrizzi R, Flamini G, Tessieri C et al. From the raw seed to choco-late: volatile profile of Blanco de Criollo in different phases ofthe processing chain. Microchem J 2017;133:474–9.

Assi-Clair BJ, Kone MK, Kouame K et al. Effect of aromapotential of Saccharomyces cerevisiae fermentation on thevolatile profile of raw cocoa and sensory attributes ofchocolate produced thereof. Eur Food Res Technol 2019;1:1–13.

Badrie N, Bekele F, Sikora E et al. Cocoa agronomy, quality, nutri-tional, and health aspects. Crit Rev Food Sci Nutr 2015;55:620–59.

Barisic V, Kopjar M, Jozinovic A et al. The chemistry behindchocolate production. Molecules 2019;24:3163.

Bartley BGD. The Genetic Diversity of Cocoa and Its Utilization.Wallingford: CABI Publishing, 2005.

Bastos VS, Santos MF, Gomes LP et al. Analysis of the coco-biota and metabolites of Moniliophthora perniciosa-resistantTheobroma cacao beans during spontaneous fermentation inSouthern Brazil. J Sci Food Agric 2018;98:4963–70.

Batista NN, Ramos CL, Dias DR et al. The impact of yeast startercultures on the microbial communities and volatile com-pounds in cocoa fermentation and the resulting sensoryattributes of chocolate. J Food Sci Technol 2016;53:1101–10.

Batista NN, Ramos CL, Ribeiro DD et al. Dynamic behavior of Sac-charomyces cerevisiae, Pichia kluyveri and Hanseniaspora uvarumduring spontaneous and inoculated cocoa fermentationsand their effect on sensory characteristics of chocolate. FoodSci Technol 2015;63:221–7.

Beckett ST. Industrial Chocolate Manufacture and Use. Chichester:John Wiley & Sons, 2009.

Bertazzo A, Comai S, Brunato I et al. The content of protein andnon-protein (free and protein-bound) tryptophan in Theo-broma cacao beans. Food Chem 2011;124:93–6.

Bittenbender HC, Gautz LD, Seguine E et al. Microfermentationof cacao: the CTAHR bag system. HortTechnol 2017;27:690–4.

Bortolini C, Patrone V, Puglisi E et al. Detailed analyses of thebacterial populations in processed cocoa beans of differentgeographic origin, subject to varied fermentation conditions.Int J Food Microbiol 2016;236:98–106.

Bourdichon F, Casaregola S, Farrokh C et al. Food fermentations:microorganisms with technological beneficial use. Int J FoodMicrobiol 2012;154:87–97.

Braga SCGN, Oliveira LF, Hashimoto JC et al. Study of volatile pro-file in cocoa nibs, cocoa liquor and chocolate on productionprocess using GC×GC-QMS. Microchem J 2018;141:353–61.

Buamah R, Dzogbefia VP, Oldham JH. Pure yeast culture fer-mentation of cocoa (Theobroma cacao L): effect on yield ofsweatings and cocoa bean quality. World J Microbiol Biotechnol1997;13:457–62.

Bucheli P, Rousseau G, Alvarez M et al. Developmental varia-tion of sugars, carboxylic acids, purine alkaloids, fatty acids,and endoproteinase activity during maturation of Theobromacacao L. seeds. J Agric Food Chem 2001;49:5046–51.

Buyukpamukcu E, Goodall DM, Hansen CE et al. Characterizationof peptides formed during fermentation of cocoa bean. J AgricFood Chem 2001;49:5822–7.

Caligiani A, Marseglia A, Prandi B et al. Influence of fermentationlevel and geographical origin on cocoa bean oligopeptide pat-tern. Food Chem 2016;211:431–9.

Camu N, De Winter T, Addo SK et al. Fermentation of cocoabeans: influence of microbial activities and polyphenol con-centrations on the flavour of chocolate. J Sci Food Agric2008b;88:2288–97.

Camu N, De Winter T, Verbrugghe K et al. Dynamics and biodiver-sity of populations of lactic acid bacteria and acetic acid bac-teria involved in spontaneous heap fermentation of cocoabeans in Ghana. Appl Environ Microbiol 2007;73:1809–24.

Camu N, Gonzalez A, De Winter T et al. Influence of turningand environmental contamination on the dynamics of pop-ulations of lactic acid and acetic acid bacteria populationsinvolved in spontaneous cocoa bean heap fermentation inGhana. Appl Environ Microbiol 2008a;74:86–98.

Dow


ic.oup.com/fem


ber 2020


Castro-Alayo EM, Idrogo-Vasquez G, Siche R et al. Formationof aromatic compounds precursors during fermentation ofCriollo and Forastero cocoa. Heliyon 2019;5:e01157.

Cempaka L, Aliwarga L, Purwo S et al. Dynamics of cocoa beanpulp degradation during cocoa bean fermentation: effectsof yeast starter culture addition. J Math Fundam Sci 2014;46:14–25.

Cevallos-Cevallos JM, Gysel L, Mariduena-Zavala MG et al. Time-related changes in volatile compounds during fermentationof bulk and fine-flavor cocoa (Theobroma cacao) beans. J FoodQual 2018;2018:1758381.

Chetschik I, Kneubuhl M, Chatelain K et al. Investigations onthe aroma of cocoa pulp (Theobroma cacao L.) and its influ-ence on the odor of fermented cocoa beans. J Agric Food Chem2018;66:2467–72.

Cleenwerck I, Camu N, Engelbeen K et al. Acetobacter ghanensis sp.nov., a novel acetic acid bacterium isolated from traditionalheap fermentations of Ghanaian cocoa beans. Int J Syst EvolMicrobiol 2007;57:1647–52.

Cleenwerck I, Gonzalez A, Camu N et al. Acetobacter fabarum sp.nov., an acetic acid bacterium from a Ghanaian cocoa beanheap fermentation. Int J Syst Evol Microbiol 2008;58:2180–5.

Coe SD, Coe MD. The True History of Chocolate. London: Thamesand Hudson, 2019.

Copetti MV, Iamanaka BT, Frisvad JC et al. Mycobiota of cocoa:from farm to chocolate. Food Microbiol 2011;28:1499–504.

Copetti MV, Iamanaka BT, Pitt JI et al. Fungi and mycotoxins incocoa: from farm to chocolate. Int J Food Microbiol 2014;178:13–20.

Copetti MV, Pereira JL, Iamanaka BT et al. Ochratoxigenic fungiand ochratoxin A in cocoa during farm processing. Int J FoodMicrobiol 2010;143:67–70.

Counet C, Ouwerx C, Rosoux D et al. Relationship between pro-cyanidin and flavor contents of cocoa liquors from differentorigins. J Agric Food Chem 2004;52:6243–9.

Crafack M, Keul H, Eskildsen CE et al. Impact of starter culturesand fermentation techniques on the volatile aroma and sen-sory profile of chocolate. Food Res Int 2014;63:306–16.

Crafack M, Mikkelsen MB, Saerens S et al. Influencing cocoaflavour using Pichia kluyveri and Kluyveromyces marxianus ina defined mixed starter culture for cocoa fermentation. Int JFood Microbiol 2013;167:103–16.

Cruz JFM, Leite PB, Soares SE et al. Assessment of the fermen-tative process from different cocoa cultivars produced inSouthern Bahia, Brazil. Afr J Biotechnol 2013;12:5218–25.

Cuellar LM, Espinosa CMO, Sanchez KA et al. Organoleptic qual-ity assessment of Theobroma cacao L. in cocoa farms in North-ern Huila, Colombia. Acta Agron 2018;67:46–52.

D’Souza RN, Grimbs A, Grimbs S et al. Degradation of cocoa pro-teins into oligopeptides during spontaneous fermentation ofcocoa beans. Food Res Int 2018;109:506–16.

D’Souza RN, Grimbs S, Behrends B et al. Origin-based polyphe-nolic fingerprinting of Theobroma cacao in unfermented andfermented beans. Food Res Int 2017;99:550–9.

Daniel H-M, Vrancken G, Takrama JF et al. Yeast diversity ofGhanaian cocoa bean heap fermentations. FEMS Yeast Res2009;9:774–83.

de Brito ES, Garcıa NHP, Gallao M et al. Structural and chemicalchanges in cocoa (Theobroma cacao L.) during fermentation,drying and roasting. J Sci Food Agric 2000;81:281–8.

De Bruyne K, Camu N, De Vuyst L et al. Lactobacillus fabifer-mentans sp. nov. and Lactobacillus cacaonum sp. nov., isolatedfrom Ghanaian cocoa fermentations. Int J Syst Evol Microbiol2009;59:7–12.

De Bruyne K, Camu N, De Vuyst L et al. Weissella fabaria sp. nov.,from a Ghanaian cocoa fermentation. Int J Syst Evol Microbiol2010;60:1999–2005.

De Bruyne K, Camu N, Lefebvre K et al. Weissella ghanensis sp.nov., isolated from a Ghanaian cocoa fermentation. Int J SystEvol Microbiol 2008;58:2721–5.

De Taeye C, Evina VJE, Caullet G et al. Fate of anthocyaninsthrough cocoa fermentation. Emergence of new polypheno-lic dimers. J Agric Food Chem 2016;64:8876–85.

De Vuyst L, Camu N, De Winter T et al. Validation of the (GTG)5-rep-PCR fingerprinting technique for rapid classification andidentification of acetic acid bacteria, with a focus on isolatesfrom Ghanaian fermented cocoa beans. Int J Food Microbiol2008;125:79–90.

De Vuyst L, Weckx S. The cocoa bean fermentation process: fromecosystem analysis to starter culture development. J ApplMicrobiol 2016b;121:5–17.

De Vuyst L, Weckx S. The functional role of lactic acid bacte-ria in cocoa bean fermentation. In: Mozzi F Raya RR VignoloGM (eds.). Biotechnology f Lactic Acid Bacteria: Novel Applica-tions. Chichester: John Wiley & Sons Ltd., 2016a, 248–78.

Diab J, Hertz-Schunemann R, Streibel T et al. On-line measure-ment of volatile organic compounds released during roastingof cocoa beans. Food Res Int 2014;63:344–52.

Diomande D, Antheaume I, Leroux M et al. Multi-element, multi-compound isotope profiling as a means to distinguish thegeographical and varietal origin of fermented cocoa (Theo-broma cacao L.) beans. Food Chem 2015;188:576–82.

Djaafar TF, Palupi LO, Marwati T et al. Characteristic of choco-late candy produced from fermented cocoa bean with Lacto-bacillus plantarum HL-15 starter culture for inhibition growthof mycotoxin-producing fungi. J Agric Sci Technol 2019;B9:128–34.

Dzialo MC, Park R, Steensels J et al. Physiology, ecology andindustrial applications of aroma formation in yeast. FEMSMicrobiol Rev 2017;41:S95–S128.

Dzogbefia VP, Buamah R, Oldham JH. The controlled fermen-tation of cocoa (Theobroma cacao L) using yeasts: enzymaticprocess and associated physicochemical changes in cocoasweatings. Food Biotechnol 1999;13:1–12.

Engeseth NJ, Pangan MFA. Current context on chocolate flavordevelopment — A review. Curr Opin Food Sci 2018;21:84–91.

Essia Ngang J-J, Yadang G, Sado Kamdem SL et al. Antifun-gal properties of selected lactic acid bacteria and appli-cation in the biological control of ochratoxin A producingfungi during cocoa fermentation. Biocontrol Sci Tech 2015;25:245–59.

Evina VJE, De Taeye C, Niemenak N et al. Influence of aceticand lactic acids on cocoa flavan-3-ol degradation throughfermentation-like incubations. Food Sci Technol 2016;68:514–22.

Figueroa-Hernandez C, Mota-Gutierrez J, Ferrocino I et al. Thechallenges and perspectives of the selection of startercultures for fermented cocoa beans. Int J Food Microbiol2019;301:41–50.

Frauendorfer F, Schieberle P. Changes in key aroma compoundsof Criollo cocoa beans during roasting. J Agric Food Chem2008;56:10244–51.

Frauendorfer F, Schieberle P. Identification of the key aromacompounds in cocoa powder based on molecular sensorycorrelations. J Agric Food Chem 2006;54:5521–9.

Frauendorfer F, Schieberle P. Key aroma compounds in fer-mented Forastero cocoa beans and changes induced byroasting. Eur Food Res Technol 2019;245:1907–15.

Dow


ic.oup.com/fem


ber 2020


Ganeswari I, Khairul Bariah S, Amizi MA et al. Effects of differentfermentation approaches on the microbiological and physic-ochemical changes during cocoa bean fermentation. Int FoodRes J 2015;22:70–6.

Garcia-Armisen T, Papalexandratou Z, Hendrickx H et al. Diver-sity of the total bacterial community associated with Ghana-ian and Brazilian cocoa bean fermentation samples asrevealed by a 16S rRNA gene clone library. Appl MicrobiolBiotechnol 2010;87:2281–92.

Garcıa-Alamilla P, Lagunes-Galvez LM, Barajas-Fernandez J et al.Physicochemical changes of cocoa beans during roastingprocess. J Food Qual 2017;2017:2969324.

Gibe AJG, Pangan JO. Evaluation of Pichia mexicana and Bacilluspumilus in hastening fermentation process and its resultingeffect of the quality of dried cacao beans. Asian J Appl Sci2015;3:1–7.

Guehi ST, Dingkuhn M, Cros E et al. Impact of cocoa processingtechnologies in free fatty acids formation in stored raw cocoabeans. Afr J Agric Res 2008;3:174–9.

Guehi TS, Dadie AT, Koffi KPB et al. Performance of differentfermentation methods and the effect of their duration onthe quality of raw cocoa beans. Int J Food Sci Technol 2010;45:2508–14.

Guehi TS, Konan YM, Koffi-Nevry R et al. Enumeration and iden-tification of main fungal isolates and evaluation of fermen-tation’s degree of Ivorian raw cocoa beans. Austr J Basic ApplSci 2007;1:479–86.

Guilloteau M, Laloi M, Michaux S et al. Identification and charac-terisation of the major aspartic proteinase activity in Theo-broma cacao seeds. J Sci Food Agric 2005;85:549–62.

Galvez LS, Loiseau G, Paredes JL et al. Study on the microfloraand biochemistry of cocoa fermentation in the DominicanRepublic. Int J Food Microbiol 2007;114:124–30.

Ganzle MG. Lactic metabolism revisited: metabolism of lacticacid bacteria in food fermentations and food spoilage. CurrOpin Food Sci 2015;2:106e117.

Hamdouche Y, Guehi T, Durand N et al. Dynamics of microbialecology during cocoa fermentation and drying: towardsthe identification of molecular markers. Food Control2015;48:117–22.

Hamdouche Y, Meile JC, Lebrun M et al. Impact of turn-ing, pod storage and fermentation time on microbial ecol-ogy and volatile composition of cocoa beans. Food Res Int2019;119:477–91.

Hammond J, van Wijk MT, Smajgl A et al. Farm types and farmermotivations to adapt: implications for design of sustainableagricultural interventions in the rubber plantations of SouthWest China. Agri Syst 2017;154:1–12.

Hinneh M, Abotsi EE, Van de Walle D et al. Pod storage with roast-ing: a tool to diversifying the flavor profiles of dark chocolatesproduced from ‘bulk’ cocoa beans? (Part I: aroma profiling ofchocolates). Food Res Int 2019a;119:84–98.

Hinneh M, Semanhyia E, Van de Walle D et al. Assessing the influ-ence of pod storage on sugar and free amino acid profilesand the implications on some Maillard reaction related fla-vor volatiles in Forastero cocoa beans. Food Res Int 2018;111:607–20.

Hinneh M, Van de Walle D, Tzompa-Sosa DA et al. Tuningthe aroma profiles of FORASTERO cocoa liquors by vary-ing pod storage and bean roasting temperature. Food Res Int2019b;125:108550.

Ho VTT, Fleet GH, Zhao J. Unravelling the contribution of lac-tic acid bacteria and acetic acid bacteria to cocoa fermenta-tion using inoculated organisms. Int J Food Microbiol 2018;279:43–56.

Ho VTT, Zhao J, Fleet G. The effect of lactic acid bacteria on cocoabean fermentation. Int J Food Microbiol 2015;205:54–67.

Ho VTT, Zhao J, Fleet G. Yeasts are essential for cocoa bean fer-mentation. Int J Food Microbiol 2014;174:72–87.

Huang Y, Barringer SA. Monitoring of cocoa volatiles producedduring roasting by selected ion flow tube-mass spectrometry(SIFT-MS). J Food Sci 2011;76:C279–86.

Hue C, Gunata Z, Breysse A et al. Impact of fermentation onnitrogenous compounds of cocoa beans (Theobroma cacao L.)from various origins. Food Chem 2016;192:958–64.

Hutkins RW. Microbiology and Technology of Fermented Foods. Hobo-ken: Wiley Blackwell, 2019.

Illeghems K, De Vuyst L, Papalexandratou Z et al. Phyloge-netic analysis of a spontaneous cocoa bean fermentationmetagenome reveals new insights into its bacterial and fun-gal community diversity. PLoS ONE 2012;7:e38040.

Illeghems K, De Vuyst L, Weckx S. Comparative genome anal-ysis of the candidate functional starter culture strains Lac-tobacillus fermentum 222 and Lactobacillus plantarum 80 forcontrolled cocoa bean fermentation processes. BMC Genom2015a;16:766.

Illeghems K, De Vuyst L, Weckx S. Complete genome sequenceand comparative analysis of Acetobacter pasteurianus 386B, astrain well-adapted to the cocoa bean fermentation ecosys-tem. BMC Genom 2013;14:526.

Illeghems K, Pelicaen R, De Vuyst L et al. Assessment of thecontribution of cocoa-derived strains of Acetobacter ghanen-sis and Acetobacter senegalensis to the cocoa bean fermen-tation process through a genomic approach. Food Microbiol2016;58:68–78.

Illeghems K, Weckx S, De Vuyst L. Applying meta-pathway anal-yses through metagenomics to identify the functional prop-erties of the major bacterial communities of a single sponta-neous cocoa bean fermentation process sample. Food Micro-biol 2015b;50:54–63.

Janek K, Niewienda A, Wostemeyer J et al. Dataset of cocoa aspar-tic protease cleavage sites. Data in Brief 2016b;8:700–8.

Janek K, Niewienda A, Wostemeyer J et al. The cleavage speci-ficity of the aspartic protease of cocoa beans involved in thegeneration of the cocoa-specific aroma precursors. Food Chem2016a;211:320–8.

Jespersen L, Nielsen DS, Hønholt S et al. Occurrence and diver-sity of yeasts involved in fermentation of West African cocoabeans. FEMS Yeast Res 2005;5:441–53.

Jinap S, Ikrawan Y, Bakar J et al. Aroma precursors andmethylpyrazines in underfermented cocoa beans induced byendogenous carboxypeptidase. J Food Sci 2008;73:H141–7.

John WA, Bottcher NL, Asskamp M et al. Forcing fermentation:profiling proteins, peptides and polyphenols in lab-scalecocoa bean fermentation. Food Chem 2019;278:786–94.

John WA, Bottcher NL, Behrends B et al. Experimen-tally modelling cocoa bean fermentation reveals keyfactors and their influences. Food Chem 2020;302:125335.

John WA, Kumari N, Bottcher NL et al. Aseptic artificial fermenta-tion of cocoa beans can be fashioned to replicate the peptideprofile of commercial cocoa bean fermentations. Food Res Int2016;89:764–72.

Kadow D, Bohlmann J, Philips W et al. Identification of main fineor flavour components in two genotypes of the cocoa tree(Theobroma cacao L.). J Appl Bot Food Qual 2013;86:90–98.

Kadow D, Niemenak N, Rohn S et al. Fermentation-like incu-bation of cocoa seeds (Theobroma cacao L.) – Reconstructionand guidance of the fermentation process. Food Sci Technol2015;62:357–61.

Dow


ic.oup.com/fem


ber 2020


Kedjebo KBD, Guehi TS, Kouakou B et al. Effect of post-harvest treatments on the occurrence of ochratoxinA in raw cocoa beans. Food Add Cont 2015; DOI:10.1080/19440049.2015.1112038.

Koffi O, Samagaci L, Goualie B et al. Diversity of yeasts involvedin cocoa fermentation of six major cocoa-producing regionsin Ivory Coast. Eur Sci J 2017;13:496–516.

Koffi O, Samagaci L, Goualie B et al. Screening of potential yeaststarters with high ethanol production for a small-scale cocoafermentation in Ivory Coast. Food Environ Saf 2018;17:130–9.

Kongor JE, Hinneh M, Van de Walle D et al. Factors influencingquality variation in cocoa (Theobroma cacao) bean flavour pro-file — A review. Food Res Int 2016;82:44–52.

Konstantas A, Jeswani HK, Stamford L et al. Environmentalimpacts of chocolate production and consumption in the UK.Food Res Int 2018;106:1012–25.

Kone MK, Guehi ST, Durand N et al. Contribution of predominantyeasts to the occurrence of aroma compounds during cocoabean fermentation. Food Res Int 2016;89:910–7.

Kostinek M, Ban-Koffi L, Ottah-Atikpo M et al. Diversity of pre-dominant lactic acid bacteria associated with cocoa fermen-tation in Nigeria. Curr Microbiol 2008;56:306–14.

Kouame LM, Goualie BG, Adom JN et al. Diversity of micro-bial strains involved in cocoa fermentation from Sud-Comoe(Cote d’Ivoire) based on biochemical properties. Eur Sci J2015b;11:69–85.

Kouame LM, Koua GAY, Niamke JA et al. Cocoa fermentationfrom Agneby-Tiassa: biochemical study of microflora. Am JBioSci 2015a;3:203–11.

Kratzer U, Frank R, Kalbacher H et al. Subunit structure of thevicilin-like globular storage protein of cocoa seeds and theorigin of cocoa- and chocolate-specific aroma precursors.Food Chem 2009;113:903–13.

Kresnowati MTAP, Suryani L, Affifah M. Improvement of cocoabeans fermentation by LAB starter addition. J Med Bioeng2013;2:274–8.

Krahmer A, Engel A, Kadow D et al. Fast and neat – Determina-tion of biochemical quality parameters in cocoa using nearinfrared spectroscopy. Food Chem 2015;181:152–9.

Kumari N, Grimbs A, D’Souza RN et al. Origin and varietal basedproteomic and peptidomic fingerprinting of Theobroma cacaoin non-fermented and fermented cocoa beans. Food Res Int2018;111:137–47.

Kumari N, Kofi KJ, Grimbs S et al. Biochemical fate of vicilin stor-age protein during fermentation and drying of cocoa beans.Food Res Int 2016;90:53–65.

Laloi M, McCarthy J, Morandi O et al. Molecular and biochemi-cal characterisation of two aspartic proteinases TcAP1 andTcAP2 from Theobroma cacao seeds. Planta 2002;215:754–62.

Leal GA, Gomes LH, Efraim P et al. Fermentation of cacao (Theo-broma cacao L.) seeds with a hybrid Kluyveromyces marxianusstrain improved product quality attributes. FEMS Yeast Res2008;8:788–98.

Lee AH, Neilson AP, O’Keefe SF et al. A laboratory-scale modelcocoa fermentation using dried, unfermented beans andartificial pulp can simulate the microbial and chemicalchanges of on-farm cocoa fermentation. Eur Food Res Technol2019;245:511–9.

Lefeber T, Gobert W, Vrancken G et al. Dynamics and speciesdiversity of communities of lactic acid bacteria and aceticacid bacteria during spontaneous cocoa bean fermentationin vessels. Food Microbiol 2011a;28:457–64.

Lefeber T, Janssens M, Camu N et al. Kinetic analysis of strainsof lactic acid bacteria and acetic acid bacteria in cocoa

pulp simulation media toward development of a starterculture for cocoa bean fermentation. Appl Environ Microbiol2010;76:7708–16.

Lefeber T, Janssens M, Moens F et al. Interesting starter culturestrains for controlled cocoa bean fermentation revealed bysimulated cocoa pulp fermentations of cocoa-specific lacticacid bacteria. Appl Environ Microbiol 2011b;77:6694–8.

Lefeber T, Papalexandratou Z, Gobert W et al. On-farm imple-mentation of a starter culture for improved cocoa bean fer-mentation and its influence on the flavour of chocolates pro-duced thereof. Food Microbiol 2012;30:379–92.

Leroy F, De Vuyst L. Lactic acid bacteria as functional starter cul-tures for the food fermentation industry. Trends Food Sci Tech-nol 2004;15:67–78.

Lima LJR, Almeida MH, Nout MJR et al. Theobroma cacao L., “thefood of the gods”: quality determinants of commercial cocoabeans, with particular reference to the impact of the fermen-tation. Crit Rev Food Sci Nutr 2011;51:731–61.

Ludlow CL, Cromie GA, Garmendia-Torres C et al. Independentorigins of yeast associated with coffee and cacao fermenta-tion. Curr Biol 2016;26:965–71.

Mahazar NH, Sufian NF, Meor Hussin AS et al. Candida sp. as astarter culture for cocoa (Theobroma cacao L.) beans fermen-tation. Int J Food Res 2015;22:1783–7.

Marco ML, Heeney D, Binda S et al. Health benefits of fer-mented foods: microbiota and beyond. Curr Opin Biotechnol2017;44:94–102.

Marseglia A, Sforza S, Faccini A et al. Extraction, identificationand semi-quantification of oligopeptides in cocoa beans.Food Res Int 2014;63:382–9.

Maura YF, Balzarini T, Borges PC et al. The environmental andintrinsic yeast diversity of Cuban cocoa bean heap fermen-tations. Int J Food Microbiol 2016;233:34–43.

Mayorga-Gross AL, Quiros-Guerrero LM, Fourny G et al. An untar-geted metabolomic assessment of cocoa beans during fer-mentation. Food Res Int 2016;89:901–9.

Meersman E, Steensels J, Mathawan M et al. Detailed analysisof the microbial population in Malaysian spontaneous cocoapulp fermentations reveals a core and variable microbiota.PLoS ONE 2013;8:81559.

Meersman E, Steensels J, Paulus T et al. Breeding strategy to gen-erate robust yeast starter cultures for cocoa pulp fermenta-tions. Appl Environ Microbiol 2015;81:6166–76.

Meersman E, Steensels J, Struyf N et al. Tuning chocolate flavorthrough development of thermotolerant Saccharomyces cere-visiae starter cultures with increased acetate ester produc-tion. Appl Environ Microbiol 2016;82:732–46.

Meersman E, Struyf N, Kyomugasho C et al. Characterization anddegradation of pectic polysaccharides in cocoa pulp. J AgricFood Chem 2017;65:9726–34.

Megıas-Perez R, Grimbs S, D’Souza RN et al. Profiling, quantifica-tion and classification of cocoa beans based on chemomet-ric analysis of carbohydrates using hydrophilic interactionliquid chromatography coupled to mass spectrometry. FoodChem 2018;258:284–94.

Megıas-Perez R, Moreno-Zambrano M, Behrends B et al. Mon-itoring the changes in low molecular weight carbohy-drates in cocoa beans during spontaneous fermentation: achemometric and kinetic approach. Food Res Int 2020;128:108865.

Menezes AGT, Batista NN, Ramos CL et al. Investigation of choco-late produced from four different Brazilian varieties of cocoa(Theobroma cacao L.) inoculated with Saccharomyces cerevisiae.Food Res Int 2016;81:83–90.

Dow


ic.oup.com/fem


ber 2020


Miescher Schwenninger S, Freimuller Leischtfeld S, Gantenbein-Demarchi C. High-throughput identification of the microbialbiodiversity of cocoa bean fermentation by MALDI-TOF MS.Lett Appl Microbiol 2016;63:347–55.

Miguel MGCP, Reis LVC, Efraim P et al. Cocoa fermentation:microbial identification by MALDI-TOF MS, and sensory eval-uation of produced chocolate. Food Sci Technol 2017;77:362–9.

Misnawi S. Physico-chemical changes during cocoa fermenta-tion and key enzymes involved. Rev Penelitian Kopi dan Kakao2008;24:47–64.

Moens F, Lefeber T, De Vuyst L. Oxidation of metabolites high-lights the microbial interactions and role of Acetobacter pas-teurianus during cocoa bean fermentation. Appl Environ Micro-biol 2014;80:1848–57.

Moreira IMV, Miguel MGCP, Duarte WF et al. Microbial succes-sion and the dynamics of metabolites and sugars during thefermentation of three different cocoa (Theobroma cacao L.)hybrids. Food Res Int 2013;54:9–17.

Moreira IMV, Miguel MGCP, Ramos CL et al. Influence of cocoahybrids on volatile compounds of fermented beans, micro-bial diversity during fermentation and sensory characteris-tics and acceptance of chocolates. J Food Qual 2016;39:839–49.

Moreira IMV, Vilela LF, Miguel MGCP et al. Impact of a microbialcocktail used as a starter culture on cocoa fermentation andchocolate flavor. Molecules 2017;22:766.

Moreira IMV, Vilela LF, Santos C et al. Volatile compounds andprotein profiles analyses of fermented cocoa beans andchocolates from different hybrids cultivated in Brazil. FoodRes Int 2018;109:196–203.

Moreno-Zambrano M, Grimbs S, Ullrich MS et al. A mathemat-ical model of cocoa bean fermentation. Royal Soc Open Sci2018;5:180964.

Mota-Gutierrez J, Barbosa-Pereira L, Ferrocino I et al. Traceabilityof functional volatile compounds generated on inoculatedcocoa fermentation and its potential health benefits. Nutri-ents 2019b;11:884.

Mota-Gutierrez J, Botta C, Ferrocino I et al. Dynamics and biodi-versity of bacterial and yeast communities during fermenta-tion of cocoa beans. Appl Environ Microbiol 2018;84:e01164–18.

Mota-Gutierrez J, Ferrocino I, Rantsiou K et al. Metataxonomiccomparison between internal transcribed spacer and 26Sribosomal large subunit (LSU) rDNA gene. Int J Food Microbiol2019a;290:132–40.

Motamayor JC, Lachenaud P, Da Silva e Mota JW et al. Geo-graphic and genetic population differentiation of the Ama-zonian chocolate tree (Theobroma cacao L). PLoS ONE 2008;3:3311.

Mounjouenpou P, Gueule D, Ntoupka M et al. Influence ofpost-harvest processing on ochratoxin A content in cocoaand on consumer exposure in Cameroon. World Mycotoxin J2010;4:141–6.

Munoz MS, Cortina JR, Vaillant FE et al. An overview of the phys-ical and biochemical transformation of cocoa seeds to beansand to chocolate: flavor formation. Crit Rev Food Sci Nutr 2019;https://doi.org/10.1080/10408398.2019.1581726.

Nazaruddin R, Seng LK, Hassan O et al. Effect of pulp precondi-tioning on the content of polyphenols in cocoa beans (Theo-broma cacao) during fermentation. Ind Crops Prod 2006;24:87–94.

Ndife J, Bolaji P, Atoyebi D et al. Production and quality evalua-tion of cocoa products (plain cocoa powder and chocolate).Am J Food Nutr 2013;3:31–8.

Nielsen DS, Honholt S, Tano-Debrah K et al. Yeast popula-tions associated with Ghanaian cocoa fermentations anal-ysed using denaturing gradient gel electrophoresis (DGGE).Yeast 2005;22:271–84.

Nielsen DS, Jakobsen M, Jespersen L. Candida h almiae sp. nov.,Geotrichum ghanense sp. nov. and Candida awuaii sp. nov.,isolated from Ghanaian cocoa fermentations. Int J Syst EvolMicrobiol 2010;60:1460–5.

Nielsen DS, Schillinger U, Franz CMAP et al. Lactobacillus gha-nensis sp. nov., a motile lactic acid bacterium isolatedfrom Ghanaian cocoa fermentations. Int J Syst Evol Microbiol2007b;57:1468–72.

Nielsen DS, Teniola OD, Ban-Koffi L et al. The microbiologyof Ghanaian cocoa fermentations analysed using culture-dependent and culture-independent methods. Int J FoodMicrobiol 2007a;114:168–86.

Ooi TS, Sepiah M, Khairul Bariah S. Effect of yeast species ontotal soluble solids, total polyphenol content and fermenta-tion index during simulation study of cocoa fermentation. IntJ Innov Sc Eng Technol 2016; 3:477–83.

Ooi TS, Ting ASY, Siow LF. Influence of selected native yeaststarter cultures on the antioxidant activities, fermentationindex and total soluble solids of Malaysia cocoa beans: a sim-ulation study. Food Sc Technol 2020;122:108977.

Ouattara DH, Ouattara HG, Goualie BG et al. Biochemical andfunctional properties of lactic acid bacteria isolated from Ivo-rian cocoa fermenting beans. J Appl Biosci 2014;77:6489–99.

Ouattara HD, Ouattara HG, Droux M et al. Lactic acid bacte-ria involved in cocoa beans fermentation from Ivory Coast:species diversity and citrate lyase production. Int J Food Micro-biol 2017;256:11–9.

Ouattara HG, Koffi BL, Karou GT et al. Implication of Bacillus sp.in the production of pectinolytic enzymes during cocoa fer-mentation. World J Microbiol Biotechnol 2008;24:1753–60.

Ouattara HG, Reverchon S, Niamke SL et al. Molecular identifica-tion and pectate lyase production by Bacillus strains involvedin cocoa fermentation. Food Microbiol 2011;28:1–8.

Owusu M, Petersen MA, Heimdal H. Effect of fermentationmethod, roasting and conching conditions on the aromavolatiles of dark chocolate. J Food Process Pres 2012;36:1–11.

Owusu M, Petersen MA, Heimdal H. Relationship of sensoryand instrumental aroma measurements of dark chocolate asinfluenced by fermentation method, roasting and conchingconditions. J Food Sci Technol 2013;50:909–17.

Ozturk G, Young GM. Food evolution: the impact of society andscience on the fermentation of cocoa beans. Compr Rev FoodSci Food Safety 2017;16:431–55.

Paoletti R, Poli A, Conti A et al. Chocolate and Health. Milan:Springer-Verlag, 2012.

Papalexandratou Z, Camu N, Falony G et al. Comparison of thebacterial species diversity of spontaneous cocoa bean fer-mentations carried out at selected farms in Ivory Coast andBrazil. Food Microbiol 2011a;28:964–73.

Papalexandratou Z, Cleenwerck I, De Vos P et al. (GTG)5-PCR ref-erence framework for acetic acid bacteria. FEMS Microbiol Lett2009;301:44–9.

Papalexandratou Z, De Vuyst L. Assessment of the yeastspecies composition of cocoa bean fermentations in differ-ent cocoa-producing regions using denaturing gradient gelelectrophoresis. FEMS Yeast Res 2011;11:564–74.

Papalexandratou Z, Falony G, Romanens E et al. Species diver-sity, community dynamics, and metabolite kinetics of the

Dow


ic.oup.com/fem


ber 2020

https://doi.org/10.1080/10408398.2019.1581726


microbiota associated with traditional Ecuadorian spon-taneous cocoa bean fermentations. Appl Environ Microbiol2011c;77:7698–714.

Papalexandratou Z, Kaasik K, Kauffmann LV et al. Linking cocoavarietals and microbial diversity of Nicaraguan fine cocoabean fermentations and their impact on final cocoa qualityappreciation. Int J Food Microbiol 2019;304:106–18.

Papalexandratou Z, Lefeber T, Bahrim B et al. Hanseniasporaopuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, andAcetobacter pasteurianus predominate during well-performedMalaysian cocoa bean box fermentations, underlining theimportance of these microbial species for a successful cocoabean fermentation process. Food Microbiol 2013;35:73–85.

Papalexandratou Z, Nielsen DS. It’s gettin’ hot in here: breedingrobust yeast starter cultures for cocoa fermentation. TrendsMicrobiol 2016;24:168–70.

Papalexandratou Z, Vrancken G, De Bruyne K et al. Spontaneousorganic cocoa bean box fermentations in Brazil are charac-terized by a restricted species diversity of lactic acid bacteriaand acetic acid bacteria. Food Microbiol 2011b;28:1326–38.

Pelicaen R, Gonze D, De Vuyst L et al. Genome-scale metabolicreconstruction of Acetobacter pasteurianus 386B, a candidatefunctional starter culture for cocoa bean fermentation. FrontMicrobiol 2019;10:2801.

Pereira GV, Magalhaes-Guedes KT, Schwan RF. rDNA-basedDGGE analysis and electron microscopic observation ofcocoa beans to monitor microbial diversity and distributionduring the fermentation process. Food Res Int 2013b;53:482–6.

Pereira GV, Magalhaes KT, de Almeida EG et al. Spontaneouscocoa bean fermentation carried out in a novel design stain-less steel tank: influence on the dynamics of microbial pop-ulations and physical-chemical properties. Int J Food Microbiol2013a;161:121–33.

Pereira GVM, Alvarez JP, Neto DPC et al. Great intraspecies diver-sity of Pichia kudriavzevii in cocoa fermentation highlights theimportance of yeast strain selection for flavor modulation ofcocoa beans. Food Sci Technol 2017;84:290–7.

Pereira GVM, Miguel MGCP, Ramos CL et al. Microbiologicaland physicochemical characterization of small-scale cocoafermentations and screening of yeast and bacterial strainsto develop a defined starter culture. Appl Environ Microbiol2012;78:5395–405.

Pereira GVM, Soccol VT, Soccol CR. Current state of research oncocoa and coffee fermentations. Curr Opin Food Sci 2016;7:50–7.

Qaim M, Krattiger AF, Von Braun J. Agricultural Biotechnology inDeveloping Countries: Towards Optimizing the Benefits for thePoor. Norwell: Kluwer Academic Publishers, 2000.

Qin X-W, Lai J-X, Tan L-H et al. Characterization of volatile com-pounds in Criollo, Forastero, and Trinitario cocoa seeds (Theo-broma cacao L.) in China. Int J Food Prop 2017;20:2261–75.

Racine KC, Lee AH, Wiersema BD et al. Development and char-acterization of a pilot-scale model cocoa fermentation sys-tem suitable for studying the impact of fermentation onputative bioactive compounds and bioactivity of cocoa. Foods2019;8:102.

Ramos CL, Dias DR, Miguel MGCP et al. Impact of different cocoahybrids (Theobroma cacao L.) and S. cerevisiae UFLA CA11 inoc-ulation on microbial communities and volatile compoundsof cocoa fermentation. Food Res Int 2014;64:908–18.

Rawel HM, Huschek G, Tchewonpi Sagu S et al. Cocoa beanproteins – Characterization, changes and modifications dueto ripening and post-harvest processing. Nutrients 2019;11:428.

Rezac S, Kok CR, Heermann M et al. Fermented foods as a dietarysource of live organisms. Front Microbiol 2018;9:1785.

Rodrigues F, Ludovico P, Leao C. Sugar metabolism in yeasts:an overview of aerobic and anaerobic glucose catabolism.In: Rosa CA Gabor P (eds.). The Yeast Handbook: Biodiver-sity and Ecophysiology of Yeasts. Berlin: Springer-Verlag, 2006,101–22.

Rodriguez-Campos J, Escalona-Buendıa HB, Contreras-RamosSM et al. Effect of fermentation time and drying temperatureon volatile compounds in cocoa. Food Chem 2012;132:277–88.

Rodriguez-Campos J, Escalona-Buendıa HB, Orozco-Avila I et al.Dynamics of volatile and non-volatile compounds in cocoa(Theobroma cacao L.) during fermentation and drying pro-cesses using principal components analysis. Food Res Int2011;44:250–8.

Rohsius C, Matissek R, Lieberei R. Free amino acid amountsin raw cocoas from different origins. Eur Food Res Technol2006;222:432–8.

Romanens E, Leischtfeld SF, Volland A et al. Screening of lacticacid bacteria and yeast strains to select adapted antifungalco-cultures for cocoa bean fermentation. Int J Food Microbiol2019;290:262–72.

Romanens E, Naf R, Lobmaier T et al. A lab-scale model sys-tem for cocoa bean fermentation. Appl Microbiol Biotechnol2018;102:3349–62.

Romero-Cortes T, Robles-Olvera V, Rodriguez-Jimenes G et al.Isolation and characterization of acetic acid bacteria in cocoafermentation. Afr J of Microbiol Res 2012;6:339–47.

Romero-Cortes T, Salgado-Cervantes MA, Garcıa-Alamilla P et al.Relationship between fermentation index and other bio-chemical changes evaluated during the fermentation ofMexican cocoa (Theobroma cacao) beans. J Sci Food Agric2013;93:2596–604.

Romero CT, Cuervo PJA, Ortız YG et al. Influence of acetic acidbacteria on the acidity of the cocoa beans during fermenta-tion. Fermented Food and Beverages 2012;2:497–501.

Rottiers H, Sosa DAT, De Winne A et al. Dynamics of volatile com-pounds and flavor precursors during spontaneous fermenta-tion of fine flavor Trinitario cocoa beans. Eur Food Res Technol2019;245:1917–37.

Ruf F, Kone S, Bebo B. Le boom de l’anacarde en Cote d’Ivoire:transition ecologique et sociale des systemes a base de cotonet de cacaos. Cahiers Agri 2019;28:21.

Ruggirello M, Nucera D, Cannoni M et al. Antifungal activity ofyeasts and lactic acid bacteria isolated from cocoa bean fer-mentations. Food Res Int 2019;115:519–25.

Rusere F, Mkuhlani S, Crespo O et al. Developing pathways toimprove smallholder agricultural productivity through eco-logical intensification technologies in semi-arid Limpopo,South Africa. Afr J Sci Technol Innov Dev 2019;11:543–53.

Saltini R, Akkerman R, Frosch S. Optimizing chocolate produc-tion through traceability: a review of the influence of farmingpractices on cocoa bean quality. Food Control 2013;29:167–87.

Samagaci L, Ouattara H, Niamke S et al. Pichia kudriavzevii andCandida nitrativorans are the most well-adapted and relevantyeast species fermenting cocoa in Agneby-Tiassa, a local Ivo-rian cocoa producing region. Food Res Int 2016;89:773–80.

Samagaci L, Ouattara HG, Goualie BG et al. Growth capacity ofyeasts potential starter strains under cocoa fermentationstress conditions in Ivory Coast. Emir J Food Agric 2014;26:861–70.

Samagaci L, Ouattara HG, Goualie BG et al. Polyphasic analysis ofpectinolytic and stress-resistant yeast strains isolated fromIvorian cocoa fermentation. J Food Res 2015;4:124–35.

Dow


ic.oup.com/fem


ber 2020


Samah OA, Ptih MF, Selamat J. Biochemical changes duringfermentation of cocoa beans inoculated with Saccharomycescerevisiae (wild strain). J Food Sci Technol 1992;29:341–3.

Samah OA, Puteh MF, Selemat J et al. Fermentation products incocoa beans inoculated with Acetobacter xylinum. ASEAN FoodJ 1993;8:22–5.

Sanchez J, Daguenet G, Guiraud J-P et al. A study of the yeast floraand the effect of pure culture seeding during the fermenta-tion of cocoa beans. Lebensmitt Wiss Technol 1985;18:69–76.

Sandhya MVS, Yallappa BS, Varadaraj MC et al. Inoculum ofthe starter consortia and interactive metabolic process inenhancing quality of cocoa bean (Theobroma cacao) fermen-tation. Food Sci Technol 2016;65:731–8.

Scalone GLL, Textoris-Taube K, De Meulenaer B et al. Cocoa-specific flavor components and their peptide precursors. FoodRes Int 2019;123:503–15.

Schwan RF, Fleet GH, Afoakwa EO. Cocoa and Coffee Fermentations.Boca Raton: CRC Press, 2015.

Schwan RF, Wheals AE. The microbiology of cocoa fermenta-tion and its role in chocolate quality. Crit Rev Food Sci Nutr2004;44:205–21.

Schwan RF. Cocoa fermentations conducted with a definedmicrobial cocktail inoculum. Appl Environ Microbiol1998;64:1477–83.

Servent A, Boulanger R, Davrieux F et al. Assessment of cocoa(Theobroma cacao L.) butter content and composition through-out fermentations. Food Res Int 2018;107:675–82.

Sirbu D, Grimbs A, Corno M et al. Variation of triacylglycerol pro-files in unfermented and dried fermented cocoa beans of dif-ferent origins. Food Res Int 2018;111:361–70.

Snauwaert I, Papalexandratou Z, De Vuyst L et al. Characteriza-tion of strains of Weissella fabalis sp. nov. and Fructobacillustropaeoli from spontaneous cocoa bean fermentations. Int JSyst Evol Microbiol 2013;63:1709–16.

Soumahoro S, Ouattara HG, Goualie BG et al. Occurrence of highacetic acid-producing bacteria in Ivorian cocoa fermentationand analysis of their response to fermentative stress. Am JBioSci 2015;3:70–9.

Sousa LS, Rocha FS, Silveira PTS et al. Enzymatic activity of pro-teases and its isoenzymes in fermentation process in cul-tivars of cocoa (Theobroma cacao L.) produced in SouthernBahia, Brazil. Food Sci Technol Campinas 2016;36:656–63.

Suazo Y, Davidov-Pardo G, Arozarena I. Effect of fermentationand roasting on the phenolic concentration and antioxidantactivity of cocoa from Nicaragua. J Food Qual 2014;37:50–6.

Sukha DA, Butler DR, Comissiong EA et al. The impact of pro-cessing location and growing environment on flavor in cocoa(Theobroma cacao L.) – Implications for “terroir” and certi-fication – Processing location study. Acta Hortic 2014;1047:255–62.

Sukha DA, Umaharan P, Butler DR. The impact of pollen donoron flavor in cocoa. J Am Soc Hortic Sci 2017;142:13–9.

Sanchez-Hervas M, Gil JV, Bisbal F et al. Mycobiota and myco-toxin producing fungi from cocoa beans. Int J Food Microbiol2008;125:336–40.

Tamang JP, Watanabe K, Holzapfel WH. Review: diversity ofmicroorganisms in global fermented foods and beverages.Front Microbiol 2016;7:377.

Thompson SS, Miller KB, Lopez A et al. Cocoa and coffee. In:Doyle MP Beuchat RL (eds.). Food Microbiology: Fundamentalsand Frontiers. Washington DC: ASM Press, 2013, 881–9.

Tran PD, Van de Walle D, De Clercq N et al. Assessing cocoaaroma quality by multiple analytical approaches. Food Res Int2015;77:657–66.

Trognitz B, Cros E, Assemat S et al. Diversity of cacao trees inWaslala, Nicaragua: associations between genotype spectra,product quality and yield potential. PLoS ONE 2013;8:e54079.

Tuenter E, Foubert K, Pieters L. Mood components in cocoa andchocolate: the mood pyramid. Planta Med 2018;84:839–44.

Utrilla-Vazquez M, Rodrıguez-Campos J, Avendano-Arazate CHet al. Analysis of volatile compounds of five varieties of Mayacocoa during fermentation and drying processes by Venndiagram and PCA. Food Res Int 2020;129:108834.

Visintin S, Alessandria V, Valente A et al. Molecular identifica-tion and physiological characterization of yeasts, lactic acidbacteria and acetic acid bacteria isolated from heap and boxcocoa bean fermentations in West Africa. Int J Food Microbiol2016;216:69–78.

Visintin S, Ramos L, Batista N et al. Impact of Saccharomyces cere-visiae and Torulaspora delbrueckii starter cultures on cocoabeans fermentation. Int J Food Microbiol 2017;257:31–40.

Voigt J, Janek K, Taube KT et al. Partial purification and character-isation of the peptide precursors of the cocoa-specific aromacomponents. Food Chem 2016;192:706–13.

Voigt J, Taube KT, Wostemeyer J. pH-dependency of the prote-olytic formation of cocoa- and nutty-specific aroma precur-sors. Food Chem 2018;255:209–15.

von Wright A, Axelsson L. Lactic acid bacteria: an introduction.In: Vinderola G Ouwehand A Salminen S et al. (eds.). Lac-tic Acid Bacteria: Microbiological and Functional Aspects. BocaRaton: CRC Press, 2019, 1–16.

Vasquez-Ovando A, Chacon-Martınez L, Betancur-Ancona Det al. Sensory descriptors of cocoa beans from cultivatedtrees of Soconusco, Chiapas, Mexico. Food Sci Technol Camp-inas 2015;35:285–90.

Vasquez ZS, Neto DPC, Pereira GVM et al. Biotechnologicalapproaches for cocoa waste management: a review. WasteManagement 2019:90:72–83.

Wessel M, Quist-Wessel PMF. Cocoa production in West-Africa,a review and analysis of recent developments. Wageningen JLife Sci 2015;74-75:1–7.

Wolfe BE, Dutton RJ. Fermented foods as experimentallytractable microbial ecosystems. Cell 2015;161:49–55.

Wollgast J, Anklam E. Review on polyphenols in Theobroma cacao:changes in composition during the manufacture of chocolateand methodology for identification and quantification. FoodRes Int 2000;33:423–47.

Yao W, Doue G, Ouattara H et al. Selection of potential Bacillusstarters for cocoa beans fermentation improvement. AUDJG– Food Technol 2017a;41:131–46.

Yao W, Goualie BG, Honore G et al. Growth capacity of Bacil-lus potential starter strains isolated from cocoa beans fer-mentation under culture stress conditions. St Cerc St CICBIA2017b;18:201–11.

Yao W, Ouattara HG, Goualie B et al. Analysis of some functionalproperties of acetic acid bacteria involved in Ivorian cocoafermentation. J Appl Biosc 2014;75:6282–90.

Yusep I, Jinap S, Jamilah B et al. Influence of carboxypeptidaseon free amino acid, peptide and methylpyrazine contentsin under-fermented cocoa beans. J Sci Food Agric 2002;52:1584–92.

Zarrillo S, Gaikwad N, Lanaud C et al. The use and domestica-tion of Theobroma cacao during the mid-Holocene in the upperAmazon. Nat Ecol Evol 2018;2:1879–88.

Zaunmuller T, Eichert M, Richter H et al. Variations in the energymetabolism of biotechnologically relevant heterofermenta-tive lactic acid bacteria during growth on sugars and organicacids. Appl Microbiol Biotechnol 2006;72:421–9.

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