The Role of Microbial Dynamics, Sensorial Compounds, and Producing Regions in Cocoa Fermentation

Abstract

Cocoa fermentation is a critical step in chocolate production, influencing the flavor, aroma, and overall quality of the final product. This review focuses on the microbial dynamics of cocoa fermentation, emphasizing the roles of yeasts, lactic acid bacteria (LAB), and acetic acid bacteria (AAB). These microorganisms interact in a well-defined succession, producing organoleptic compounds such as alcohols, organic acids, and esters, which are key to the sensory profile of cocoa. This article examines the impact of different fermentation methods, including spontaneous fermentation and the use of starter cultures, on microbial communities and flavor development. Advances in starter culture technology are highlighted, demonstrating how microbial control can enhance fermentation efficiency, reduce fermentation time, and improve the consistency of chocolate flavor. Patents related to cocoa fermentation further emphasize the growing interest in microbial management to meet market demands for high-quality, distinct chocolate. This review also outlines future research directions, including the identification of new microbial strains, optimization of fermentation conditions, and the potential of biotechnological advancements to improve the fermentation process. Understanding microbial dynamics in cocoa fermentation offers significant potential for enhancing chocolate quality, sustainability, and the development of new, region-specific flavor profiles.

1. Introduction

Cocoa beans (CBs), mostly recognized for their use in chocolate, have seen increasing value throughout the years due to growing consumer demand. The global cocoa market is projected to grow from an estimated value of USD 15.15 billion in 2023 to USD 23.98 billion by 2033, reflecting a compound annual growth rate (CAGR) of approximately 4.7% during this period [1,2]. Such a scenario is attributed mainly to recent trends towards a preference for premium and artisanal cocoa-based products, which show consumers being willing to pay more for higher quality that offers more distinct flavor experiences [3]. Additionally, cocoa is increasingly used across various sectors beyond confectionery, including pharmaceuticals, cosmetics, and functional foods. This diversification is enhancing its market appeal and driving growth [1].

The fermentation of CBs is a critical step in the high-quality production of cocoa-based products, as it directly impacts the flavor, aroma, and overall sensorial qualities of the final product [4]. Furthermore, during fermentation, bioactive molecules with health-inducing properties, such as antioxidant and anti-inflammatory effects, are released [5]. This process is mainly carried out by yeasts, lactic acid bacteria (LAB), and acetic acid bacteria (AAB). The dynamic interplay of microorganisms is essential to reach high-quality end products, as each species contributes uniquely to the sensory attributes [6]. Understanding these microbial communities allows producers to optimize fermentation conditions for improved flavor and quality in cocoa products.

Different fermentation techniques can lead to distinct sensory profiles. There are primarily two: spontaneous fermentation and fermentation with starter cultures. The former happens naturally without the addition of specific microorganisms. The microorganisms responsible for spontaneous fermentations come from the surrounding environment, such as the soil and air of the producing region, and the equipment used during processing. The variability, while oftentimes posing a challenge, can also be looked at as an opportunity to discover new sensory profiles. Fermentations with starter cultures, on the other hand, involve the intentional addition of specific strains to initiate and control the process. The use of specific strains can lead to desired flavor characteristics, such as reduced bitterness and enhanced fruity or floral notes [7,8]. Each method has distinct characteristics, advantages, and implications for the final product, and, as consumer preferences shift toward unique flavor experiences, it becomes increasingly important to leverage this microbial diversity in fermentation to meet market demands.

In this context, this article explores the intricate relationships between microbial dynamics, sensorial compounds, and the diverse producing regions of cocoa. By examining how these factors interact during fermentation, we aim to highlight their significance in shaping the global cocoa market.

2. Cocoa Fermentation Processes

The fermentation of cocoa beans is a crucial step in chocolate production, significantly influencing the flavor and quality of the final product [9]. Prior to fermentation, cocoa pods are harvested from the cacao tree and the ripe pods are cut open to extract the cocoa beans along with their surrounding pulp. The extracted beans and pulp are placed in fermentation containers, which can vary from traditional wooden boxes to modern fermenters. There are three main groups of microorganisms predominant in CB fermentation: yeasts, lactic acid bacteria, and acetic bacteria [10]. For the first 24–48 h, the mixture ferments anaerobically due to the high sugar content in the pulp, primarily facilitated by yeasts. This step generates heat, raising the temperature of the mass. The temperature should ideally reach between 32 °C and 60 °C for optimal fermentation. If this temperature is not achieved within 48 h, it may indicate issues with the fermentation process. After initial yeast activity, lactic acid bacteria proliferate, converting sugars into lactic acid, which further lowers the pH and enhances flavor development. Acetic acid bacteria then take over, oxidizing ethanol produced by yeasts into acetic acid, which is crucial for flavor development and seed viability [7,11].

Fermentation typically lasts between 5 and 7 d. Advancements in controlled fermentation techniques allow for shorter durations without compromising quality, showcasing the importance of managing microbial dynamics throughout the process. Recent studies indicate that under optimized conditions, cocoa fermentation can be completed in 2 to 3 days. This reduction is achieved through the use of specific starter cultures and controlled environmental factors such as temperature and humidity [12,13]. The management of microbial dynamics is crucial in these controlled processes. By introducing specific strains of yeasts and bacteria at the beginning of fermentation, researchers have observed enhanced fermentation efficiency and flavor development. For instance, inoculating with a cocktail of yeasts and lactic acid bacteria has been shown to increase fermentation rates by up to 24% compared to spontaneous fermentation, while also improving the organoleptic properties of the beans [14].

The strategic use of microbial starter cultures not only accelerates fermentation but also enhances the complexity of flavors in the cocoa beans. This is due to the precise control over the metabolic activities of yeasts and bacteria, which produce desirable compounds more efficiently than spontaneous fermentation [12,13]. By managing the microbial community throughout the fermentation process, producers can ensure a more consistent quality in their cocoa beans. This is particularly important for fine-flavor cocoa varieties, where specific flavor profiles are sought after in chocolate production [15].

During fermentation, the microbial community evolves, contributing to the complex flavor precursors essential for high-quality chocolate [16]. After fermentation, cocoa beans are dried. The drying process is a crucial step in producing high-quality cocoa, as it reduces the water activity and moisture content of the beans, thereby preventing fungal growth and the development of undesirable flavors. The target moisture content for dried cocoa beans is typically between 5% and 8%, which is essential for their market readiness. Inadequate drying can lead to fragile beans that are prone to fungal contamination, affecting both quality and safety [17,18]. Once dried, the beans are stored in a cool, dry place until they are processed further into chocolate or other products [19]. Each of these steps is vital for developing the organoleptic properties of cocoa, including its color, flavor, and aroma, ultimately influencing the quality of chocolate produced from these beans.

3. Microorganisms

3.1. Yeasts

Yeasts are the most abundant microorganisms during the first 24 h period in cocoa fermentation and initiate a complex biochemical process that leads to the development of desirable sensory characteristics in the final product. Through the fermentation of sugars naturally present in the fruit’s pulp, namely fructose and glucose, yeasts are able to produce ethanol, heat—making the temperature rise from ambient temperature (25–30 °C) to 35–40 °C within 48 h—and carbon dioxide, creating an anaerobic environment that will be indispensable for further microbial activity in the beans [7,20]. Moreover, the produced ethanol is the precursor of acetic acid to be produced by bacteria, an important metabolite in defining cocoa’s sensory profile. This combination of metabolites is responsible for penetrating and killing the bean embryo, which allows for several biochemical reactions to happen [10]. Another important characteristic of yeasts is that they exhibit pectinolytic activity to break down the pectin in cocoa pulp. Pectin degradation facilitates aeration within the substrate, contributing to the growth of acetic acid bacteria [21].

Collectively, these actions highlight the crucial role of yeasts in establishing the environment for complex interactions among microorganisms that ultimately influence the flavor and quality of the final cocoa product. In addition to creating favorable conditions for other microorganisms, yeasts produce direct metabolic byproducts that shape the cocoa’s flavor and aroma profile. Ethanol, one of the primary products of yeast fermentation, not only acts as a precursor for acetic acid production but also serves as a solvent for volatile compounds, enhancing the perception of these sensory molecules [22]. These direct byproducts of yeast metabolism, aside from ethanol, include higher alcohols, ketones, aldehydes, and non-volatile compounds, each adding layers of complexity to cocoa’s sensory profile [23,24].

Higher alcohols, such as 2-phenylethanol and 3-methylbutanol, are direct byproducts of yeast metabolism via the Ehrlich pathway, which converts amino acids into aromatic alcohols, adding floral and fruity notes to the cocoa beans [20]. Aldehydes, including benzaldehyde and phenylacetaldehyde, provide almond and floral notes and may also balance sweetness, alongside acids [25,26]. Ketones, such as 2-heptanone and 2-nonanone, contribute fruity and floral notes, respectively. It is important to note that the production of ketones during cocoa fermentation is a complex process, with some authors suggesting that their presence may be due to compounds already existing in cocoa beans prior to fermentation. However, yeasts may indirectly influence the concentration of ketones by creating favorable conditions for their preservation during the fermentation process [23]. Glycerol is another non-volatile byproduct of yeast metabolism, produced as an osmoprotectant and, although it does not directly contribute to aroma, it adds smoothness and body to the cocoa product. Yeasts are also responsible for producing organic acids such as succinic acid, which is produced via the Krebs cycle. This provides subtle umami and sour notes that enhance flavor complexity [22,23].

On the other hand, esters, including ethyl acetate and phenethyl acetate, are not direct yeast byproducts but result from reactions between organic acids (often produced by bacteria) and alcohols generated by yeast fermentation. These esters contribute fruity and floral notes to the cocoa’s profile [27]. Likewise, pyrazines are also not direct byproducts of yeast. Instead, they are formed during the roasting process through Maillard reactions between sugars and amino acids previously broken down by yeasts and bacteria, adding the roasted and chocolate-like notes characteristic of the final product [28]. This intricate combination of yeast-derived volatiles, non-volatile compounds, and microbially influenced acids demonstrates the depth of yeast’s influence, not only in setting the environment but also in contributing fundamental flavor compounds essential to chocolate’s sensory profile.

Among the key yeast species present in cocoa fermentation are Saccharomyces cerevisiae, Pichia kudriavzevii, and Hanseniaspora opuntiae [23]. S. cerevisiae is one of the most predominant and relevant species and is known for its high fermentative capacity. It is commonly utilized as a starter culture to improve the fermentation process and enhance the sensory qualities of fermented CBs [20]. P. kudriavzevii is also frequently found, and similar to S. cerevisiae, it exhibits high ethanol tolerance and contributes to the production of aromatic compounds important for chocolate flavor [20,29]. H. opuntiae, commonly isolated during the early stages of fermentation, is recognized for its production of esters that contribute to the fruity notes of chocolate [20]. Each yeast species possesses a unique metabolic profile, expressing different enzymes and metabolic pathways, which results in the production of various compounds in differing quantities.

The absence of yeasts during cocoa fermentation leads to significant changes in both the process and the final product. This was exemplified in the work of Ho et al. (2014), which explored the crucial importance of yeasts in the cocoa fermentation process by using an innovative approach to determine their role in chocolate quality. The study compared cocoa fermentations with and without the addition of natamycin, a food additive that inhibits yeast growth. The absence of yeasts in cocoa fermentation resulted in several changes: (1) increased shell content in the beans; (2) reduced production of ethanol, higher alcohols, and esters during fermentation; and (3) a lower presence of pyrazines in the roasted product. This significantly impacted chocolate quality, as the intensity and complexity of flavor and aromas were greatly reduced. This happened mostly due to the lack of volatile compounds and smaller quantity of acetic acid produced since its generation by bacterial metabolism highly depends on ethanol concentration produced by yeasts. Beans fermented without yeasts developed a purple-violet color instead of the characteristic brown, resulting in chocolate with an acidic taste and lacking the signature chocolate flavor. This was explained by the reduced presence of pyrazines and excessive lactic acid production by bacteria, which had their growth favored in the absence of yeasts, as well as the absence of polygalacturonase and polyphenol oxidase enzyme activities, responsible for chocolate’s characteristic brown color, which depend on ethanol presence. It was also noted that the final product presented astringent notes, due to the permanence of residual sugars in the pulp not fermented by the yeasts, and a granular texture, because of the incomplete breakdown of the cocoa pulp. In contrast, beans fermented with yeasts showed a full brown color and produced chocolate with typical characteristics, which was preferred by sensory panels [30].

Moreover, a lack of yeast metabolism during CB fermentation leads to lower ethanol production, causing the fermentation to be slower and ultimately incomplete. Van de Voorde et al. (2023) showed that using S. cerevisiae in starter cultures greatly sped up the consumption of glucose and fructose, resulting in a faster and more efficient fermentation process compared to spontaneous fermentation, where S. cerevisiae may not always be present or active [14]. In the same context, Morales-Rodriguez et al. (2024) examined how incorporating yeasts and enzymes—polyphenol oxidase (PPO) and pectinase (PTE)—during fermentation affected the physicochemical quality of fine-flavor cocoa beans. The findings indicated that using S. cerevisiae was a more effective approach to improving cocoa fermentation than enzyme addition. The yeasts sped up the fermentation process and produced higher-quality beans, as shown by the cut test, reduced moisture levels, and increased protein content. The addition of enzymes resulted in less efficient fermentation, with higher moisture levels and the presence of mold. Therefore, the authors argued that the presence of yeasts inhibited the growth of undesirable microorganisms, such as filamentous fungi [21].

In the aforementioned study of Van de Voorde et al. (2023), temperature was also assessed throughout the experiments as it is a parameter that directly affects microbial growth since different organisms thrive at different temperatures. For instance, yeasts prefer moderate temperatures at the start of the process—lower than 45 °C, while acetic acid bacteria are more active at higher temperatures—between 40 and 50 °C [14]. Furthermore, temperatures above 40 °C are essential for the death of the seed embryo, a crucial step for the development of cocoa’s color and flavor, and elevated temperatures, along with the diffusion of ethanol and acetic acid into the beans, trigger endogenous enzymes that facilitate the creation of flavor precursors, which affects the product’s sensory profile. Yeast metabolism is linked to a rise in temperature, particularly due to two exothermic reactions: alcoholic fermentation and ethanol oxidation by acetic bacteria [14,31]. Hence, the presence of yeasts is crucial for maintaining these progressions of optimal temperature conditions, ensuring efficient fermentation, and enhancing the overall quality of cocoa.

3.2. Lactic Acid Bacteria

Lactic acid bacteria (LAB) play a crucial role in the fermentation of CBs by converting sugars present in the cocoa pulp into lactic acid and other metabolites, which contribute to the complex flavor profile of chocolate [6]. There are specific strains of LAB preferred for CB fermentation; Lactobacillus platarum [32], Lactobacillus rhamnosus [33], Lactobacillus fermentum [32], Leuconostoc mesenteroides [33], and Pediococcus spp. [34] are examples of these strains. Their unique properties contribute significantly to the flavor, aroma, and overall quality of chocolate products. The ideal environment for the development of LAB occurs between 24 and 72 h of fermentation, as the pulp is drained and more air enters the mass of cocoa pulp during fermentation [7].

The compounds produced by LAB during fermentation have been intensively studied due to their ability to inhibit or prevent contamination, such as that caused by fungal growth. This effect is attributed to their production of various antimicrobial compounds, including organic acids, low-molecular-weight substances, hydroxyl fatty acids, and protein-based metabolites such as bacteriocins [35]. Lactic acid (LA) is the main fermentation product of these bacteria, formed from the metabolization of sugars such as glucose and fructose. LA contributes to the acidity of cocoa nibs, a crucial factor in the development of chocolate flavor precursors [36]. LA is absent in the pulp and nibs of unfermented CBs. However, as fermentation progresses, its concentration gradually rises in the pulp and then diffuses into the nibs [37]. Acidity originating mainly from LA contributes to the death of the seed embryo, which is essential for germination and allows the development of flavor precursors [6]. Furthermore, the decrease in pH caused by the production of acids activates endogenous enzymes, such as proteases, invertases, and glycosylates, which act in the degradation of proteins, sugars, and phenolic compounds [38].

LAB also produce acetic acid (AA) during the fermentation of CBs. This acid can be produced as a byproduct of heterolactic fermentation or from the utilization of citric acid present in the pulp. AA may have a greater impact than LA on the acidity of CBs [36]. LAB also produce compounds that give chocolate a buttery note, such as diacetyl and acetoins. These are volatile compounds that give chocolate a creamy note. These compounds are produced by LAB from the metabolism of pyruvate, an intermediate in the fermentation of sugars [39]. The fruity aspect is given by the production of esters. Although yeasts are the largest producers of these compounds, LAB also contribute to the formation of these compounds, which give fruity and floral notes. In the production of these compounds, an alcohol (ethanol, for example) is condensed with a carboxylic acid (AA, for example). Benzyl alcohol, which gives floral notes, almond aroma, and a touch of sweetness to chocolate, can also be produced by LAB [40].

During cocoa fermentation, LAB can carry out different metabolic pathways that directly influence the sensory quality and safety of the fermented beans. The most significant among them are homolactic fermentation and heterolactic fermentation [41]. Homolactic fermentation involves the metabolism of glucose, primarily through the glycolytic pathway, resulting almost exclusively in lactic acid. Biochemically, glucose is converted into pyruvate, generating two molecules of ATP and two molecules of NADH per molecule of glucose. Pyruvate is subsequently reduced to lactic acid by the enzyme lactate dehydrogenase, utilizing the NADH produced during glycolysis. This step regenerates NAD⁺, which is essential for the continuation of glycolysis [42]. The activation of the glycolytic pathway occurs under conditions of high sugar availability and the absence of oxidative stress [43]. These conditions create an acidic environment that inhibits the growth of undesirable microorganisms.

In contrast, heterolactic fermentation metabolizes glucose through the pentose phosphate pathway, producing a mixture of lactic acid, ethanol, and carbon dioxide. This pathway is activated under low sugar concentrations or when the bacteria metabolize sugars other than glucose [44]. The activation of the heterolactic pathway can influence the volatilization of compounds during the drying process of the beans and affect the aromatic profile of the cocoa. Other pathways, such as those leading to the production of diacetyl and acetoin, can be activated as secondary routes. Diacetyl is produced when pyruvate is redirected to form acetolactate, which undergoes spontaneous or enzymatic decarboxylation to generate diacetyl. This pathway is typically activated under conditions of low NADH availability, which limits the reduction of diacetyl to acetoin [45]. Additionally, ester production can occur through the esterification of organic acids with alcohols. This pathway becomes more active under higher oxygen levels or in the presence of free alcohols derived from yeasts associated with the fermentation process [20].

The proportion of compounds produced during cocoa fermentation is essential to defining the sensory profile of the beans, directly affecting flavor, texture, and the aromatic complexity of the final chocolate. LA contributes a smooth acidity that can enhance bean complexity; however, as it is non-volatile, excess LA leads to a lingering acidity that can overwhelm the palate and mask other essential notes [6]. This delicate balance is often modulated through the interaction of LAB and yeasts, which produce various esters and aromatic compounds. Acetic acid production adds volatile acidity [41], amplifying flavor intensity but, if exceeding 10 mg/g in nibs, can impart overly acidic, vinegar-like notes, compromising chocolate smoothness and aroma [36]. The ideal ratio between LA and AA varies depending on the type of chocolate desired. Diacetyl, produced by both LAB and yeasts, offers mild buttery notes at low concentrations, enhancing aromatic depth; yet in high amounts, it may seem artificial, overshadowing the beans’ natural character [46]. Thus, fermentation control and careful monitoring of these compound levels are crucial for achieving a harmonious final product, balancing acidity, smoothness, and complex aromatic nuances.

The optimal fermentation conditions for CBs using LAB include maintaining temperatures between 25 and 30 °C to support LAB growth and metabolism. The fermentation typically lasts 4–7 d, allowing complete pulp degradation and the development of flavor-enhancing compounds [47]. Optimizing the fermentation process is essential for controlling and directing microbial activity to produce homogeneous, high-quality fermented cocoa beans. The addition of specific LAB strains as starter cultures can increase the presence of desirable volatiles, promoting complex and uniform flavor profiles. Such controlled fermentations ensure balanced production of lactic and acetic acids, diacetyl, and esters, contributing to a consistent and high-quality chocolate sensory profile [10]. Kresnowati et al. (2013) utilized L. plantarum starter cultures in cocoa fermentation and observed a significant increase in the concentration of lactic acid, ethanol, and acetic acid compared to standard fermentation processes. The study highlights how the introduction of specific LAB starter cultures can enhance key metabolic compounds, potentially improving the sensory quality and consistency of the final product by promoting desirable flavor profiles [48].

3.3. Acetic Acid Bacteria

Acetic acid bacteria (AAB) constitute a group of Gram-negative, obligate aerobic microorganisms that are distinguished by their capacity to oxidize sugars or alcohols to organic acids through acetic fermentation [49,50]. This process entails the conversion of ethanol to acetaldehyde by the catalytic action of pyrroloquinoline quinone-dependent alcohol dehydrogenase (PQQQ-ADH), which is followed by the oxidation of acetaldehyde to acetic acid, catalyzed by aldehyde dehydrogenase (ALDH). In this metabolic pathway, oxygen serves as the final electron acceptor, resulting in the generation of H₂O₂ or H₂O and the synthesis of adenosine triphosphate (ATP), which releases energy [51,52]. These metabolic properties have been exploited in various industrial applications, particularly in the food sector.

This group of microorganisms, which primarily comprises the genera Acetobacter, Gluconobacter, and Komagataeibacter, is widely distributed in environments with high concentrations of sugars and acidic conditions, such as fermented foods and fruits [13,53,54]. Their capacity to withstand these extreme conditions confers upon them a remarkable adaptive advantage. Moreover, these bacteria are capable of producing a variety of secondary compounds, including bacterial cellulose and aromatic compounds, which present a wide range of potential applications in industry [55,56].

During the final stage of microbial succession, AAB plays a role in the fermentation process of cocoa. The presence of specific compounds, including mannitol, ethanol, and lactic acid, which have been previously produced by yeasts and lactic acid bacteria (LAB), is conducive to this process. These compounds create a favorable environment for the proliferation of these bacteria due to their capacity for oxidative metabolism of ethanol [45]. Furthermore, the periodic mixing of cocoa beans creates an environment with elevated oxygen levels, which enhances the metabolic activity of ABB [57,58]. As a consequence of this oxidative process, the primary products are acetic acid and, to a lesser extent, lactic acid [59].

Acetic acid, produced by ABB, plays a pivotal role in the fermentation of cocoa beans. This compound penetrates cotyledons, destabilizing the cell structure and altering key components such as proteins, polyphenols, and lipids [45]. These modifications exert a profound influence on essential bean characteristics, including the formation of chocolate flavor precursors, the development of characteristic color, and the inhibition of bean germination [60,61]. Ultimately, the majority of acetic acid is eliminated through volatilization during the sun-drying process.

Recent studies have demonstrated that the utilization of starter cultures enables more precise control of the cocoa fermentation process, which can offer significant advantages. Such benefits include an improvement in the organoleptic characteristics of the final product, an increase in the productivity of the process, and a reduction in the time required to complete fermentation. Furthermore, the utilization of starter cultures enables more regulated management of the process, reducing the impact of external variables and facilitating a more comprehensive analysis of the compounds produced by the involved microorganisms.

In a recent study, Falconi et al. (2023) demonstrated that a starter culture comprising two yeasts (Torulaspora delbrueckii and Hanseniaspora uvarum), a lactic acid bacterium (Limosilactobacillus plantarum), and an acetic bacterium (Acetobacter ghanensis) resulted in notable alterations in the sugar and acidity content, accompanied by an increase in polyphenol concentration and alterations in pH and temperature levels. Furthermore, A. ghanensis demonstrated a beneficial impact on the fermentation process by inhibiting external microorganisms, such as filamentous fungi, which impair the flavor and quality of the grains. This effect is attributed to the bacterium’s production of acetic acid [14].

These results substantiate the assertion that ABB plays a pivotal role in cocoa fermentation, functioning as “aseptic” agents. Chang et al. (2024) corroborated this assertion through genomic and metabolomic analyses, which revealed a notable decline in the abundance of other microbial taxa in the final stages of fermentation following bean oxygenation. Conversely, there was an exponential proliferation of species belonging to the genus Acetobacter. This phenomenon can be attributed to the tolerance of these microorganisms to acidic pH and compounds such as acetic acid, conditions that are inhospitable to other microbial groups [62].

Furthermore, Acetobacter spp. have been shown to contribute to the synthesis of dipeptides, which influence the development of the characteristic flavor of cocoa [63], as well as dethiobiotin, a precursor of biotin, an essential vitamin in various metabolic processes [64]. Furthermore, it has been documented that these bacteria are responsible for the reduction in compounds such as theobromine and caffeoyl-aspartic acid. The former is an alkaloid, while the latter is a phenolic compound. Both are associated with both the characteristic bitterness of cocoa and its antioxidant properties [65].

In summary, cocoa fermentation relies on a dynamic microbial environment, where yeasts, LAB, and AAB each play distinct yet complementary roles in developing the flavor, aroma, and quality of the final chocolate product, as shown in Figure 1. During fermentation, different microorganisms succeed one another in a dynamic process. Yeasts, such as Saccharomyces cerevisiae and Torulaspora delbrueckii, initiate fermentation by converting sugars into ethanol and producing various flavor compounds. They create an anaerobic environment that supports their metabolic processes and promotes the growth of beneficial bacteria. Yeasts also produce ethanol and aid in the breakdown of pectin, which improves aeration and nutrient availability for subsequent microbial populations. This sets the stage for the production of essential compounds, such as acetic acid, which drive the distinctive flavor development in cocoa.

LAB then take over the fermentation process, converting sugars into lactic acid and stabilizing the environment. This process supports the growth of AAB, which play a crucial role in the later stages of fermentation. LAB also produce aromatic compounds, such as diacetyl and esters, which elevate the sensory profile of chocolate. In addition to their contribution to flavor complexity, LAB like Lactobacillus plantarum and Leuconostoc mesenteroides contribute to the overall acidity of cocoa beans. Furthermore, controlled fermentation using LAB starter cultures optimizes the production of these compounds, ensuring balanced acidity and uniformity in high-quality chocolate.

Finally, AAB, particularly those from the genera Acetobacter, Gluconobacter, and Komagataeibacter, oxidize ethanol into acetic acid, which is vital for flavor development and bean preservation. This step facilitates the breakdown of proteins, polyphenols, and lipids, shaping the beans’ flavor and color and reducing bitterness. AAB also thrive in acidic environments and produce secondary metabolites like bacterial cellulose, further underscoring their importance in fermentation.

The interplay between these microbial groups leads to the production of various metabolites, contributing to the complex and rich flavors in chocolate. For instance, LAB contribute to flavor through the production of organic acids and sugar alcohols, while AAB are essential for creating the acidic environment that facilitates further biochemical transformations within cocoa beans. Together, these microorganisms work in synergy to ensure a well-balanced fermentation process, which directly influences the sensory qualities and high quality of the final chocolate product.

4. Regional Influence in the Sensorial Profile of Fermented Cocoa

Recent studies have highlighted the critical role of regional microbial populations in shaping cocoa fermentation processes. For instance, Koné et al. (2016) identified key yeast strains, such as Saccharomyces cerevisiae, Candida tropicalis, Pichia kudriavzevii, Pichia galeiformis, Geotrichum geotrichum, and Wickerhamomyces anomalus, as dominant during the early stages of fermentation. In later stages, Pichia kudriavzevii and G. geotrichum prevailed [66]. Similarly, Koff et al. (2017) examined six cocoa-producing regions in Ivory Coast, observing a decline in sugar and yeast concentrations after 72 h of fermentation. Predominant yeasts included P. kudriavzevii, Pichia kluyveri, Saccharomyces cerevisiae, and Hanseniaspora uvarum [67].

Microbial diversity varies significantly across regions. In southern Indonesia, Jamili et al. (2016) isolated seven yeast types over three days of fermentation, including Saccharomyces bayanus, Candida tropicalis, and Kloeckera sp. [64]. Research in Ivory Coast revealed yeast strains and 15 genera of lactic acid bacteria (LAB), with Lactobacillus plantarum and Acetobacter pasteurianus ubiquitous across all regions [68]. Mendoza Salazar and Lizarazo-Medina (2021) noted regional differences in Colombia, where Hanseniaspora opuntiae and Saccharomyces sp. dominated [69].

In Hainan, China, studies reported the predominance of Hanseniaspora yeasts and Acetobacter bacteria, influenced by aerobic conditions. In Ecuador, researchers identified 50 bacterial and 20 yeast species during spontaneous fermentation of fine cocoa. Dominant microorganisms included S. cerevisiae, Candida metapsilosis, and Acetobacter pasteurianus. Culture-independent methods revealed even greater microbial diversity, detecting 136 bacterial genera and 42 fungal genera [70].

Advancements in microbial research have facilitated the development of starter cultures aimed at stabilizing and replicating dominant strains from fermentation. These selected strains are vital for producing volatile compounds associated with high-quality chocolate [24]. For instance, Pichia kluyveri starter cultures enhance phenylacetaldehyde production, imparting floral and fruity aromas [71,72].

Cocoa’s sensory profile is shaped by various volatile compounds, including esters, higher alcohols, and organic acids. Ho et al. (2015) documented significant increases in these compounds during spontaneous fermentation, enhancing chocolate’s aroma [73]. Key compounds such as linalool (found in fine cocoa varieties) and phenylethyl acetate contribute distinct floral and fruity notes, distinguishing premium chocolate from bulk varieties [74,75].

The fermentation process significantly influences cocoa’s flavor and aroma. Microbial activity, coupled with physical and biochemical changes, leads to enzyme release and the formation of aroma precursors like polyphenols and free amino acids. Optimal fermentation reduces bitterness and astringency, enhancing nutty and fruity notes [76,77]. Control of parameters such as temperature and pH is essential for consistent quality. These factors directly affect enzyme activity and microbial metabolism, underscoring the importance of rigorous monitoring during fermentation [38,72].

The sensory profile of cocoa is a key indicator of its quality, as it directly affects the product’s texture, aroma, color, and flavor. These attributes are essential for creating premium-quality cocoa-based products [78]. The development of cocoa’s aroma can be traced to multiple sources. Some aromas are naturally present in the cocoa pulp, while others are formed during processing steps such as fermentation, roasting, and chocolate production. The physical and biochemical transformations from harvest to processing play a pivotal role in defining the final flavor [76,77].

Producing high-quality cocoa with a rich aroma starts with choosing the right varieties for fermentation. Criollo and Trinitario genotypes, known as fine cocoa varieties, are distinguished by their higher terpene levels, especially linalool, which ranges from 1.2 to 4.7 mg·kg⁻¹. This monoterpene imparts a floral aroma, a signature characteristic of chocolate derived from these varieties. In comparison, Forastero genotypes, with linalool levels below 0.2 mg·kg⁻¹, are classified as bulk cocoa.

In addition to linalool, other compounds, such as 2-phenyl-ethyl acetate and benzaldehyde, are initially found in the cocoa pulp and persist throughout production. Some, like alpha-terpineol, develop exclusively during fermentation [72,79,80,81,82,83,84].

The key aroma and color precursors—polyphenols (e.g., epicatechin), reducing sugars (e.g., glucose, fructose), and free amino acids (e.g., leucine, alanine, phenylalanine, tyrosine)—formed during fermentation are further developed during roasting. Without these precursors, the resulting chocolate would have floral and honey-like aromas but lack the characteristic chocolate flavor [85,86]. A study in Colombia revealed that fermentation reduces polyphenols by up to 40%, thereby decreasing bitterness and astringency while enhancing desirable aromas such as nutty, fruity, and cocoa notes. Low levels of epicatechin, catechin, and procyanidins are associated with reduced bitterness, intensified chocolate color, and greater aromatic precursor availability [76].

Cocoa contains bioactive compounds such as methylxanthines (caffeine, theobromine) and flavan-3-ols (epicatechin, catechin), which show potential for geographical identification of cocoa origins [87]. The abundance of polypeptides and free amino acids varies geographically; for example, cocoa from the Americas tends to have higher levels of these compounds compared to cocoa from Southeast Asia. Soil fertilization, pH, and seasonal variations also influence nitrogen concentrations in seeds [88].

Microorganisms play an essential role in the fermentation process and significantly influence cocoa’s sensory qualities. Yeasts are especially important, as their absence results in reduced ethanol synthesis, leading to poorly fermented, purple-colored beans that lack the characteristic chocolate aroma. Non-Saccharomyces yeasts dominate cocoa fermentation, with genera like Candida, Hanseniaspora, and Pichia commonly found in cocoa-producing regions. Candida species are noted for producing higher alcohols like isoamyl alcohol, which adds fruity, banana-like notes to cocoa flavor [71,89]. Pichia species, including Pichia manshurica and Pichia kluyveri, are widely used as starter cultures to enhance flavor, with some showing high polygalacturonase activity for pulp degradation [90]. These species significantly boost the concentrations of key aromatic compounds, such as phenylacetaldehyde, during fermentation.

Bacteria also play a pivotal role in fermentation by producing organic acids and aroma precursors. Dominant bacterial genera include Lactobacillus, Acetobacter, and Gluconobacter, which are prevalent in most cocoa-producing regions, except in some parts of Asia where Gluconobacter is absent. Acetobacter pasteurianus, known for its resilience to extreme conditions, dominates in many regions due to its ability to tolerate high acidity and moderate temperatures. Additionally, species like Komagataeibacter hansenii and Roseomonas sp. have been identified in specific regions, such as Brazil’s Amazon [91].

Microbial diversity in cocoa fermentation varies greatly by geographical location and fermentation conditions. Although certain microorganisms have potential as geographical indicators, specific relationships between microbial species and their regions of origin remain underexplored (Figure 2). A recent meta-analysis identified over 1700 microbes in cocoa fermentations, with Brazil reporting the highest microbial diversity (612 species), followed by Côte d’Ivoire (237) and Ghana (257). However, no direct correlations between microbes and geographic location have been established [92].

During roasting, temperatures of 120 to 170 °C transform fermentation-derived aroma precursors into the characteristic aromas of chocolate. Heat triggers non-enzymatic browning reactions, including sugar caramelization and the Maillard reaction, which produce key intermediate compounds like aldehydes and pyrazines. These compounds provide the dominant roasted cocoa aroma, with pyrazines accounting for about 40% of the characteristic chocolate flavor. Well-fermented cocoa exhibits specific ratios of pyrazines, such as a tetramethylpyrazine-to-trimethylpyrazine ratio of 20.94, whereas poorly fermented cocoa shows nearly zero ratios [93].

Figure 2. (A) Diversity of yeasts in cocoa fermentations from different geographic locations. (B) Diversity of bacteria in cocoa fermentations from different geographic locations. Sources by continent: Africa—[53,94,95,96,97,98,99,100]; America—[72,80,91,96,101,102,103,104,105,106]; Asia—[72,107,108]; and Australia/Oceania—[37,109].

Figure 2. (A) Diversity of yeasts in cocoa fermentations from different geographic locations. (B) Diversity of bacteria in cocoa fermentations from different geographic locations. Sources by continent: Africa—[53,94,95,96,97,98,99,100]; America—[72,80,91,96,101,102,103,104,105,106]; Asia—[72,107,108]; and Australia/Oceania—[37,109].

Several classes of non-volatile and volatile chemical compounds are responsible for the composition of cacao flavor. Phenolic compounds such as catechins (catechin and epicatechin), procyanidines, and alkaloids are important precursors of aromas produced during fermentation. Aldehydes, ketones, esters, alcohols, pyrazines, quinoxalines, furans, pyrones, lactones, pyrroles, and diketopiperazines are compounds that contribute to the formation of cocoa flavor [78].

The profiles of volatile compounds have been investigated for use as markers of origin and geographical identification to differentiate them based on the production location [110,111,112]. Literature studies have revealed that the highest content of volatile compounds throughout the cocoa production chain was found in samples from the Americas (n = 150), followed by Africa (n = 131) and Southeast Asia (n = 69) (Figure 2), with predominant classes being alcohols, esters, terpenes, and phenols.

Figure 3 illustrates the volatile compounds reported in the literature across the cocoa production chain. Cocoa pulp predominantly contains terpenes. During fermentation, esters, alcohols, and terpenes are the main compounds. Unroasted cocoa beans exhibit esters, alcohols, aldehydes, and pyrazines as the predominant compounds. In roasted cocoa beans, there is an increase in pyrazines, characteristic of the roasting process, along with the presence of esters, alcohols, aldehydes, and furans/pyrans. In chocolate, esters, alcohols, and aldehydes are predominant, accompanied by terpenes, organic acids, hydrocarbons, furans/pyrans, and others.

Figure 3. Map of volatile compounds detected during chocolate production by geographic location. The numbers represent the quantity of volatile compounds classified by class. (This figure was created from data in the Supplementary Material Table S1.) Source: [113].

Figure 3. Map of volatile compounds detected during chocolate production by geographic location. The numbers represent the quantity of volatile compounds classified by class. (This figure was created from data in the Supplementary Material Table S1.) Source: [113].

Cambrai et al. (2010) investigated the aromatic profile of chocolates produced in African, Caribbean, and Madagascan countries and found that each group presented distinct volatile compounds. In the African group, the major volatile compounds identified were benzaldehyde (hazelnut aroma), linalool (floral aroma), and phenylacetaldehyde (honey aroma). In the Caribbean samples, (E,E)-2,4-nonadienal and (E,E)-2,4-decadienal were the predominant compounds, likely derived from the degradation of milk components during the conching process. In Madagascan samples, aldehydes such as 2-phenyl-2-butenal and 4-methyl-2-phenyl-pentenal, which impart the characteristic chocolate aroma, were identified as products of roasting [114].

In Brazil, a study also tracked volatile compounds in chocolates produced with cocoa from different regions according to consumer preferences. The presence of compounds such as 3-methyl butanal, phenethyl acetate, 2-phenyl-5-methyl-2-hexenal, methyl pyrazine, phenethyl acetate, 2-phenyl-5-methyl-2-hexenal, and tetramethyl pyrazine was deemed ideal for higher consumer acceptability. Volatile compounds like 2,3-methyl pyrazine, methyl pyrazine (nutty, chocolatey), and 2,3-butanediol (sweet, flowery) are important for positively influencing consumer preferences. In contrast, (Z)-2-heptenal and 2-pentyl furan are compounds most associated with chocolate rejection [115].

Figure 4 shows the aroma descriptors of volatile compounds according to geographical regions. Higher volatile compounds with fruity and floral notes were found in cocoa produced in America, followed by Africa and Southeast Asia. A systematic review analyzed the existing literature on sensory attributes to describe FFC beans and chocolate and suggested that although the same cacao variety may share similar flavor notes among varieties from different regions, there are specific flavors that are unique to certain origins. Criollo varieties from Mexico showed nutty and caramel characteristics, while those in Peru were found to be only nutty. Trinitario varieties from Colombia had a predominant fruity note, as well as those in the Dominican Republic and Indonesia, including notes of fresh, red, and yellow fruits [116].

Regional variations in microbial populations—spanning yeast, lactic acid bacteria, and acetic acid bacteria—are critical for producing the volatile compounds that define high-quality chocolate, as underscored by recent studies showing the pivotal role of microbial diversity in shaping cocoa fermentation. Advances in microbial research, such as starter cultures and culture-independent methods, offer exciting opportunities to standardize fermentation processes while preserving the unique flavor profiles tied to geographical origins. Understanding these dynamics provides valuable insights for enhancing cocoa quality, differentiating regional varieties, and meeting the growing demand for premium chocolate.

5. Starter Cultures in CB Fermentation

The use of starter cultures for cocoa pulp fermentation is becoming increasingly widespread. These cultures help achieve a controlled fermentation process by shortening fermentation times, maintaining regulated conditions, and ensuring consistent quality. Commonly used microorganisms in starter cultures include yeasts such as Saccharomyces cerevisiae and Hanseniaspora uvarum, along with certain species of Candida and Pichia [21].

In spontaneous fermentation, microbial dynamics are influenced by the natural competition between indigenous yeasts, LAB, and AAB, with the balance shifting as the fermentation progresses [117,118]. These microorganisms naturally compete for resources such as sugars and organic acids, leading to varied fermentation outcomes. Early-stage yeasts typically dominate due to their rapid sugar consumption, while LAB may take over as the pH drops and lactic acid accumulates. These microbial interactions directly influence the flavor profile by determining which metabolites are produced. High microbial diversity can enhance flavor complexity but may also lead to inconsistencies if microbial competition is not balanced [58,113].

Moreover, microbial communities can vary significantly depending on regional environmental conditions and the genetic diversity of cocoa plants. This variation results in differences in microbial succession and, consequently, in the flavor profiles of the final cocoa beans [119,120]. Understanding these regional and genetic differences is essential when selecting starter cultures, as it suggests the need for strains tailored to the unique characteristics of each cocoa-producing area. Recent studies highlighted that using dedicated starter cultures could improve both on-farm and controlled fermentation outcomes. For example, controlled fermentations in Japan underscored the importance of stable microbial populations to ensure consistent cocoa product quality [119].

In controlled fermentation with starter cultures, microbial competition is regulated, ensuring the dominance of specific strains that contribute desirable characteristics. For instance, Saccharomyces cerevisiae strains have been shown to perform well under controlled conditions, maintaining a pH of 3.8 even at temperatures as high as 48 °C. This contrasts with spontaneous fermentation, where the pH typically drops to 3.4, altering the growth conditions of microorganisms involved [121].

Currently, microorganisms are selected for their technological advantages in industrial processes, where the goal is to reduce costs and production times. Effective starter cultures in cocoa fermentation should combine microbial diversity with rapid fermentation capabilities, control over chemical transformations, antifungal properties, and enhancement of sensory attributes to produce high-quality cocoa products [14,122,123]. Furthermore, the use of robust starter cultures leads to more consistent microbial profiles across batches, which is essential for maintaining product quality and reducing variability associated with spontaneous fermentation [124].

Chagas Junior et al. (2021) showed that combining Saccharomyces cerevisiae and Pichia kudriavzevii strains improved the fermentation process by increasing methylxanthine levels and phenolic compounds, which enhanced cocoa’s antioxidant properties and mood modulation. This combination accelerated microbial succession, reduced fermentation time by one day, and inhibited the production of putrefactive amines [125]. Additionally, studies by Papalexandratou et al. (2019) also emphasized the importance of inoculating cocoa beans with thermotolerant yeast strains to achieve superior aromatic properties. Cocoa fermented with hybrid strains not only produced better sensory profiles but also inhibited mold and reduced mycotoxin risks [126].

Furthermore, the introduction of starter cultures like Lactobacillus plantarum has been shown to reduce residual fructose during the acetic acid fermentation stage, improving flavor and aroma profiles [127]. When combined with Pediococcus acidilactici strains, these cultures produce higher levels of ethanol, lactic acid, acetic acid, 2-methyl-1-butanol, isoamyl acetate, and ethyl acetate, further enhancing the flavor of fermented cocoa. In Ghana, studies on depulping cocoa beans to varying extents showed that 50% depulping followed by spontaneous fermentation in piles resulted in optimal acidification, improved flavor scores, and reduced drying times from 144 h to 4.3 h [128]. The use of a Hanseniaspora opuntiae and Kluyveromyces marxianus (1:1) mix as a starter culture also reduced fermentation time by 20% compared to spontaneous fermentation. This combination lowered acetic acid content and produced a floral flavor profile [12].

While specific starter cultures can enhance the fermentation process, careful consideration must be given to their impacts on the natural microbial community. Starter cultures can offer consistent fermentation outcomes by ensuring that beneficial strains dominate and reduce undesirable microbial activity. However, relying on them may diminish the diversity of the indigenous microbiome, potentially causing a loss of unique regional flavors. This reduction in biodiversity could impact the quality and character of cocoa from specific regions [10,129].

The introduction of starter cultures can significantly alter the naturally occurring microbiome. Starter cultures may outcompete indigenous strains, leading to a decline in beneficial microorganisms that contribute to flavor complexity. Over time, repeated use of certain starter cultures may shift the microbial community structure, reducing biodiversity and altering traditional fermentation practices [120].

Thus, while combinations of yeast strains, LAB, and AAB are essential for successful cocoa fermentation, balancing the benefits of enhanced efficiency with the preservation of indigenous microbial diversity is crucial. The integration of modern starter cultures with traditional fermentation methods presents an opportunity to preserve cultural heritage while optimizing outcomes. This balance is crucial for maintaining regional identities in cocoa production while also meeting global market standards. The use of starter cultures can help standardize flavor profiles across different batches, ensuring that producers can deliver consistent quality [130,131].

Table 1 presents various studies on starter cultures and their results, highlighting the diverse impact of different cultures on fermentation dynamics, flavor profiles, and microbial health. These studies emphasize the importance of selecting the appropriate strains for specific fermentation goals.

In summary, understanding microbial dynamics during cocoa fermentation is essential for optimizing the process and improving cocoa quality. The competition and succession of microbial species, from yeasts to LAB and AAB, are crucial in determining the fermentation outcome. By controlling these dynamics by employing starter cultures, it is possible to enhance the fermentation process, reduce variability, and produce cocoa with consistent and high-quality flavor profiles. Recent advances in microbiology have led to the development of specialized strains that thrive under challenging conditions, such as high temperatures and low pH, making fermentation faster and more reliable. These innovations not only improve efficiency but also highlight the potential for tailoring fermentation processes to meet the needs of both artisanal and industrial chocolate production [132]. The integration of modern starter cultures with traditional methods presents opportunities to preserve cultural heritage while optimizing fermentation outcomes. The future of cocoa fermentation lies in striking this balance to produce high-quality cocoa that satisfies both regional identity and global market standards.

Table 1. Relevant studies with starter cultures and their main outcomes.

Starter Microorganisms	Study Objective	Key Findings	Unique Outcome	Ref.
Saccharomyces cerevisiae, Torulaspora delbrueckii	Effect of mixed cultures on Brazilian hybrid cocoa clones	T. delbrueckii dominated early; S. cerevisiae dominated later, enhancing flavor and aroma profiles.	Improved flavor	[133]
Pichia kudriavzevii, Hanseniaspora thailandica, Hanseniaspora opuntiae, Wickerhamomyces spp., S. cerevisiae	Impact of native yeast starters on antioxidant activity in Malaysian cocoa	Higher polyphenol and flavonoid content in H. thailandica and P. kudriavzevii compared to control.	Increased antioxidants	[134]
S. cerevisiae IMDO 050523, P. kudriavzevii IMDO 020508	Monitoring starter cultures during fermentation and drying stages	The use of starter cultures extended fermentation by one additional day but increased production of desired metabolites, especially volatile organic compounds.	Enhanced aroma	[135]
Lactobacillus plantarum, Pediococcus acidilactici, Pichia fermentans	Co-culturing fructophilic lactic acid bacteria and yeast to enhance sugar metabolism and aroma formation during fermentation	Mixed cultures reduced residual sugars and suppressed wild microflora, suggesting these microbes optimize fermentation and aromatic profiles.	Enhanced color and flavor	[127]
S. cerevisiae, Pichia kudriavzevii	Effect of yeast strains on volatile compounds in Colombian cocoa	P. kudriavzevii increased desirable compounds in chocolate, suggesting mixed cultures could improve cocoa quality.	Unique regional identity	[136]
L. plantarum, S. cerevisiae, Acetobacter pasteurianus	Physiological characterization of starter cultures in cocoa fermentation	Microbial populations increased rapidly under controlled fermentation conditions, achieving higher concentrations by day 2–3 compared to natural fermentation.	Faster microbial growth	[137]
Torulaspora delbrueckii, Hanseniaspora uvarum, Limosilactobacillus plantarum, Acetobacter ghanensis	Inoculation of native microbial starter cultures to optimize fine-aroma cocoa bean fermentation	Starter cultures at various stages increased polyphenol content, improved efficiency by 24%, and decreased sugars and acids, allowing drying in 96 h. They also reduced filamentous fungi that could negatively affect flavor and aroma.	Decreased drying time and enhanced flavor	[14]
Hanseniaspora opuntiae, S. cerevisiae	Multiphase analysis of the chocolate production chain to assess the functionality of starter cultures	S. cerevisiae demonstrated superior functionality, resulting in better flavor profiles and acidic notes and outperforming Hanseniaspora opuntiae, which led to under-fermented beans.	Superior flavor profiles	[138]
S. cerevisiae, W. anomalus, C. tropicalis, P. kudriavzevii	Effect of solar predrying treatments and inoculation with yeast starter cultures on the chemical composition of cocoa beans from Colombia	Starter cultures and predrying treatments altered chemical composition, reducing polyphenol, caffeine, and lactic acid content.	Improved sensory attributes	[139]
S. cerevisiae	Amino acid profile during Criollo cocoa fermentation	Starter cultures increased essential amino acids (63.4% vs. 61.8% in spontaneous fermentation) and reduced fermentation time by 3–4 d.	Improved flavor and aroma profiles.	[140]
Lactiplantibacillus plantarum subsp. plantarum HL-15	Alternative cocoa bean fermentation method using L. plantarum HL-15 as a starter culture and valorizing cocoa mucilage byproducts	Pulp removal and inoculation with L. plantarum HL-15 improved bean quality, suppressed fungal growth, and enhanced sensory qualities.	Reduced fungi and improved flavor	[141]
Issatchenkia orientalis, S. cerevisiae, W. anomalus, H. thailandica, P. kluyveri, Candida oleophila	Sensory and chemical selection of indigenous yeasts in cocoa fermentation	Different strains imparted unique sensory notes, with H. thailandica and P. kluyveri recognized by the panel for their outstanding flavor profiles.	Distinct flavor profiles	[142]

Table 1. Relevant studies with starter cultures and their main outcomes.

Starter Microorganisms	Study Objective	Key Findings	Unique Outcome	Ref.
Saccharomyces cerevisiae, Torulaspora delbrueckii	Effect of mixed cultures on Brazilian hybrid cocoa clones	T. delbrueckii dominated early; S. cerevisiae dominated later, enhancing flavor and aroma profiles.	Improved flavor	[133]
Pichia kudriavzevii, Hanseniaspora thailandica, Hanseniaspora opuntiae, Wickerhamomyces spp., S. cerevisiae	Impact of native yeast starters on antioxidant activity in Malaysian cocoa	Higher polyphenol and flavonoid content in H. thailandica and P. kudriavzevii compared to control.	Increased antioxidants	[134]
S. cerevisiae IMDO 050523, P. kudriavzevii IMDO 020508	Monitoring starter cultures during fermentation and drying stages	The use of starter cultures extended fermentation by one additional day but increased production of desired metabolites, especially volatile organic compounds.	Enhanced aroma	[135]
Lactobacillus plantarum, Pediococcus acidilactici, Pichia fermentans	Co-culturing fructophilic lactic acid bacteria and yeast to enhance sugar metabolism and aroma formation during fermentation	Mixed cultures reduced residual sugars and suppressed wild microflora, suggesting these microbes optimize fermentation and aromatic profiles.	Enhanced color and flavor	[127]
S. cerevisiae, Pichia kudriavzevii	Effect of yeast strains on volatile compounds in Colombian cocoa	P. kudriavzevii increased desirable compounds in chocolate, suggesting mixed cultures could improve cocoa quality.	Unique regional identity	[136]
L. plantarum, S. cerevisiae, Acetobacter pasteurianus	Physiological characterization of starter cultures in cocoa fermentation	Microbial populations increased rapidly under controlled fermentation conditions, achieving higher concentrations by day 2–3 compared to natural fermentation.	Faster microbial growth	[137]
Torulaspora delbrueckii, Hanseniaspora uvarum, Limosilactobacillus plantarum, Acetobacter ghanensis	Inoculation of native microbial starter cultures to optimize fine-aroma cocoa bean fermentation	Starter cultures at various stages increased polyphenol content, improved efficiency by 24%, and decreased sugars and acids, allowing drying in 96 h. They also reduced filamentous fungi that could negatively affect flavor and aroma.	Decreased drying time and enhanced flavor	[14]
Hanseniaspora opuntiae, S. cerevisiae	Multiphase analysis of the chocolate production chain to assess the functionality of starter cultures	S. cerevisiae demonstrated superior functionality, resulting in better flavor profiles and acidic notes and outperforming Hanseniaspora opuntiae, which led to under-fermented beans.	Superior flavor profiles	[138]
S. cerevisiae, W. anomalus, C. tropicalis, P. kudriavzevii	Effect of solar predrying treatments and inoculation with yeast starter cultures on the chemical composition of cocoa beans from Colombia	Starter cultures and predrying treatments altered chemical composition, reducing polyphenol, caffeine, and lactic acid content.	Improved sensory attributes	[139]
S. cerevisiae	Amino acid profile during Criollo cocoa fermentation	Starter cultures increased essential amino acids (63.4% vs. 61.8% in spontaneous fermentation) and reduced fermentation time by 3–4 d.	Improved flavor and aroma profiles.	[140]
Lactiplantibacillus plantarum subsp. plantarum HL-15	Alternative cocoa bean fermentation method using L. plantarum HL-15 as a starter culture and valorizing cocoa mucilage byproducts	Pulp removal and inoculation with L. plantarum HL-15 improved bean quality, suppressed fungal growth, and enhanced sensory qualities.	Reduced fungi and improved flavor	[141]
Issatchenkia orientalis, S. cerevisiae, W. anomalus, H. thailandica, P. kluyveri, Candida oleophila	Sensory and chemical selection of indigenous yeasts in cocoa fermentation	Different strains imparted unique sensory notes, with H. thailandica and P. kluyveri recognized by the panel for their outstanding flavor profiles.	Distinct flavor profiles	[142]

6. Patent Analysis on the Production of Organoleptic Compounds by the Fermentation of Cacao Beans

To better evaluate the increase in the use of starter microorganisms that produce organoleptic compounds from the fermentation of cocoa beans, a historical analysis of patents over the last 5 years (2020–2024) was conducted. For this purpose, the Derwent Innovations Index^® database was used. The algorithm applied for this patent search was as follows: TS = (Cocoa fermentation) OR TS = (Cacao fermentation) OR TS = (Cocoa bean fermentation) OR TS = (Cacao bean fermentation) OR TS = (Cocoa fermentation process) OR TS = (Cacao fermentation process) OR TS = (Cocoa fermentation microbiota) OR TS = (Cocoa microbiome) OR TS = (Cocoa microbial fermentation) OR TS = (Yeasts in cocoa fermentation) OR TS = (Lactic acid bacteria in cocoa fermentation) OR TS = (Acetic acid bacteria in cocoa fermentation) AND Derwent Manual Code = (D03-E07) AND International Patent Classification (IPC) = (A23G-001/00), respectively.

The 70 documents were exported to MS Excel^® and then manually selected by analyzing the title and summary of each work. A total of 21 patent documents were selected. This analysis showed that the main patent holders for the generation of organoleptic compounds from cocoa bean fermentations were from Brazil and the Philippines, holding 19.05% of patents each. This reflects their focus on enhancing chocolate flavor through microbial processes, which are central to fermentation dynamics (Figure 5).

The majority of the patents analyzed involved filamentous fungi and fermentative yeasts, which play a pivotal role in microbial communities during cocoa fermentation. A significant proportion of the patents analyzed focused on the development of organoleptic compounds with the objective of enhancing the aromas and flavors of beverages such as brandy and cocoa bean-based chocolates. Additionally, the patents sought to protect new technologies for the production of antioxidant compounds that could be used as additives in alcoholic beverages and high-value chocolates.

A more detailed examination of the data obtained from the patent database revealed a greater degree of variability in the organoleptic compounds generated and their diverse applications. Of the applications, 38% focused on the utilization of microorganisms (yeasts) for the fermentation of selected portions of the cocoa bean, including cocoa beans and cocoa honey, with the objective of generating a superior quality raw material that could be employed in the production of chocolate with enhanced organoleptic properties. This is due to the fact that microorganisms are capable of enhancing the properties of cocoa, including its acidity, bitterness, and astringency.

It is also noteworthy that fermented cocoa can be employed as a source of alcohol, as evidenced by the 20% of patents that focused on this particular use. The resulting products include cocoa liquor, brandy, and wine. In both cases, cocoa honey is fermented by yeasts. However, it is important to note that none of the patents explicitly claimed the ability to produce large quantities of alcoholic beverages on a commercial scale. This suggests that the economic and production viability of this process is not yet sufficient to meet the high demand for alcoholic beverages.

The remaining applications are primarily categorized into either polyphenol or antioxidant production (14%) or polysaccharide production (19%). These organoleptic compounds are employed to enhance the quality of chocolate, particularly in regard to its nutritional and compositional attributes. The following section will present a selection of patents to facilitate a more comprehensive understanding of the subject matter.

It should also be noted that most of the patents identified utilized microorganisms, including lactic acid bacteria, acetic acid bacteria, and yeasts. Nevertheless, 24% of the patents identified employed a consortium of these microorganisms. A combination of strains belonging to the genera Saccharomyces, Lactobacillus, and Acetobacter has been observed to facilitate more efficient fermentation of cocoa, with potential applications in food and beverage production.

These patents highlight the significance of elucidating the intricacies of microbial interactions in cocoa fermentation. By capitalizing on this knowledge to refine the fermentation process, cocoa producers can engineer distinctive flavor profiles and achieve superior-quality, more uniform products. The innovations showcased in these patents are directly informed by the microbial dynamics of fermentation, exemplifying how the manipulation of microbial communities can result in enhanced organoleptic outcomes in cocoa-based products. Examples of patents and their applications are shown in

Table 2. Overview of patents analyzed.

Patent	Category	Objective	Ingredients/Organisms	Technological Approach	Key Benefits	Ref.
ID202000442-U1	Mevastatin	Produce Mevastatin-rich quail feed	Cocoa husks, tofu pulp, Pleurotus ostreatus	Fermentation using agricultural byproducts	Low cholesterol, omega-3 enriched feed; promotes sustainability by reducing food waste	[143]
CN110800994-A	Polysaccharides	Produce selenium-enriched oligosaccharide tablets	Aspergillus niger, mushroom powder, barley grass, deer antler	Low-temperature mixing, ethanol granulation, TCM principles	High selenium, natural health benefits for heart, liver, immunity	[144]
CN110810602-A	Exopolysaccharide	Produce heat-resistant chocolate	Cocoa butter, lard, palm oil, Bifidobacterium, Lactobacillus strains	Emulsifiers, thickening agents (fenugreek gum), non-traditional grinding	Increased melting point and shelf life, suitable for variable storage conditions	[145]
BR102019020655-A2	Alcohols	Produce cacao-based aguardente	Cacao fruit, Saccharomyces cerevisiae, Saccharomyces bayanus	Enzyme-yeast sequencing, optimized must fermentation	Improved fermentation efficiency; quality alcoholic beverage derived from cacao	[146]
WO2022046913-A1	Enzymatic Digestion	Produce myceliated cacao with improved flavor	Cacao beans, pulp, pods, filamentous fungi	Natural fermentation with biotechnological enhancement	Flavorful cacao without artificial sweeteners, appealing to natural food markets	[147]
IN202221007331-A	Polyphenols and Antioxidants	Produce cacao with high polyphenol content	Cacao beans (fat removal, preserved polyphenols)	Efficient extraction and preservation methods	Nutrient-dense products with high polyphenol content for improved health benefits	[148]
DE102019123661-A1	Vitamins and Tannins	Develop fermented cacao drink with optimized flavor	Cacao seeds, natural microorganisms	Controlled fermentation, seed drying and soaking	Enhanced flavor profile in cacao drink, potential for new beverage blends like coffee infusions	[149]

.

As can be observed, cocoa fermentation is capable of producing a considerable variety of organoleptic compounds. A variety of compounds, including oligosaccharides, polysaccharides, alcohols, polyphenols, antioxidants, and vitamins, are employed in diverse applications within the food and beverage industry. Nevertheless, the therapeutic area represents a potential avenue for future applications. To enhance the intelligibility and informational content of this subject with respect to its applications, a selection of representative patents will be presented in the following section.

To illustrate an example of an application, patent ID202000442-U1, filed by Andalas University in 2022, showed how cocoa could be fermented to help produce animal feed. The document stated that cocoa pods were fermented by the fungus Pleurotus ostreatus, which ultimately generated a fermented residue rich in a statin called mevastatin, crude proteins, and minerals. This residue was incorporated into the feed of birds such as quail, to help them produce eggs with a lower cholesterol content and a better concentration of omega-3s. This demonstrates the nutraceutical potential of cocoa fermentation applied to animal feed [120].

The patent, CN110800994-A, filed by Wuhan Polytechnic University in 2020, extended beyond the conventional scope of applications to encompass a method of producing a functional food designed to assist in the reduction in blood and urine sugar levels. As detailed in the document, the production of this functional food entailed a fermentation process involving cocoa powder. The fermentation process was conducted using the Aspergillus niger strain. Ultimately, the starch content of the cocoa powder was diminished while its flavonoid content was augmented. In this manner, the cocoa powder and additional compounds were combined with a peptide derived from the balsam pear plant, thereby yielding a functional foodstuff that exerted therapeutic effects on the blood [130].

In 2020, Jieshou Meierli Chocolate Food Co. Ltd. filed Patent CN110810602-A, which developed a method for producing chocolate with greater heat resistance. The objective was to develop a product that would not deform easily, would not leave stains or smudges on the surfaces it was placed on, and would be more suitable for storage and transportation without causing issues or additional costs for the producer. During the chocolate-making process, cornstarch was added and then fermented by lactic acid bacteria, specifically Lactobacillus delbrueckii and Lactobacillus bulgaricus. During the fermentation process, the bacteria produced an extracellular polysaccharide that was attached to the chocolate, thereby enhancing its resistance to heat. The patent did not provide precise details regarding the specific exopolysaccharide in question [131].

It is also noteworthy to mention the BR102019020655-A2 patent. In contrast to the majority of patents identified, which pertain to the food industry and chocolate, this particular patent was concerned with the development of an alcoholic beverage derived from the fermentation of cocoa juice extracted from the fruit. The patent, filed in 2021, outlined a method for producing brandy through the inoculation of pectinolytic, proteolytic, and cellulolytic enzymes with Saccharomyces cerevisiae yeasts. The yeasts transformed the cocoa juice into wine or must, rapidly absorbing the existing fermentable sugars with the assistance of the enzymes. The final products were ethanol and carbon dioxide, which were released into the atmosphere. This process yielded the raw material required for brandy production [132].

The aforementioned examples illustrate the considerable innovative potential of organoleptic compounds derived from cocoa fermentation. This has significant implications for both the food and beverage industries, as well as the therapeutic sector, particularly in the development of functional foods for both humans and animals.

7. Bioprospection of Secondary Compounds from Cocoa Fermentation

For a bioprospecting study of secondary components produced during cocoa fermentation by known microorganisms, a systematic search was performed using the following search algorithm: (TITLE-ABS-KEY)“Cocoa fermentation” OR “Cocoa fermentation” OR “Cocoa bean fermentation” OR “Cocoa bean fermentation” OR “Cocoa fermentation process” OR “Cocoa fermentation process” OR “Cocoa fermentation microbiota” OR “Cocoa microbiome” OR “Cocoa microbial fermentation” OR “Yeasts in cocoa fermentation” OR “Lactic acid bacteria in cocoa fermentation” OR “Acetic acid bacteria in cocoa fermentation”) AND TITLE- ABS-KEY(“Secondary metabolites” OR “Secondary compounds” OR “Volatile compounds” OR “Organic acids” OR “Phenolic compounds” OR “Alcohol production” OR “Esters” OR “Amino acids” OR “Metabolic by-products” OR “Flavor precursors” OR “Biochemical changes” OR “Biochemical changes” OR “Biochemical changes” OR “Biochemical changes” OR “Biochemical changes” OR “Biochemical changes” OR “Biochemical changes” OR “Biochemical changes” OR “Biochemical changes OR “Biochemical changes” OR “Aroma compounds” OR “Compounds produced during fermentation”) AND TITLE-ABS-KEY(“Cocoa beans” OR “Cacao beans”)) AND PUBYEAR > 2019 AND PUBYEAR < 2025. This query was designed with the objective of obtaining information for a more precise bibliometric analysis that provides scientific articles related to secondary compounds obtained during coffee fermentation, as well as more current information, i.e., from the last 5 years.

According to the analysis performed, a wide variety of compounds produced during cocoa fermentation were identified. These compounds were grouped into 14 broad categories, based on their chemical nature and functional class. The categories include alcohols, aldehydes, alkaloids, amines and nitrogenous compounds, aromatic compounds, esters, flavonoids and polyphenols, ketones, natural polymers, organic acids, peptides and proteins, phenolic compounds, pyrazines, and reducing sugars. In Figure 6, it is shown that about 56 different compounds have been reported, among which acetic acid, lactic acid, and ethanol stand out for their high frequency. This is because they are the main products generated during fermentation, in direct association with acetic acid bacteria (AAB), lactic acid bacteria (LAB), and yeasts, as previously mentioned.

On the other hand, one of the most recurrent compounds according to the literature is 2-phenylethanol (2-phenylethanol), an aromatic alcohol characterized by its distinctive floral aroma. This volatile compound plays a crucial role in cocoa fermentation, as it contributes significantly to the aromatic profile of fermented beans and, consequently, to the final flavor of chocolate [20]. Its production has been mainly associated with yeasts of the Saccharomyces genus and is the result of the metabolism of the amino acid phenylalanine through the Ehrlich pathway. This process has also been described in the literature [150].

In addition to its relevance in cocoa fermentation, 2-phenylethanol has considerable potential for industrial applications, especially as a flavoring or flavoring agent in the food and cosmetics industry. Its floral and sweet notes can be complemented and enhanced in combination with other aromatic compounds, which broadens its possibilities for use in high-value-added products [151].

Similarly, 2-phenylethyl acetate is an ester that shares characteristics with 2-phenylethanol. Both contribute to the aromatic profile during cocoa fermentation. However, 2-phenylethyl acetate is distinguished by its slightly fruitier aroma [152]. This compound is formed via an esterification reaction of 2-phenylethanol, resulting in a more complex and intense aromatic profile, as well as greater volatility. Due to these properties, 2-phenylethyl acetate has applications in various industries, including food, cosmetics, fragrances, and pharmaceuticals [151]. Additionally, its production can be optimized through biotechnological approaches to maximize its performance and usefulness in industrial processes.

Ethyl acetate is a volatile ester that is formed as a result of microbial metabolism, particularly by the action of yeasts and acetic acid bacteria [12]. This occurs through an esterification reaction between ethanol and acetic acid, which is produced by both microbial groups [153]. This compound is of particular relevance in cocoa fermentation due to its influence on the aromatic profile. It contributes fruity and sweet notes to cocoa beans [154]. It is considered one of the most important aromatic compounds, as its balanced concentrations can be decisive in the sensory quality of chocolate [155].

Ethyl acetate is employed in a multitude of industries, including food, cosmetics, and pharmaceuticals, among others [88]. Furthermore, as it is a compound that is produced naturally during the fermentation process, its production can be optimized by using renewable substrates, which represents a sustainable alternative for its production [154].

Figure 6 illustrates the additional compounds present during cocoa fermentation. Although these compounds are present in lower concentrations according to the literature, their identification and exploitation could expand the list of bioactive and value-added compounds, which could benefit conventional fermentation processes such as cocoa fermentation. These compounds include aldehydes, such as phenylacetaldehyde, which contribute a sweet, floral, honey-like aroma [156]; alkaloids, such as theobromine, which, although structurally related to caffeine, exerts a milder effect on the nervous system and is abundantly present in cocoa beans [125], contribute to the characteristic bitter taste of chocolate, which is also attributed to the presence of amines and nitrogenous compounds, such as putrescine, generally regarded as an undesirable compound in cocoa fermentation due to its potential to generate off-flavors and accelerate product deterioration [157]. However, putrescine has been the subject of studies in fields such as cancer treatment, due to its ability to promote cell proliferation [158], and in the agricultural industry, where it is used as a fertilizer and plant biostimulant [159].

Finally, it can be concluded that the most abundant groups found in the bibliography correspond to the group of amines and nitrogen compounds; most of the compounds present here correspond to essential amino acids produced during cocoa fermentation, followed by esters, alcohols, and aldehydes.

The number of patents related to the use of secondary compounds from the biological fermentation of cocoa is slowly but gradually increasing. This conclusion was observed by Nascimento et al. (2024), who carried out a recent data search and obtained 49 patents, where the vast majority of the inventions found were related to the field of food sciences [160]. These results show that one factor that has contributed to the objectives of the research and inventions carried out is the growing consumer demand for quality and low prices. As a result, it is clear and self-explanatory why most of the patents found follow the trend of developing and implementing better cocoa fermentation techniques to improve quality, create excellent products, and generate new bioproducts in a sustainable and economically viable way [47].

The preceding topic illustrates the emphasis on enhancing the quality of cocoa through the utilization of fermenting microorganisms, including yeasts and lactic acid bacteria. These strains are capable of modifying the chemical composition of cocoa beans, thereby improving attributes such as flavor, nutritional value, and resistance to physical factors. The generation of compounds, including volatile flavorings, polyphenols, and antioxidants, contributes to this quality [161].

It is also noteworthy to consider the increasing evidence of bioactive compounds present in cocoa that can be extracted or produced. The patents identified indicate that cocoa contains a range of compounds, including polyphenols, alcohols, antioxidants, polysaccharides, oligosaccharides, and statins. As previously documented, polyphenols have been employed to enhance the astringency and bitterness of cocoa and chocolate through oxidation. During the fermentation of cocoa, the total phenol content is extracted and reduced by the action of enzymes produced by microorganisms that promote the oxidation of polyphenol [162]. Another illustrative example is the production of alcohols, as evidenced in patent BR102019020655-1A2. In this instance, enzymes and yeast strains were employed in a combined fermentation process to transform fermentable sugars into ethanol, utilizing cocoa as the primary substrate [163].

In addition to food trends, potential applications in pharmaceutical and cosmetic industries must also be considered. As previously stated, technological innovations have concentrated on the extraction of the polyphenolic compounds present in cocoa, which can exhibit concentrations that exceed those observed in other food and beverage sources, including green tea, wine, soy, and blueberries [164]. The pivotal point is that flavonoids are antioxidants with salutary effects on human health. Flavonoids have been demonstrated to possess anti-cancer, anti-diabetic, anti-obesity, and immune-enhancing properties. Nevertheless, this is a nascent field of research that is still in its infancy, without a significant number of patents yet published. However, it represents a promising avenue for future investigation and development [165].

8. Conclusions and Future Perspectives

Cocoa fermentation is a complex and crucial process that significantly impacts the flavor, aroma, and overall quality of chocolate. This review has highlighted the intricate microbial dynamics that govern cocoa fermentation, focusing on the roles of yeasts, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) in shaping the sensory profile of cocoa. These microorganisms interact in a well-defined succession that is essential for producing the desirable organoleptic compounds that give chocolate its distinctive flavor characteristics.

The microbial communities involved in cocoa fermentation play a critical role in the production of key metabolites, including alcohols, organic acids, and esters, which contribute to the fruity, floral, and acidic notes found in high-quality chocolate. Additionally, the strategic use of starter cultures—carefully selected microbial strains—has emerged as an effective method to control fermentation dynamics, improve efficiency, and ensure consistency in product quality. Patents related to microbial fermentation techniques underscore the growing recognition of microbial dynamics as a key factor in producing chocolate with unique and desirable flavor profiles. These innovations demonstrate the potential for manipulating microbial communities to create specific, regionally distinct flavors and improve the overall fermentation process.

Understanding and controlling microbial succession during fermentation not only enhances flavor but also helps optimize fermentation efficiency. This is particularly important in light of the increasing consumer demand for high-quality, premium chocolate with unique flavor profiles. The ability to influence the fermentation process through the use of starter cultures and the control of environmental conditions opens up new opportunities for the cocoa industry to meet market demands while improving sustainability and reducing the time required for fermentation.

Future research should continue to explore the microbial interactions that occur during cocoa fermentation, focusing on the identification of novel microorganisms, the development of more efficient starter cultures, and the optimization of fermentation conditions. Additionally, advancements in biotechnological approaches, such as genetic engineering and metagenomics, offer promising avenues for improving fermentation efficiency and creating chocolates with even more distinct and enhanced flavor profiles. The integration of these technologies, along with an ongoing understanding of microbial dynamics, will undoubtedly drive the next generation of innovations in cocoa fermentation, contributing to the growth of the global chocolate industry.

In conclusion, microbial dynamics in cocoa fermentation are central to the creation of high-quality products. The ongoing exploration and manipulation of these microbial communities will not only improve the consistency and sensory characteristics of cocoa products but also open new doors for future advancements in the field. As the chocolate industry continues to evolve, the role of microbial dynamics in fermentation will remain a critical area of research and innovation, shaping the future of cocoa production and its applications in various industries.