# Prospective yeast species with enzymatic, aromatic, and antifungal applications isolated from cocoa fermentation in various producing areas in Côte d'Ivoire

Alfred Koffi Yao, Kouame Guy Florent Amien, Brice Judicaël Assi-Clair, Nabounou Koné, Mai Koumba Koné, Kevin Bethune, Isabelle Maraval, Vincent Chochois, Jean-Christophe Meile, Renaud Boulanger, et al.

<figure><img src="image_1.png" /></figure>

## ► To cite this version:

Alfred Koffi Yao, Kouame Guy Florent Amien, Brice Judicaël Assi-Clair, Nabounou Koné, Mai Koumba Koné, et al.. Prospective yeast species with enzymatic, aromatic, and antifungal applications isolated from cocoa fermentation in various producing areas in Côte d'Ivoire. Microbiology Research, 2025, 16 (12), pp.256. 10.3390/microbiolres16120256. [hal-05484764](https://hal.science/hal-05484764v1)

HAL Id: hal-05484764

https://hal.science/hal-05484764v1

Submitted on 30 Jan 2026

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
Article

# Prospective Yeast Species with Enzymatic, Aromatic, and Antifungal Applications Isolated from Cocoa Fermentation in Various Producing Areas in Côte d'Ivoire

Alfred Koffi Yao<fnref n="1" />, Guy Florent Kouamé Amien<fnref n="1" />, Brice Judicaël Assi-Clair<fnref n="1" />, Nabounou Koné<fnref n="1" />, Mai Koumba Koné<fnref n="2" />, Kevin Bethune<fnref n="3" />,<fnref n="4" />, Isabelle Maraval<fnref n="3" />,<fnref n="4" />, Vincent Chochois<fnref n="3" />,<fnref n="4" /> ID, Jean-Christophe Meile<fnref n="3" />,<fnref n="4" /> ID, Renaud Boulanger<fnref n="3" />,<fnref n="4" /> ID and Simplice Tagro Guéhi<fnref n="1" />,* ID

<figure><img src="image_3.png" /></figure>

check for
updates

Academic Editor: Ligang Zhou

Received: 29 September 2025

Revised: 23 October 2025

Accepted: 29 October 2025

Published: 8 December 2025

**Citation:** Yao, A.K.; Amien, G.F.K.; Assi-Clair, B.J.; Koné, N.; Koné, M.K.; Bethune, K.; Maraval, I.; Chochois, V.; Meile, J.-C.; Boulanger, R.; et al. Prospective Yeast Species with Enzymatic, Aromatic, and Antifungal Applications Isolated from Cocoa Fermentation in Various Producing Areas in Côte d'Ivoire. *Microbiol. Res.* **2025**, *16*, 256. https://doi.org/10.3390/microbiolres16120256

**Copyright:** © 2025 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license
(https://creativecommons.org/
licenses/by/4.0/).

Abstract

This research study investigated the potential biotechnological applications of yeast species obtained from cocoa fermentation performed in Côte d'Ivoire. A total of 279 yeast isolates were molecularly identified and then screened for their antifungal ability against various *Aspergillus* species and for the production of aromatic compounds and extracellular enzymes. Thirty-one yeast species belonging to nineteen genera, dominated by Pichia, Candida, Hanseniaspora, and Rhodotorula, were isolated from fermented cocoa beans. All extracellular enzymes screened were produced by most yeast species, except β-glucanase and esterase activity, whereas the most common enzyme was β-glucosidase. Yeasts of the Pichia, Saccharomyces, Candida, Clavispora, and Hanseniaspora genera produced various enzymes, including xylanase, β-glucosidase, polygalacturonase, invertase, pectinase, and chitinase. The 88 aromatic compounds produced were grouped into five main chemical families, including esters, alcohols, acids, aldehydes, and ketones. *Wickerhamomyces anomalus* was the highest producer of major desirable aromatic compounds, including alcohols, ketones, and esters. All yeast species showed a specific antagonistic effect against the growth of various Aspergillus species, but *Candida incommunis*, *Saccharomyces cerevisiae*, and *Torulaspora delbrueckii* recorded the greatest antifungal ability. These yeast species could be used to develop promising starter cultures to improve the organoleptic quality of various fermented foods and beverages.

**Keywords:** yeasts; enzymes; biotechnology; applied microbiology; flavor

1. Introduction

The biotechnological approach mainly involves the selection of native yeast strains [1]. Yeasts are protist fungi and include a heterogeneous group of microorganisms representing the phyla Ascomycetes and Basidiomycetes, which have biotechnological potential

<footnote n="1">¹ Unité de Formation et de Recherche Sciences et Technologies des Aliments, Université Nangui Abrogoua, Abidjan 02 BP 801, Côte d'Ivoire; alfredyao94@gmail.com (A.K.Y.); abaamien1er@gmail.com (G.F.K.A.); clairassi@gmail.com (B.J.A.-C.); nabounou95@gmail.com (N.K.)</footnote>

<footnote n="2">² Institut National Polytechnique Félix Houphouët-Boigny, Yamoussoukro BP 1093, Côte d'Ivoire; mahikoumba@yahoo.fr</footnote>

<footnote n="3">³ Centre de Coopération Internationale en Recherche Agronomique pour le Développement—CIRAD, UMR QualiSud, TA B 96/16, 75 Av. JF Breton, 34398 Montpellier, France; kevin.bethune@cirad.fr (K.B.); isabelle.maraval@cirad.fr (I.M.); meile@cirad.fr (J.-C.M.); renaud.boulanger@cirad.fr (R.B.)</footnote>

<footnote n="4">⁴ Qualisud, Université de Montpellier, CIRAD, Université d'Avignon, Université de la Réunion, Montpellier SupAgro, 1101, Avenue Agropolis, 34090 Montpellier, France</footnote>

<footnote>* Correspondence: g.tagro09@gmail.com</footnote>
during spontaneous food fermentation. Yeasts have a rich history and a bright future in biotechnology [2]. Their involvement and importance in traditional food fermentation are unparalleled by other organisms of biotechnological relevance. These biotechnological applications of interest include alcohol production, production and utilization of organic acids, improvement in flavor, aroma, and texture, enhancement of nutritional properties, and reduction in anti-nutritional factors and toxins [3]. Yeasts produce a wide range of fermented products, ranging from alcoholic beverages prepared using various biochemical substrates to condiments [4]. Yeast species isolated and characterized from various traditionally fermented foods have been successfully applied as a starter/co-starter for the production of functional foods at the industrial level [5]. The use of most yeast species in biotechnological processes is accelerating due to their nonpathogenic effect on humans and animals [6]. Cocoa beans sourced from Côte d'Ivoire are subject to various quality defects [7], including low aromatic quality due to the absence of aromas and fine flavors [8] and high levels of ochratoxin A [9] due to mold contamination [10]. In order to improve the aromatic quality of fermented foods, flavor-producing yeasts providing a strong aroma to foods and thus playing a great role in the modern fermentation industry can be used [11]. Also, the use of biological alternatives, including antifungal microorganisms, for reducing the growth and production of mold mycotoxins in fermented foods is being increasingly promoted [12,13]. Fortunately, both aroma-producing and fungal antagonist yeast species are some of the main ingredients responsible for flavor precursor production [14,15] and the reduction in both free fatty acid and ochratoxin A content [7] associated with chocolate quality. The main research questions were as follows: (i) What are the species of yeast and the communities involved in cocoa fermentation carried out in Côte d'Ivoire? (ii) Are the yeasts bioactive against the growth of molds? (iii) What are the key enzymes and aroma compound-producing yeast species from this microbiome? This study aimed to screen potential yeast species with biotechnological applications of interest usable in the food industry. In the present work, we highlighted the ability of yeast species involved in cocoa fermentation carried out in Côte d'Ivoire (i) to inhibit the growth of molds and (ii) to produce some key enzymes and (iii) aromatic compounds.

## 2. Materials and Methods

### 2.1. Cocoa Fermentation and Yeast Isolation

Cocoa fermentation was carried out on small-scale farms located in eight main cocoa-producing regions of Côte d'Ivoire (Figure 1) during the major cocoa harvest seasons of 2023 and 2024 using about 50 kg of fresh cocoa beans placed in wooden boxes. The bottom and each side of the boxes were lined with a layer of banana leaves. Fermentation lasted 6 days with manual stirring at 48 and 96 h [16]. Samples (10 g) were taken aseptically from the fermenting cocoa mass every 24 h for 3 days and were homogenized in 90 mL of sterile 0.1% peptone solution for 5 min. Serial dilutions in tenths were prepared up to $10^{-9}$ in sterile physiological water and 0.1 mL aliquots were spread on chloramphenicol Sabouraud agar incubated at 30 °C for 48 h. Distinct yeast colonies were selected, purified by successive subculturing on the same medium as previously described by Koné et al. [17]. The pure cultures of 279 yeast isolates were stored at -80 °C in cryotubes. Prior to testing, each yeast was reactivated by growing at 30 °C on YPDA (yeast extract-peptone-dextrose-agar) for 24 h.
<figure><img src="image_4.png" /><figcaption>**Figure 1.** Mapping of the areas where cocoa fermentation trials were carried out on the farm-level in Côte d'Ivoire.</figcaption></figure>

## 2.2. Reference Ochratoxinogenic Molds Strains

Three reference strains were used: Previously ochratoxigenic Aspergillus including *Aspergillus carbonarius* (voucher strain), *Aspergillus niger*, and *Aspergillus ochraceus* with respective GenBank accession numbers NR_111094.1, MT582749, KX610750, respectively, isolated from dry fermented cocoa beans.

## 2.3. Molecular Identification of Yeasts

Genomic DNA was extracted from single yeast colonies grown on YPDA medium using the protocol of Csutak and Csutak [18]. DNA quality was checked using nanodrop (NanoQuant plate, Infinite® M200, TECAN, Männedorf, Switzerland) and concentration was assessed using nanodrop Qubit 3.0 (Life Technologies, Invitrogen, Thermo Fisher Scientific, Singapore). The ITS1-5.8S-ITS2 region of the rDNA gene was amplified by PCR using universal fungal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), with a 30 bp GC-clamp added to the forward primer [19,20]. Amplification conditions included an initial denaturation at 94 °C for 3 min, followed by 30 cycles of 95 °C for 1 min, 52 °C for 1 min, 72 °C for 1 min, and a final extension at 72 °C for 10 min. Amplicons were visualized on 2% agarose gel stained with TBE buffer, and fragment sizes were analyzed using PyElph software 1.4 [18]. Sanger sequencing was performed by CeMIA (Larissa, Greece), and configs were assembled using DNA Dragon 1.4.1. The obtained sequences were compared to GenBank using the Basic Local Alignment Search Tool (BLAST 2.17.0) algorithm, and yeast isolates showing ≥97% identity were considered to belong to the same species.

## 2.4. Production of Extracellular Hydrolytic Enzymes: Ability of Yeast Species

Yeast extracellular enzymes were qualitatively screened on solid media with specific substrates. For each assay, cell suspension at 10⁴ CFU of each yeast species was aseptically and carefully prepared from 24 h, 30 °C cultures on yeast extract, peptone, dextrose, and adenine (YPDA) medium for all identified species except those of *Kluyveromyces*, *Geotrichum*,
*Cyberlindnera*, and *Rhodotorula* genera. Harvested cells by centrifugation were washed twice in sterile water, and adjusted by optic density (620 nm) and microscopy (Malassez counting chamber). Ten microliters of each cell suspension were spot-inoculated onto selective agar and incubated at 30 °C for 5–10 days.

**Pectinase activity:** Pectinase activity was assessed on a specific medium containing 20 g/L of Potato Dextrose Agar (PDA), 10 g/L of citrus pectin, and 5 g/L of peptone. The pH was adjusted to 7.0 prior to autoclaving. Ten microliters of yeast cell suspension (10⁴ CFU/mL) were spread on the medium and then incubated at 30 °C. After incubation, the plates were flooded with a solution of hexadecyltrimethylammonium bromide (CTAB, 10 g/L). The formation of a clear halo surrounding the colonies indicated pectinase activity [21].

**Chitinase activity:** Chitinase activity was determined on a chitin agar medium composed of 20 g/L of Yeast Extract Agar (YEA) and 10 g/L of colloidal chitin. Ten microliters of yeast suspension (10⁴ CFU/mL) were plated and incubated at 30 °C. After incubation, plates were stained with 0.1% Congo red for 30 min at room temperature and then rinsed with 1 M NaCl for 1 h. Chitinase activity was visualized as yellowish-clear zones against the red background [22].

**Xylanase activity:** Xylanase production was tested using a medium containing 5 g/L of xylan, 5 g/L of peptone, 1 g/L of KH2PO4, 0.2 g/L of MgSO4·7H2O, 5 g/L of yeast extract, and 20 g/L of agar. Ten microliters of yeast suspension (10⁴ CFU/mL) were inoculated and incubated at 30 °C. Plates were then flooded with 2% Congo red for 15 min and washed with 1 M NaCl. Clear halos around colonies indicated xylanase activity [23].

**Invertase activity:** Invertase activity was detected using a medium containing 10 g/L of peptone, 3 g/L of yeast extract, 1 g/L of NaCl, 20 g/L of agar, and 16 g/L of sucrose. Ten microliters of yeast suspension (10⁴ CFU/mL) were spread onto the medium and incubated at 30 °C. Invertase-producing yeasts were identified by the presence of clear halos around them.

**Esterase activity:** Esterase activity was evaluated using Sierra medium supplemented with Tween 20. The medium contained 10 g/L of peptone, 1 g/L of NaCl, 0.5 g/L of CaCl₂·H₂O, 20 g/L of agar, and 10 mL/L of Tween 20. The pH was adjusted to 7.4. After inoculation with 10 µL of yeast suspension (10⁴ CFU/mL), plates were incubated at 30 °C. Esterase production was confirmed by the formation of opaque precipitation zones around the colonies.

**Protease activity:** Protease production was tested on skim milk agar containing 10 g/L of peptone, 1 g/L of NaCl, 3 g/L of yeast extract, 20 g/L of casein, and 20 g/L of agar. Yeast suspensions (10⁴ CFU/mL) were inoculated and incubated at 30 °C. Proteolytic activity was indicated by the appearance of clear zones surrounding the yeast colonies.

**β-glucosidase activity:** β-Glucosidase activity was assessed on a medium (pH 5.0) composed of 6.7 g/L yeast nitrogen base (YNB, Difco, Nantes, France), 5 g/L arbutin (Sigma, Saint Louis, MO, USA), and 20 g/L agar. Before pouring the medium, 2 mL of a 1% (v/v) ferric ammonium citrate solution (filtered with 0.22 µm Millipore membrane) was added per 100 mL. Ten microliters of yeast suspension (10⁴ CFU/mL) were inoculated, and β-glucosidase activity was indicated by the dark brown discoloration of the medium around colonies.

**Polygalacturonase activity:** Polygalacturonase production was determined following the method of Strauss et al. [24]. The medium contained 12.5 g/L of polygalacturonic acid (Sigma), 6.8 g/L of potassium phosphate buffer (pH 3.5), 6.7 g/L of YNB without ammonium sulfate (Difco), 10 g/L of glucose, and 20 g/L of agar. After yeast inoculation and incubation, colonies were rinsed with deionized water and stained with 0.1%
ruthenium red. Polygalacturonase activity was visualized by violet halo formation around the colonies.

**Polyphenol Oxidase (PPO) activity:** PPO activity was tested on LBM medium composed of 1 g of KH₂PO₄, 0.5 g of hexamethylenetetramine, 0.01 g of MgSO₄·7H₂O, 0.01 g of CaCl₂·2H₂O, 0.001 g of yeast extract, 0.001 g of CuSO₄·5H₂O, 0.001 g of Fe₂(SO₄)₃, and 0.001 g of MnSO₄. The medium was supplemented with 0.05% (w/v) Remazol Brilliant Blue R (RBBR) and 1.6% (w/v) agar. After sterilization, 10 mL of sterile 20% (w/v) glucose solution was added. PPO activity was inferred from the decolorization of RBBR in the surrounding agar [23].

## 2.5. Production of Aroma Compounds

Flavor compounds production was assessed by cultivating yeasts in a Cocoa Pulp-Simulating Medium broth (CPSM). The composition of the CPSM medium (pH 5.8) is provided in Table 1 [25]. A 100 mL (10×) concentrated nutritious broth (6.7 g/L yeast nitrogen base, 5 g/L glucose) was prepared and sterilized by filtration (0.22 µm). Four single fresh colonies of each yeast were inoculated into 250 mL Erlenmeyer flasks containing 100 mL of 1× nutritious broth and incubated at 30 °C, 100 rpm for 24 h (pre-culture). Pre-cultures were centrifuged (6000 × g, 5 min), washed in sterile saline, and resuspended in CPSM maintained at +4 °C. Cell density was estimated by OD (620 nm) (UV-1800, Shimadzu Corporation, Colombia, MD, USA) and microscopy (Malassez counting chamber), establishing calibration curves of CFU/mL vs. optic density after several decimal dilutions in CPSM [26]. Based on the calibration curves established for each yeast species, 100 mL of suspension at 10⁴ CFU/mL was prepared in CPSM and distributed into eighteen sterile 10 mL vials. Three milliliters of inoculum was transferred into each hermetically sealed vial and incubated (30 °C, 100 rpm) for 72 h. At 24 h intervals, sub-samples were withdrawn: three vials were measured for optic density (620 nm) and the other three were frozen at -20 °C for volatile extraction. Volatile compounds in the headspace were extracted by dynamic headspace sampling (DHS) and analyzed by gas chromatography-mass spectrometry (GC-MS) according to Waehrens et al. [27] and Owusu et al. [28]. Identifications were made using the NIST mass spectral library and linear retention indices. Peak areas were quantified using Agilent MassHunter software (v12.0, Agilent Technology, Santa Clara, CA, USA) according to the method described by Llano et al. [29] and Cruz-Rojas et al. [30]. Semi-quantification was achieved by comparing it to a 3-heptanol internal standard using the following equation:

$$qi (\mu g \cdot mL^{-1}) = \frac{A_i \times 0.818}{A_e \times V_i}$$

*qi*: Amount of compound *i* released into the headspace, expressed in µg·mL⁻¹;

*A*<sub>i</sub>: Peak area of compound *i*;

*V*<sub>i</sub>: Volume of sample suspension introduced into the vial (mL);

*A*<sub>e</sub>: Peak area of the internal standard (3-heptanol);

0.818: Mass of the internal standard in µg.

<table><caption>**Table 1.** Composition of cocoa pulp similar medium (CPSM).</caption><thead><tr><td>Components</td><td>Concentration (g·L<sup>-1</sup>)</td></tr></thead><tbody><tr><td>Yeast nitrogen base (YNB)</td><td>2.0</td></tr><tr><td>Fructose</td><td>30.0</td></tr><tr><td>Glucose</td><td>20.0</td></tr><tr><td>Pectin</td><td>10.0</td></tr><tr><td>Sucrose</td><td>6.7</td></tr></tbody></table>
<table><caption>**Table 1. Cont.**</caption><thead><tr><th>Components</th><th>Concentration (g·L<sup>-1</sup>)</th></tr></thead><tbody><tr><td>Acetic acid</td><td>1.4</td></tr><tr><td>Lactic acid</td><td>1.0</td></tr><tr><td>Citric acid</td><td>1.0</td></tr><tr><td>Ammonium sulfate (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub></td><td>2.8</td></tr><tr><td>Monobasic potassium phosphate (KH<sub>2</sub>PO<sub>4</sub>)</td><td>1.0</td></tr><tr><td>Dibasic potassium phosphate (K<sub>2</sub>HPO<sub>4</sub>)</td><td>6.0</td></tr><tr><td>Magnesium sulfate heptahydrate (MgSO<sub>4</sub>·7H<sub>2</sub>O)</td><td>0.5</td></tr><tr><td>Iron (II) sulfate heptahydrate (FeSO<sub>4</sub>·7H<sub>2</sub>O)</td><td>0.01</td></tr><tr><td>Analytic Ethanol</td><td>2.0</td></tr><tr><td>Polyphenols (catechin 1136 mg·L<sup>-1</sup>, epicatechin 1441 mg·L<sup>-1</sup>, isoquercetin 539 mg·L<sup>-1</sup>)</td><td>0.051%</td></tr></tbody></table>

## 2.6. Preparation of Yeast and Fungal Spore Suspensions

Conidia of *Aspergillus carbonarius*, *A. ochraceus*, and *A. niger* were aseptically collected by scraping 7-day-old cultures grown on Sabouraud chloramphenicol agar using a 2% Tween 20 solution. The conidial suspensions were adjusted to concentrations of 105 and 107 conidia/mL in sterile distilled water using a Malassez counting chamber [31]. For yeast preparation, a colony from each species grown on YPDA was inoculated into 8 mL of nutrient broth and incubated at 30 °C for 24 h. The yeast cell concentration was adjusted to 107 cells/mL using a Malassez counting chamber.

### 2.6.1. Antifungal Activity of Yeasts by Direct Confrontation

The antifungal potential of yeasts via direct confrontation was assessed using the double-layer technique. YPDA agar plates were centrally spotted with 100 µL of yeast suspension (10⁷ cells/mL) and incubated for 24 h at 30 °C. Each plate was then overlaid with 10 mL of CYA medium containing fungal conidia (10⁴ conidia/mL). After incubation at 30 °C for 5 days, fungal mycelial growth was evaluated by measuring the colony diameter. Three types of controls were included: (i) positive control with YPDA + yeast, (ii) YPDA + yeast + CYA without mold, and (iii) negative control with YPDA + CYA + mold without yeast. Growth inhibition was determined by comparing the mycelial diameter to that of the controls.

### 2.6.2. Antifungal Activity of Non-Volatile Yeast Metabolites

To evaluate the antifungal activity of non-volatile metabolites, 100 µL of each yeast suspension (107 cells/mL) was spotted in the center of YPDA agar. After 24 h of incubation at 30 °C, the surface was overlaid with CYA medium. Then, 10 µL of fungal conidial suspension (10⁵ conidia/mL) was inoculated in the center of each plate. The plates were incubated at 30 °C for 6 days. The transverse diameters of the fungal colonies were measured, and all tests were performed in duplicate. The results were expressed as the arithmetic mean of inhibition zone diameters (mm). The inhibition rate (Ti) of mold growth was calculated according to the following formula:

$$Ti (\%) = \frac{D_t - D_e}{D_t} \times 100$$

where Ti is the mycelial growth inhibition rate (%), Dt is the colony diameter of the control (mm), and De is the colony diameter of the yeast-treated sample (mm).
## 2.7. Statistical Analysis

Quantitative data, such as volatile compound concentrations, were analyzed using one-way ANOVA with XLSTAT (v4.2, 2024) and Fisher's LSD test (α = 0.05) to determine significant differences between yeast strains.

# 3. Results

## 3.1. *Yeast Species Diversity*

The sequencing of PCR or polymerase chain reaction products with various sizes, ranging from 375 to 880 bp, allowed us to identify 31 yeast species belonging to 19 genera and one unidentified yeast isolate (Table 2). The genus *Pichia* includes eight species, such as *Pichia sporocuriosa*, *P. terricola*, *P. manshurica*, *P. bruneiensis*, *P. kluyveri*, *P. pseudolambica*, *P. ethanolica*, and *P. kudriavzevii*. The genus *Candida* comprises four species such as *Candida berthetii*, *C. boidinii*, *C. incommunis*, and *C. tropicalis*. Two species each of *Hanseniaspora* (*Hanseniaspora opuntiae* and *H. pseudoguillermondii*) and *Rhodotorula* (*Rhodotorula mucilaginosa* and *Rhodotorula paludigena*) genera were found. Fifteen other genera were each represented by a single species (e.g., *Wickerhamomyces anomalus*, *Torulaspora delbrueckii*, *Kluyveromyces marxianus*, etc.). The cocoa fermentation carried out in Agnibilékrou's area recorded a wide diversity of yeasts with 20 species, while only 5 yeast species were involved in that carried out in the areas of Adzopé or Maféré. *P. kudriavzevii* and *Saccharomyces cerevisiae* were the most yeast species isolated from cocoa fermentation, followed by *C. tropicalis* and *Hanseniaspora opuntiae*, regardless of the producing area. However, *C. incommunis*, *C. boidinii*, *Cyberlindnera* sp., *Saccharomycopsis amapae*, and *Starmera stellimalicola* were found in Agniblékrou's area. Five yeast species, including *Clavispora lusitaniae*, *Geotrichum candidum*, *P. sporocuriosa*, *Rhodotorula paludigena*, and *R. mucilaginous*, were specific to Biankouma's area. *P. pseudolambica* and *H. pseudoguilliermondii* were detected in Divo's area. *Kluyveromyces marxianus*, *Starmerella bacillaris* in Méagui's area, while only *Torulaspora delbrueckii* were isolated from the area of Bonon.

## 3.2. *Extracellular Hydrolytic Enzyme Produced by Identified Yeast Species*

Screening for extracellular hydrolases showed that most yeast species secreted multiple enzymes, while other species produced fewer enzymes (Table 3). *Candida incommunis*, *Pichia manshurica*, and *Saccharomyces cerevisiae* were the greater hydrolytic enzyme-producing yeast species with 8 activities, while *Nakaseomyces glabratus* and *Saccharomyces ludwigii* produced only 2 enzyme activities among 10 tested enzymes (Figure 2). Across all tested hydrolytic activities, the most frequently detected enzymes were β-glucosidase (positive in 87.5% of yeast species), xylanase (75%), invertase (66.66%), chitinase (66.66%), and polyphenol oxidase (62.5%). In contrast, only β-glucanase was produced by *Starmera stellimalicola*, while esterase was produced by fewer yeast species (18.2%), including two species of *Pichia* (*P. bruneiensis* and *P. terricola*), two species of *Candida* (*C. incommunis* and *C. boidinii*), and *Starmerella bacillaris*. Many yeast species, particularly within *Pichia*, *Candida*, *Clavispora*, and *Hanseniaspora* genera, produced a broad spectrum of enzymes. For example, within *Pichia* genus, six species, including *P. bruneiensis*, *P. kluyveri*, *P. kudriavzevii*, *P. manshurica*, *P. sporocuriosa*, and *P. terricola*, commonly produce chitinase, xylanase, protease, β-Glucosidase, polyphenol oxidase, and pectinase activities. All identified yeast species of the *Candida* genus produced some key xylanase, invertase, β-Glucosidase, and polyphenol oxidase activities.
<table><caption>**Table 2.** Distribution of yeast species isolated from cocoa fermentation carried out in 8 main producing areas in Côte d'Ivoire.</caption><thead><tr><th colspan="2">Yeasts Isolated</th><th colspan="8">Cocoa Fermentations Areas</th><th rowspan="2">Frequence (%)</th><th rowspan="2">Dominance (%)</th><th rowspan="2">% Identity</th><th rowspan="2">GenBank Accession Number</th></tr><tr><th>Genera</th><th>Species</th><th>Adzopé</th><th>Agnibilékrou</th><th>Biankouma Bonon</th><th>Divo</th><th>Guiberoua</th><th>Maféré</th><th>Meagui</th></tr></thead><tbody><tr><td rowspan="4">Candida</td><td>berthetii</td><td>1</td><td></td><td></td><td></td><td>1</td><td></td><td></td><td></td><td>25</td><td>0.72</td><td>99</td><td>NR_077118.1</td></tr><tr><td>boidinii</td><td></td><td>3</td><td></td><td></td><td></td><td></td><td></td><td></td><td>13</td><td>1.08</td><td>100</td><td>KY445950.1</td></tr><tr><td>incommunis</td><td></td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td>13</td><td>0.36</td><td>100</td><td>MZ506857.1</td></tr><tr><td>tropicalis</td><td>1</td><td>3</td><td>5</td><td></td><td>3</td><td>1</td><td>2</td><td></td><td>75</td><td>5.38</td><td>100</td><td>LC317537.1</td></tr><tr><td>Clavispora</td><td>lusitaniae</td><td></td><td></td><td>8</td><td></td><td></td><td></td><td></td><td></td><td>13</td><td>2.87</td><td>100</td><td>KP674846.1</td></tr><tr><td>Cyberlindnera</td><td>sp.</td><td></td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td>13</td><td>0.36</td><td>100</td><td>PP112176.1</td></tr><tr><td>Geotrichum</td><td>candidum</td><td></td><td></td><td>3</td><td></td><td></td><td></td><td></td><td></td><td>13</td><td>1.08</td><td>99</td><td>MT071788.1</td></tr><tr><td rowspan="2">Hanseniaspora</td><td>opuntiae</td><td></td><td>5</td><td>5</td><td>2</td><td>8</td><td>2</td><td>2</td><td>2</td><td>88</td><td>9.32</td><td>100</td><td>FM199955.1</td></tr><tr><td>pseudoguilliermondii</td><td></td><td></td><td></td><td></td><td></td><td>2</td><td></td><td></td><td>13</td><td>0.72</td><td>100</td><td>MW990151.1</td></tr><tr><td>Kluyveromyces</td><td>marxianus</td><td></td><td></td><td></td><td></td><td></td><td></td><td>2</td><td></td><td>13</td><td>0.72</td><td>100</td><td>MT187614.1</td></tr><tr><td>Kurtzmanella</td><td>quercitrusa</td><td></td><td>4</td><td></td><td></td><td>2</td><td></td><td></td><td></td><td>25</td><td>2.15</td><td>100</td><td>MF574303.1</td></tr><tr><td>Meyerozyma</td><td>caribbica</td><td></td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td>13</td><td>0.36</td><td>100</td><td>MT312818.1</td></tr><tr><td rowspan="10">Pichia</td><td>glabratus</td><td></td><td>1</td><td></td><td>1</td><td></td><td>1</td><td></td><td></td><td>38</td><td>1.08</td><td>100</td><td>MK998697.1</td></tr><tr><td>bruneiensis</td><td></td><td>1</td><td></td><td></td><td>1</td><td></td><td></td><td>1</td><td>38</td><td>1.08</td><td>98</td><td>LC431631.2</td></tr><tr><td>ethanolica</td><td></td><td>1</td><td></td><td></td><td></td><td>5</td><td>1</td><td></td><td>38</td><td>2.51</td><td>100</td><td>KM368822.1</td></tr><tr><td>kluyveri</td><td>1</td><td>2</td><td>1</td><td>1</td><td>1</td><td></td><td></td><td></td><td>63</td><td>2.15</td><td>100</td><td>KM982973.1</td></tr><tr><td>kudriavzevii</td><td>5</td><td>16</td><td>18</td><td>7</td><td>8</td><td>13</td><td>15</td><td>11</td><td>100</td><td>33.33</td><td>100</td><td>JQ808004.1</td></tr><tr><td>manshurica</td><td></td><td></td><td>4</td><td>2</td><td></td><td></td><td></td><td>2</td><td>38</td><td>2.87</td><td>100</td><td>OR554031.1</td></tr><tr><td>pseudolambica</td><td></td><td></td><td></td><td></td><td>1</td><td></td><td></td><td></td><td>13</td><td>0.36</td><td>100</td><td>OP418367.1</td></tr><tr><td>sporocuriosa</td><td></td><td></td><td>1</td><td></td><td></td><td></td><td></td><td></td><td>13</td><td>0.36</td><td>100</td><td>FJ153179.1</td></tr><tr><td>terricola</td><td></td><td>1</td><td>1</td><td></td><td></td><td></td><td></td><td></td><td>25</td><td>0.72</td><td>100</td><td>KY495764.1</td></tr><tr><td>mucilaginosa</td><td></td><td></td><td>2</td><td></td><td></td><td></td><td></td><td></td><td>13</td><td>0.72</td><td>100</td><td>MT465994.1</td></tr><tr><td>paludigena</td><td></td><td></td><td>1</td><td></td><td></td><td></td><td></td><td></td><td>13</td><td>0.36</td><td>100</td><td>MN515011.1</td></tr><tr><td>Saccharomyces</td><td>cerevisiae</td><td>4</td><td>9</td><td>12</td><td>1</td><td>3</td><td>6</td><td>6</td><td>3</td><td>100</td><td>15.77</td><td>100</td><td>KY816904.1</td></tr><tr><td>Saccharomycodes</td><td>ludwigii</td><td></td><td>1</td><td></td><td></td><td></td><td></td><td>2</td><td></td><td>25</td><td>1.08</td><td>100</td><td>OR554100.1</td></tr><tr><td>Saccharomycopsis</td><td>amapae</td><td></td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td>13</td><td>0.36</td><td>97</td><td>HG939418.1</td></tr><tr><td>Saturnispora</td><td>diversa</td><td></td><td>3</td><td>2</td><td></td><td></td><td></td><td></td><td></td><td>25</td><td>1.79</td><td>96</td><td>KT175190.1</td></tr><tr><td>Starmera</td><td>stellimalicola</td><td></td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td>13</td><td>0.36</td><td>100</td><td>KM982969.1</td></tr></tbody></table>
<table><caption>Table 2. Cont.</caption><thead><tr><th colspan="2">Yeasts Isolated</th><th colspan="8">Cocoa Fermentations Areas</th><th rowspan="2">Frequence (%)</th><th rowspan="2">Dominance (%)</th><th rowspan="2">% Identity</th><th rowspan="2">GenBank Accession Number</th></tr><tr><th>Genera</th><th>Species</th><th>Adzopé</th><th>Agnibilékrou</th><th>Biankouma Bonon</th><th>Divo</th><th>Guiberoua</th><th>Maféré</th><th>Meagui</th></tr></thead><tbody><tr><td>Starmerella</td><td>bacillaris</td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td><td>13</td><td>0.36</td><td>100</td><td>KT029752.1</td></tr><tr><td>Torulaspora</td><td>delbrueckii</td><td></td><td></td><td></td><td></td><td>1</td><td></td><td></td><td>13</td><td>0.36</td><td>100</td><td>MK267708.1</td></tr><tr><td>Wickerhamomyces</td><td>anomalus</td><td></td><td>4</td><td>7</td><td></td><td>4</td><td></td><td></td><td>38</td><td>5.38</td><td>100</td><td>HM044864.1</td></tr><tr><td>Unidentified yeast isolates</td><td></td><td></td><td>3</td><td>1</td><td>1</td><td></td><td>2</td><td>2</td><td>2</td><td>75</td><td>3.94</td><td>N/I</td><td>N/A</td></tr><tr><td>Total of yeast isolates</td><td></td><td>12</td><td>62</td><td>71</td><td>16</td><td>34</td><td>30</td><td>28</td><td>26</td><td></td><td></td><td></td><td></td></tr><tr><td>Total of identified yeast species</td><td></td><td>5</td><td>20</td><td>14</td><td>7</td><td>11</td><td>6</td><td>5</td><td>8</td><td></td><td></td><td></td><td></td></tr></tbody></table>

<table><caption>Table 3. Hydrolytic enzyme activity produced by each yeast species isolated from cocoa fermentation carried out in 8 main producing areas in Côte d'Ivoire.</caption><thead><tr><th colspan="2">Yeasts Isolated</th><th colspan="10">Hydrolytic Enzymes Activity</th><th rowspan="2">Total of Hydrolytic Enzyme Activity Produced</th></tr><tr><th>Genera</th><th>Species</th><th>Chitinase</th><th>β-Glucanase</th><th>Xylanase</th><th>Pectinase</th><th>Invertase</th><th>Esterase</th><th>Protease</th><th>β-Glucosidase</th><th>Polygalacturonase</th><th>Polyphenol Oxidase</th></tr></thead><tbody><tr><td rowspan="4">Candida</td><td>berthetii</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>4</td></tr><tr><td>boidinii</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>6</td></tr><tr><td>incommunis</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>8</td></tr><tr><td>tropicalis</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>5</td></tr><tr><td>Clavispora</td><td>lusitaniae</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>7</td></tr><tr><td>Cyberlindnera</td><td>sp.</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Geotrichum</td><td>candidum</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2">Hanseniaspora</td><td>opuntiae</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>4</td></tr><tr><td>pseudoguilliermondii</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>3</td></tr><tr><td>Kluyveromyces</td><td>marxianus</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Kurtzmanella</td><td>quercitrusa</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>7</td></tr><tr><td>Meyerozyma</td><td>caribbica</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Nakaseomyces</td><td>glabratus</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>2</td></tr></tbody></table>
<table><caption>Table 3. Cont.</caption><thead><tr><th colspan="2">Yeasts Isolated</th><th colspan="10">Hydrolytic Enzymes Activity</th><th rowspan="2">Total of Hydrolytic Enzyme Activity Produced</th></tr><tr><th>Genera</th><th>Species</th><th>Chitinase</th><th>β-Glucanase</th><th>Xylanase</th><th>Pectinase</th><th>Invertase</th><th>Esterase</th><th>Protease</th><th>β-Glucosidase</th><th>Polygalacturonase</th><th>Polyphenol Oxidase</th></tr></thead><tbody><tr><td rowspan="6">Pichia</td><td>bruneiensis<br>ethanolica</td><td>+</td><td>—</td><td>+</td><td>+</td><td>—</td><td>+</td><td>+</td><td>Not tested</td><td>+</td><td>—</td><td>+</td><td>7</td></tr><tr><td>kluyveri</td><td>+</td><td>—</td><td>+</td><td>+</td><td>+</td><td>—</td><td>+</td><td>+</td><td>+</td><td>—</td><td>+</td><td>7</td></tr><tr><td>kudriavzevii</td><td>+</td><td>—</td><td>+</td><td>+</td><td>+</td><td>—</td><td>+</td><td>+</td><td>+</td><td>—</td><td>+</td><td>7</td></tr><tr><td>manshurica<br>pseudolambica</td><td>+</td><td>—</td><td>+</td><td>+</td><td>+</td><td>—</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>8</td></tr><tr><td>sporocuriosa</td><td>+</td><td>—</td><td>+</td><td>+</td><td>+</td><td>—</td><td>+</td><td>+</td><td>+</td><td>—</td><td>+</td><td>7</td></tr><tr><td>terricola</td><td>+</td><td>—</td><td>—</td><td>—</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>—</td><td>+</td><td>6</td></tr><tr><td>Rhodotorula</td><td>mucilaginosa<br>paludigena</td><td colspan="10">Not tested<br>Not tested</td></tr><tr><td>Saccharomyces</td><td>cerevisiae</td><td>+</td><td>—</td><td>+</td><td>+</td><td>+</td><td>—</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>8</td></tr><tr><td>Saccharomycodes</td><td>ludwigii</td><td>—</td><td>—</td><td>+</td><td>—</td><td>—</td><td>—</td><td>—</td><td>+</td><td>—</td><td>—</td><td>+</td><td>2</td></tr><tr><td>Saccharomycopsis</td><td>amapae</td><td>—</td><td>—</td><td>+</td><td>+</td><td>+</td><td>—</td><td>—</td><td>+</td><td>+</td><td>+</td><td>+</td><td>6</td></tr><tr><td>Saturnispora</td><td>diversa</td><td>—</td><td>—</td><td>+</td><td>—</td><td>—</td><td>—</td><td>—</td><td>+</td><td>—</td><td>—</td><td>+</td><td>3</td></tr><tr><td>Starmera</td><td>stellimalicola</td><td>—</td><td>—</td><td>+</td><td>+</td><td>+</td><td>—</td><td>—</td><td>—</td><td>+</td><td>—</td><td>—</td><td>5</td></tr><tr><td>Starmerella</td><td>bacillaris</td><td>+</td><td>—</td><td>+</td><td>—</td><td>+</td><td>+</td><td>—</td><td>+</td><td>—</td><td>—</td><td>—</td><td>5</td></tr><tr><td>Torulaspora</td><td>delbrueckii</td><td>+</td><td>—</td><td>+</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>+</td><td>—</td><td>—</td><td>3</td></tr><tr><td>Wickerhamomyces anomalus</td><td></td><td>+</td><td>—</td><td>—</td><td>+</td><td>+</td><td>—</td><td>—</td><td>+</td><td>—</td><td>—</td><td>—</td><td>4</td></tr><tr><td>Unidentified yeast isolate</td><td></td><td>—</td><td>—</td><td>—</td><td>+</td><td>+</td><td>—</td><td>—</td><td>+</td><td>+</td><td>+</td><td>+</td><td>5</td></tr><tr><td>Total number of each enzyme activity producing yeast species</td><td></td><td>16</td><td>1</td><td>18</td><td>12</td><td>16</td><td>5</td><td>8</td><td>21</td><td>8</td><td>15</td><td>24</td><td></td></tr><tr><td>Number of each enzyme activity producing yeast species (%)</td><td></td><td>66.66</td><td>3.16</td><td>75.0</td><td>50.0</td><td>66.66</td><td>20.83</td><td>33.33</td><td>87.5</td><td>33.33</td><td>62.5</td><td>100</td><td></td></tr></tbody></table>
<figure><img src="image_5.png" /><figcaption>**Figure 2.** Various extracellular hydrolytic enzymes producing identified yeast species isolated per genus from cocoa fermentation carried out in different cocoa-producing locations of Côte d'Ivoire.</figcaption></figure>

### 3.3. Aromatic Compounds Produced by Identified Yeast Species

The aroma compounds produced by the tested yeast species were classified into five main chemical families, including alcohols, aldehydes, ketones, esters, and acids (Table 4). Alcohol contents ranged from 0.23 ± 0.01 to 25.62 ± 4.38 µg·mL⁻¹, and the ester concentrations ranged from 0.38 ± 0.03 to 48.33 ± 0.41 µg·mL⁻¹. In contrast, concentrations of aldehydes and acids remained below 1 µg·mL⁻¹ and 3 µg·mL⁻¹, respectively. Yeast species, including *Nakaseomyces glabratus*, *Wickerhamomyces anomalus*, *Candida tropicalis*, *Torulaspora delbrueckii*, *Kluyveromyces marxianus*, and *Saccharomyces cerevisiae*, produced alcohols at concentrations ranging between 14.27 and 25.62 µg·mL⁻¹. *Nakaseomyces glabratus* and *Wickerhamomyces anomalus* appeared as the greater alcohol producers with 25.62 ± 4.38 and 21.18 ± 0.44 µg·mL⁻¹, respectively. Similarly, *W. anomalus*, *Hanseniaspora opuntiae*, *Candida berthetii*, *Starmera stellimalicola*, *Clavispora lusitaniae*, and *Pichia kluyveri* generated ketones in the range of 5.07 to 16.00 µg·mL⁻¹. *W. anomalus* and *Hanseniaspora opuntiae* recorded higher ketone concentrations of about 15.5–16.0 µg·mL⁻¹, whereas *Pichia ethanolia* produced less ketones (0.09 µg·mL⁻¹). Esters were found at concentrations comprised between 17.26 and 48.33 µg·mL⁻¹ by *Pichia kluyveri*, *Wickerhamomyces anomalus*, *Geotrichum candidum*, *H. opuntiae*, *Saccharomycopsis amapae*, *Candida berthetii*, *Candida lusitaniae*, *K. marxianus*, and *Starmera stellimalicola*. *Pichia kluyveri* and *Wickerhamomyces anomalus* produced higher ester content, ranging from 43.69 ± 2.15 to 48.33 ± 0.41 µg·mL⁻¹, while *P. manshurica* produced lower content (0.38 ± 0.03 µg·mL⁻¹). *W. anomalus* and *H. opuntiae* produced acid concentrations that were higher by 2.70 µg·mL⁻¹, while *P. manshurica* produced less (0.07 ± 0.01 µg·mL⁻¹). The community of 32 yeast species produced higher concentrations of esters and lower concentrations of acids than other chemical families.
<table><caption>**Table 4.** Chemical families of volatile compounds produced by yeast species isolated from spontaneous cocoa fermentation carried out in different producing areas of Côte d'Ivoire.</caption><thead><tr><th rowspan="2">Yeasts Species</th><th colspan="5">Average of Concentration of Volatile Compounds per Chemical Family (µg·mL<sup>-1</sup>) Produced by Yeast Species</th></tr><tr><th>Alcohols</th><th>Aldehydes</th><th>Ketones</th><th>Esters</th><th>Acids</th></tr></thead><tbody><tr><td>Candida berthetii</td><td>6.33 ± 0.27<sup>j</sup></td><td>0.06 ± 0.01<sup>nop</sup></td><td>7.78 ± 1.21<sup>b</sup></td><td>23.16 ± 2.21<sup>de</sup></td><td>1.2 ± 0.11<sup>gh</sup></td></tr><tr><td>Candida boidinii</td><td>7.42 ± 0.47<sup>hij</sup></td><td>0.09 ± 0.01<sup>mno</sup></td><td>0.48 ± 0.02<sup>nopq</sup></td><td>2.64 ± 0.13<sup>mno</sup></td><td>1.64 ± 0.09<sup>cd</sup></td></tr><tr><td>Candida incommunis</td><td>2.13 ± 0.21<sup>lm</sup></td><td>0.61 ± 0.11<sup>a</sup></td><td>0.72 ± 0<sup>mno</sup></td><td>2.57 ± 0.46<sup>mno</sup></td><td>0.55 ± 0.03<sup>m</sup></td></tr><tr><td>Candida tropicalis</td><td>15.47 ± 1.27<sup>c</sup></td><td>0.21 ± 0.03<sup>efg</sup></td><td>1.19 ± 0.1<sup>lm</sup></td><td>12.31 ± 0.79<sup>gh</sup></td><td>1.51 ± 0.09<sup>def</sup></td></tr><tr><td>Clavispora lusitaniae</td><td>10.43 ± 0.46<sup>defg</sup></td><td>0.12 ± 0.01<sup>klm</sup></td><td>5.97 ± 0.55<sup>d</sup></td><td>23.10 ± 2.08<sup>de</sup></td><td>1.91 ± 0.02<sup>b</sup></td></tr><tr><td>Cyberlindnera sp.</td><td>9.88 ± 0.59<sup>efg</sup></td><td>0.13 ± 0.01<sup>ijklm</sup></td><td>2.20 ± 0.19<sup>hi</sup></td><td>11.67 ± 0.67<sup>h</sup></td><td>1.05 ± 0.04<sup>hijk</sup></td></tr><tr><td>Geotrichum candidum</td><td>3.62 ± 0.32<sup>kl</sup></td><td>0.21 ± 0.01<sup>ef</sup></td><td>1.45 ± 0.21<sup>kl</sup></td><td>37.31 ± 2.67<sup>c</sup></td><td>0.07 ± 0.01<sup>n</sup></td></tr><tr><td>Hanseniaspora opuntiae</td><td>8.97 ± 1.07<sup>gh</sup></td><td>0.29 ± 0.01<sup>d</sup></td><td>15.60 ± 0.85<sup>a</sup></td><td>36.45 ± 4.17<sup>c</sup></td><td>2.70 ± 0.27<sup>a</sup></td></tr><tr><td>Hanseniaspora pseudoguilliermondii</td><td>9.03 ± 0.87<sup>gh</sup></td><td>0.11 ± 0.01<sup>mn</sup></td><td>1.94 ± 0.21<sup>ijk</sup></td><td>9.89 ± 0.47<sup>hi</sup></td><td>0.96 ± 0.03<sup>ijkl</sup></td></tr><tr><td>Kluyveromyces marxianus</td><td>14.48 ± 1.11<sup>c</sup></td><td>0.22 ± 0.02<sup>ef</sup></td><td>2.97 ± 0.06<sup>g</sup></td><td>21.42 ± 1.26<sup>e</sup></td><td>0.97 ± 0.15<sup>hijk</sup></td></tr><tr><td>Kurtzmanella quercitrusa</td><td>3.88 ± 0.13<sup>k</sup></td><td>0.05 ± 0<sup>oP</sup></td><td>1.77 ± 0.05<sup>ijk</sup></td><td>3.50 ± 0.39<sup>lmn</sup></td><td>1.04 ± 0.04<sup>hijk</sup></td></tr><tr><td>Meyerozyma caribbica</td><td>8.76 ± 0.6<sup>ghi</sup></td><td>0.24 ± 0.02<sup>e</sup></td><td>1.66 ± 0.07<sup>ijkl</sup></td><td>3.68 ± 0.43<sup>klm</sup></td><td>1.29 ± 0.09<sup>fg</sup></td></tr><tr><td>Nakaseomyces glabratus</td><td>25.62 ± 4.38<sup>a</sup></td><td>0.37 ± 0.02<sup>c</sup></td><td>1.58 ± 0.13<sup>kl</sup></td><td>6.19 ± 0.65<sup>jk</sup></td><td>1.01 ± 0.2<sup>hijk</sup></td></tr><tr><td>Pichia bruneensis</td><td>10.60 ± 0.8<sup>def</sup></td><td>0.29 ± 0.02<sup>d</sup></td><td>0.32 ± 0.01<sup>nopq</sup></td><td>3.51 ± 0.42<sup>lmn</sup></td><td>1.02 ± 0.23<sup>hijk</sup></td></tr><tr><td>Pichia ethanolica</td><td>0.23 ± 0.01<sup>c</sup></td><td>0.47 ± 0.1<sup>b</sup></td><td>0.09 ± 0<sup>q</sup></td><td>0.94 ± 0.13<sup>no</sup></td><td>0.53 ± 0.07<sup>m</sup></td></tr><tr><td>Pichia kudriavzevii</td><td>12.04 ± 0.37<sup>d</sup></td><td>0.16 ± 0.01<sup>hijk</sup></td><td>4.08 ± 0.52<sup>f</sup></td><td>14.81 ± 4.79<sup>fg</sup></td><td>1.33 ± 0.06<sup>efg</sup></td></tr><tr><td>Pichia kluyveri</td><td>7.12 ± 0.84<sup>ij</sup></td><td>0.11 ± 0.01<sup>lmn</sup></td><td>5.07 ± 0.1<sup>e</sup></td><td>48.33 ± 0.41<sup>a</sup></td><td>0.92 ± 0.06<sup>ijkl</sup></td></tr><tr><td>Pichia manshurica</td><td>0.84 ± 0.04<sup>mno</sup></td><td>0.04 ± 0<sup>P</sup></td><td>0.40 ± 0.04<sup>nopq</sup></td><td>0.38 ± 0.03<sup>o</sup></td><td>0.07 ± 0.01<sup>n</sup></td></tr><tr><td>Pichia pseudolambica</td><td>2.18 ± 0.11<sup>lm</sup></td><td>0.05 ± 0<sup>P</sup></td><td>0.51 ± 0.05<sup>nopq</sup></td><td>2.41 ± 0.23<sup>mno</sup></td><td>1.02 ± 0.07<sup>hijk</sup></td></tr><tr><td>Pichia sporocuriosa</td><td>2.03 ± 0.21<sup>lmn</sup></td><td>0.10 ± 0<sup>lmn</sup></td><td>0.11 ± 0.01<sup>Pq</sup></td><td>0.46 ± 0.03<sup>o</sup></td><td>1.83 ± 0.22<sup>bc</sup></td></tr><tr><td>Pichia terricola</td><td>11.61 ± 1.19<sup>d</sup></td><td>0.11 ± 0.01<sup>lmn</sup></td><td>3.73 ± 0.19<sup>f</sup></td><td>14.99 ± 0.6<sup>f</sup></td><td>1.14 ± 0.18<sup>ghij</sup></td></tr><tr><td>Rhodotorula mucilaginosa</td><td>0.43 ± 0.04<sup>no</sup></td><td>0.05 ± 0.01<sup>oP</sup></td><td>0.42 ± 0<sup>nopq</sup></td><td>0.78 ± 0.07<sup>o</sup></td><td>0.12 ± 0.01<sup>n</sup></td></tr><tr><td>Rhodotorula paludigena</td><td>0.65 ± 0.09<sup>mno</sup></td><td>0.07 ± 0.01<sup>nop</sup></td><td>0.52 ± 0.02<sup>nopq</sup></td><td>1.18 ± 0.22<sup>mno</sup></td><td>0.90 ± 0.1<sup>kl</sup></td></tr><tr><td>Saccharomyces cerevisiae</td><td>14.27 ± 1.49<sup>c</sup></td><td>0.21 ± 0.03<sup>efg</sup></td><td>0.82 ± 0.1<sup>mn</sup></td><td>6.69 ± 0.55<sup>j</sup></td><td>1.45 ± 0.36<sup>def</sup></td></tr><tr><td>Saccharomyces amapae</td><td>10.10 ± 0.53<sup>de</sup></td><td>0.18 ± 0.01<sup>gh</sup></td><td>2.65 ± 0.03<sup>gh</sup></td><td>24.95 ± 0.45<sup>d</sup></td><td>1.29 ± 0.25<sup>fg</sup></td></tr><tr><td>Saturnispora diversa</td><td>4.28 ± 1.20<sup>k</sup></td><td>0.14 ± 0.01<sup>hijkl</sup></td><td>0.19 ± 0.11<sup>opq</sup></td><td>1.95 ± 3.81<sup>mno</sup></td><td>0.73 ± 0.01<sup>lm</sup></td></tr><tr><td>Saccharomyces ludwigii</td><td>8.19 ± 0.21<sup>hi</sup></td><td>0.14 ± 0.02<sup>hijkl</sup></td><td>0.63 ± 0.01<sup>nop</sup></td><td>7.35 ± 0.24<sup>ij</sup></td><td>1.18 ± 0.02<sup>ghi</sup></td></tr><tr><td>Starmerella bacillaris</td><td>4.39 ± 0.14<sup>k</sup></td><td>0.12 ± 0.01<sup>klm</sup></td><td>2.12 ± 0.28<sup>hij</sup></td><td>5.67 ± 0.91<sup>ijkl</sup></td><td>1.78 ± 0.07<sup>bc</sup></td></tr><tr><td>Starmera stellimalicola</td><td>1.06 ± 0.2<sup>mno</sup></td><td>0.17 ± 0.01<sup>fghi</sup></td><td>7.09 ± 0.12<sup>c</sup></td><td>17.26 ± 0.14<sup>f</sup></td><td>0.60 ± 0.35<sup>m</sup></td></tr><tr><td>Torulaspora delbrueckii</td><td>14.79 ± 0.55<sup>c</sup></td><td>0.17 ± 0<sup>ghij</sup></td><td>0.61 ± 0.05<sup>nopq</sup></td><td>6.37 ± 0.34<sup>j</sup></td><td>1.53 ± 0.04<sup>de</sup></td></tr><tr><td>Wickerhamomyces anomalus</td><td>21.18 ± 0.44<sup>b</sup></td><td>0.11 ± 0.01<sup>mn</sup></td><td>16.00 ± 0.56<sup>a</sup></td><td>43.69 ± 2.15<sup>b</sup></td><td>2.76 ± 0.14<sup>a</sup></td></tr></tbody></table>

The assigned values of the same alphabetic letter do not show any significant difference at the threshold of α = 0.05.
### 3.4. *Changes in Concentrations of Major Aromatic Compounds of Some Chemical Families Produced by Identified Yeast Species*

Figure 3 presents the concentrations of major aroma compounds of the main chemical families produced by yeast strains isolated from cocoa fermentation. Five major alcohols, including 2-propanol, ethanol, 2-methyl-1-propanol, 2-methyl-butanol, and 3-methyl-butanol, were globally produced regardless of the tested yeast species. *Wickerhamomyces anomalus* was the main species that produced propan-2-ol and ethanol at the highest concentrations of 6.67 and 6.61 µg·mL⁻¹, respectively, while *Nakaseomyces glabratus* synthesized 2-methyl-1-butanol and 3-methyl-1-butanol at 9.45 and 14.32 µg·mL⁻¹, respectively. *Saccharomycess cerevisiae* was practically associated with the production of all major alcohols. Indeed, this species produced 3-methyl-1-butaol, 2-methyl-1-butaol, and 2-methyl-1-propanol at 6.31, 3.92, and 2.81 µg·mL⁻¹. Also, *Kluyveromycess marxianus* produced various alcohols except 2-methyl-1-butanol, while appearing as the second greatest producer of 3-methyl-1-butanol at 11.48 µg·mL⁻¹. *Torulaspora delbrueckii* produced a wide range of alcohols and substantially produced 3-methyl-1-butanol and 2-methyl-1-butanol at 7.8 and 4.76 µg·mL⁻¹, respectively. All major alcohols detected were produced by Candida tropical from 0.37 µg·mL⁻¹ (ethanol) to 5.90 µg·mL⁻¹ (3-methyl-1-butanol) as indicated by Figure 3A. The production of many key esters, including ethyl acetate, ethyl propanoate, methyl butanoate, isobutyl acetate, propyl acetate, methyl butanoate, and 3 methylbutyl acetate, etc., is shown in Figure 3B. Ethyl acetate was the most ester produced by many identified yeast species. *W. anomalus* and *H. opuntiae* produced ethyl acetate at levels of 25.96 and 24.69 µg·mL⁻¹, and propyl acetate at 4.87 and 3.47 µg·mL⁻¹, respectively. Both yeasts also produced methyl butanoate at a similar level (1.68 µg·mL⁻¹), and *Geotrichum candidum* synthesized ethyl propanoate, isobutyl acetate, ethyl butanoate, and methyl thioacetate at concentrations of 5.30, 4.82, 6.65, and 4.84 µg·mL⁻¹, respectively. *Pichia kluyveri* produced various esters, including butyl acetate (3.40 µg·mL⁻¹), ethyl pentanoate (0.98 µg·mL⁻¹), 3-methylbutyl acetate (22.55 µg·mL⁻¹), ethyl 2-butenoate (3.10 µg·mL⁻¹), and 2-phenylethyl acetate (4.69 µg·mL⁻¹). *Pichia kluyveri* was the most yeast-producing of several major esters except isobutyl acetate, ethyl butanoate, and methyl thiolacetate, while it appeared as the best producer of 3-methylbutyl-acetate. Regarding the ketone family, five major compounds, including 2-butanone, 2-pentanone, 2,3-butandione, 2,3-pentandione, and 3-heptanone, were found. *H. opuntiae* was the greatest producer among all identified yeast species isolated from cocoa fermentation. Indeed, this species produced 2-butanone, 2-pentanone, and 2,3-butandione at concentrations of 8.3, 3.45, and 2.3 µg·mL⁻¹, respectively. *Candida berthetii* produced three major ketones, such as 2,3-butanedione and 2-pentanone, at similar concentrations around 3.1 µg·mL⁻¹. *W. anomalus* produced 2-butanone, 2-pentanone, and 2,3-butandione at concentrations reaching 5.34, 4.84, and 4.62 µg·mL⁻¹, respectively. In addition, *Starmera stellinalicola* mainly produced 2-pentanone and 3,3-butandione at 3.15 µg·mL⁻¹, whereas *Pichia kluyveri* produced 2,3-pentanedione and 3-heptanone at contents of 0.62 and 1.73 µg·mL⁻¹, respectively (Figure 3C). Figure 4 indicates that among all tested yeast strains, only *W. anomalus* and *H. opuntiae* produced acetic acid at substantial concentrations close to 2.70 µg·mL⁻¹, while *Candida boidinii* produced less (1.63 µg·mL⁻¹). Figure 4 indicates the concentration of acetic acid, the major compound of the acid class produced by yeast species isolated from cocoa fermentation. *W. anomalus* and *H. opuntiae* were the greater producers of acetic acid at a concentration of 2.70–2.75 µg·mL⁻¹, while *Starmerella bacillaris*, *Pichia sporocuriosa*, *Clavispora lusitaniae*, and *Candida boidinii* produced it at concentrations comprised between 1.64 and 1.97 µg·mL⁻¹.
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>**Figure 3.** Changes in total concentrations of some major aroma compounds produced by various yeast species isolated from cocoa fermentation carried out by different cocoa producers from Côte d'Ivoire: (A) Alcohol family, (B) Ketone Family, (C) Ester family. Bars are ± standard deviation. N = 4.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Figure 4.** Changes in total concentrations of acetic acid as representative of Acid family. Bars are ±standard deviation. N = 4.</figcaption></figure>

### 3.5. Antifungal Activity of Identified Yeast Species Against Ochratoxigenic Molds

In direct confrontation assays, all tested yeast species suppressed the mycelial growth of the OTA-producing *Aspergillus* strains to varying degrees (Table 5). The non-volatile metabolites of three yeast species, *Candida incommunis*, *Torulaspora delbrueckii*, and *Saccharomyces cerevisiae*, were exceptionally antifungal. Indeed, they completely inhibited the growth of all three mycotoxigenic molds tested. In contrast, metabolites from *Pichia terricola*, *Candida ethanolica*, and *Starmera stellimalicola* showed no inhibitory effect on the growth of any fungus. The remaining isolates exhibited intermediate inhibition.
<table><caption>**Table 5.** Biocontrol effects of different yeast species isolated from cocoa fermentation carried out in various cocoa-producing locations of Côte d'Ivoire on the mycelial growth of some mycotoxinogenic molds by direct confrontation.</caption><thead><tr><th rowspan="2">Mycotoxinogenic Mold Species</th><th colspan="2">Mycelial Growth of Mycotoxinogenic Molds on YPDA</th><th rowspan="2">By Direct Confrontation</th><th rowspan="2">Yeast Species<br>By Aroma Compounds</th></tr><tr><th>Controls</th><th>Antagonistic Effect</th></tr></thead><tbody><tr><td>Aspergillus carbonarius</td><td></td><td></td><td></td><td>1. Torulaspora delbrueckii<br>2. Saccharomyces cerevisiae<br>3. Candida incommunis<br>4. Pichia terricola<br>5. Candida boidinii<br>6. Hanseniaspora pseudoguillermondii</td></tr><tr><td>Aspergillus niger</td><td></td><td></td><td>All 32 tested yeast species</td><td>1. Torulaspora delbrueckii<br>2. Candida incommunis<br>3. Saccharomyces cerevisiae<br>4. Pichia bruneiensis<br>5. Candida ethanolica<br>6. Starmerella davenportii<br>7. Starmerella stellimalicola</td></tr><tr><td>Aspergillus ochraceus</td><td></td><td></td><td></td><td>All yeast species were isolated except Pichia kudriavzevii, Candida tropicalis, Wickerhamomyces anomalus, and Saccharomyces ludwigii</td></tr></tbody></table>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>
**4. Discussion**

Morphological and molecular characterization of the yeast isolates led to the identifica-
tion of 31 species belonging to 19 genera, notably *Pichia*, *Candida*, *Hanseniaspora*, *Rhodotorula*,
*Wickerhamomyces*, *Kurtzmaniella*, *Clavispora*, *Starmerella*, *Nakaseomyces*, *Meyerozyma*, *Torulas-
pora*, *Starmera*, *Saccharomycodes*, *Saccharomyces*, *Saccharomycopsis*, *Saturnispora*, *Kluyveromyces*,
*Cyberlindnera*, and *Geotrichum*. Among these, the genera Pichia, Saccharomyces, Candida,
and Hanseniaspora were particularly dominant, reflecting findings in earlier cocoa fermen-
tation studies [32]. Their high prevalence in this study may be attributed to their wide
distribution across cocoa-growing regions in Côte d'Ivoire [8,33,34]. Previous works sug-
gest that these genera are not only resilient under acidic and thermophilic conditions [35]
but also originate from diverse sources, including soil, plant material, pod surfaces, tools,
and human handling [36]. Their ability to withstand microbial competition, due to the
nutritional versatility or antifungal capacity, may also promote their dominance during fer-
mentation [37] as less abundant but still notable genera like *Wickerhamomyces*, *Kluyveromyces*,
*Torulaspora*, and *Rhodotorula*, which have been similarly detected at lower levels in other
spontaneous fermentations [38]. Despite various geographical origins, yeast communities
reported across cocoa-producing regions in Côte d'Ivoire are similar to those detected in
other cocoa-producing countries [14,17,39]. As reported by Ouattara and Niamké [33], yeast
diversity can differ regionally even within the same genus. Importantly, yeasts are known
to contribute to cocoa fermentation through the production of various extracellular hy-
drolytic enzymes, which drive pigment degradation and aroma precursor formation [40,41].
*P. kudriavzevii* and *Saccharomyces cerevisiae* [39], *C. tropicalis* [17], and *Hanseniaspora opun-
tiae* [42] were previously reported as the most common yeast species isolated from cocoa
fermentation carried out in many cocoa-producing countries.

Differences between the community of yeast species involved in cocoa fermentation
according to producing area could be ascribed to the fermentative performance of some
yeast species due to their acid tolerance and resilience to temperature [43]. The specificity
of more yeast species involved in cocoa fermentation carried out in the Biankouma region
than the others can be explained by their ability to resist the environmental stress prevalent
in this area due to its particularly mountainous terrain, the average altitude of the cocoa
plantations between 250 and 1300 m, and an average daily temperature below 28 °C
throughout the year. Indeed, according to Mager and Ferreira [44], the response of yeasts
to an environmental stress is complex, involving various aspects of cellular detection,
signal transduction, transcriptional and post-transcriptional control, protein targeting,
accumulation of protectants, and increased activity of repair functions. Consequently, the
robustness of a yeast strain depends on these biological mechanisms and allows predicting
its ability to play a key role in technological processes [45].

The enzymatic screening of yeast species revealed some substantial activities for
xylanase, β-glucosidase, polygalacturonase, invertase, pectinase, chitinase, polyphenol
oxidase, and protease. These enzymes have documented roles in cocoa pulp degradation
and aroma development. For example, β-glucosidase contributes to precursors of aromatic
compound formation [40], protease enhances amino acid and volatile profiles [46–48],
while pectinase reduces cocoa pulp viscosity, enhancing fermentation efficiency and re-
leases trapped aromatic compounds [49–51]. Xylanase has been reported to aid cocoa pulp
disintegration [52], and β-glucosidase hydrolyzes glycosides to release fermentable sugars
for both cocoa and microbial invertases [53]. Though polyphenol oxidase and invertase
are typically inactive during fermentation [40], their detection here indicates residual or
strain-specific activity. The pectinase activity observed in most yeast species is consis-
tent with previous findings highlighting its role in the degradation and liquefaction of
cocoa pulp [54]. Over half of the tested yeasts produced chitinase, a rare finding, as this
activity is generally associated with *Bacillus* spp. [55]. Chitinase may provide biocontrol
functions by degrading mold cell walls or deterring pests [56]. Protease activity may
facilitate shell softening and metabolite diffusion [41,57] while contributing to flavor and
aroma via amino acid metabolism [52]. Polyphenol oxidase activity detected in most yeast
species has been linked to the reduction in astringency and the development of aroma
precursors [58,59]. Many yeast species, particularly within *Pichia*, *Saccharomyces*, *Candida*, *Clavispora*, and *Hanseniaspora* genera, produced a broad spectrum of enzymes, indicating
their potential greater contribution to the degradation of cocoa pulp [60,61] and the release
of flavor precursors during fermentation [62,63]. The combination of all hydrolase enzymes,
including β-glucosidase, xylanase, invertase, chitinase, polygalacturonase, protease, pecti-
nase, and polyphenol oxidase, β-glucanase chitinase, xylanase, protease, β-glucosidase,
polyphenol oxidase, and pectinase produced by yeast species involved in the cocoa fermen-
tation contributes to the hydrolysis and degradation of cocoa mucilaginous pulp [54,64,65].
Also, according to Zhao and Fleet [41], yeasts are essential for cocoa fermentation for
their contribution to the generation of cocoa flavor and aroma [17]. Pichia manshurica
and Saccharomyces cerevisiae were the most hydrolytic enzyme-producing yeast species.
These results could probably explain why the two yeast species were most involved in
cocoa fermentation [66,67] and are currently used as starter cultures for it [62]. Only a
few tested yeast species exhibited esterase activity as previously reported [47,56], and
Starmera stellimalicola was the only yeast isolate producing β-glucanase, an uncommon
trait beyond *Saccharomyces* spp. [24,68]. These enzymatic functions collectively reinforce
the hypothesis that yeast's extracellular hydrolytic enzymes play a central role in aroma
compounds generation during cocoa fermentation [69–71].

Volatile compounds profiles revealed the presence of various chemical families, in-
cluding alcohols, aldehydes, ketones, esters, and acids, among which alcohols, esters, and
ketones predominated as previously reported by Koné et al. [17]. The production of those
various aroma compounds was realized not only by the yeasts but with the contribution
of lactic acid bacteria for alcohols, aldehydes, ketones, and organic acids, while ester
production was performed by only yeasts [72,73]. These results corroborate the role of
yeasts in generating cocoa aroma compounds [8,74]. Aldehydes likely arise from fatty acid
or amino acid degradation [34,38,75], while acids may result from ethanol oxidation by
*Acinetobacter* or deamination by *Bacillus* spp. [69,76]. Yeast species such as *Nakaseomyces
glabratus*, *W. anomalus*, *Candida tropicalis*, *Torulaspora delbrueckii*, *Kluyveromyces marxianus*,
and *Saccharomyces cerevisiae* were prolific producers of alcohols. In contrast, ketones were
abundantly produced therefrom *W. anomalus*, *H. opuntiae*, *Candida berthetii*, *Starmera stel-
limalicola*, *Clavispora lusitaniae*, and *Pichia kluyveri*, while the production of esters of yeast
species like *P. kluyveri*, *W. anomalus*, *Geotrichum candidum*, *Saccharomycopsis amapae*, and
*C. berthetii* was abundant. *W. anomalus* was particularly versatile, producing high levels
across alcohols, ketones, and esters [17]. The high production of esters of *W. anomalus*
would be ascribed to the combination of concentrations of 3 to 6% ethanol and 1% lactic
acid in the CPSM media. Indeed, according to Cai et al. [77], the combination of these
conditions may be ascribed to the upregulation of EAT1, ADH5, and TGL5 genes. This
yeast species' multifunctionality probably supports its selection as a starter culture to
enhance cocoa flavor [15,78]. *N. glabratus* was the main producer of 2-methylbutan-1-ol
and 3-methylbutan-1-ol, which impart chocolate and malty notes [16] while *H. opuntiae* and
*W. anomalus* produced key ketones like 2-butanone and 2,3-butanedione unlike previous
findings by Koné et al. [17,79], due to the composition differences between media. These
ketones provide fruity and buttery notes to fermented cocoa [80]. Major esters, including
ethyl acetate, propyl acetate, methyl and ethyl butanoate, and isoamyl acetate, were also
identified, likely generated by transesterification between alcohols and acetic acid [14,81].
The highest production of several positive aroma compounds of both *W. anomalus* [79] and *H. opuntiae* [82] allows us to select them as promising starter cultures for cocoa fermentation to enhance the sensory quality of chocolate.

Yeast-derived aroma compounds have also been implicated in fungi growth inhibition [79,83,84]. All tested yeast species in this research inhibited mycotoxigenic mold growth [85,86], likely due to nutrient competition, antifungal compounds secretion, or rapid yeast colony growth [87,88]. Non-volatile antifungal effects of yeasts varied by species [89–91], with *T. delbrueckii*, *C. boidinii*, *S. cerevisiae*, *H. pseudoguillermondii*, *H. guillermondii*, *S. stellimalicola*, *C. berthetii*, and *C. incommunis* showing strong inhibition of mold growth. *C. incommunis*, *T. delbrueckii*, and *S. cerevisiae* completely suppressed fungal growth, likely due to diffusible antifungal metabolites or aroma compounds [92–95].

**5. Conclusions**

This study revealed a diverse yeast microbiome composed of 31 species involved in cocoa fermentation in Côte d'Ivoire. These species belong to 19 genera, with *Pichia*, *Candida*, *Hanseniaspora*, and *Rhodotorula* being the most dominant. Yeast species from the *Pichia*, *Saccharomyces*, *Candida*, *Clavispora*, and *Hanseniaspora* genera displayed high enzymatic potential, producing xylanase, β-glucosidase, polygalacturonase, invertase, pectinase, and chitinase. *Wickerhamomyces anomalus* showed the highest capacity to produce aroma-active compounds in the chemical families of alcohols, ketones, and esters. All yeast strains demonstrated antifungal activity against mycotoxigenic molds, while *T. delbrueckii*, *C. incommunis*, *S. cerevisiae*, and *H. guillermondii* exhibited fungicidal effects capable of mitigating fungal contamination in cocoa. These findings highlight the relevant biotechnological potential of wild yeast strains to enhance cocoa fermentation processes and improve the final product's aroma quality.

**Author Contributions:** A.K.Y.: Formal analysis, Investigation, Methodology, Writing—original draft; G.F.K.A.: Formal analysis, Investigation, Methodology, Writing—original draft; B.J.A.-C.: Data curation, Investigation, Resources, Writing—original draft; N.K.: Formal analysis, Investigation; M.K.K.: Data curation, Methodology, Resources, Writing—original draft; K.B.: Data curation, Investigation, Visualization; I.M.: Project administration, Supervision, Writing—original draft; V.C.: Conceptualization, Data curation, Software; J.-C.M.: Conceptualization, Data curation, Writing—reviewing and editing; R.B.: Conceptualization, Funding acquisition, Project administration, Validation, Writing—reviewing and editing; S.T.G.: Conceptualization, Funding acquisition, Project administration, Validation, Writing—reviewing and editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by [European Union and French Agency for Development] grant number [No. FOOD/2019/412-132] and the APC was funded by [Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD)].

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

**Acknowledgments:** The project Cocoa4Future brings together a wide range of expertise to address the development challenges facing West African cocoa farming. It unites numerous partners with a shared ambition to place People and the Environment at the heart of the cocoa farming of tomorrow.

**Conflicts of Interest:** The authors declare that they have no conflicts of interest.
References

1. Iorizzo, M.; Bagnoli, D.; Vergalito, F.; Testa, B.; Tremonte, P.; Succi, M.; Pannella, G.; Letizia, F.; Albanese, G.; Lombardi, S.J. Coppola Raffaele Diversity of Fungal Communities on Cabernet and Aglianico Grapes from Vineyards Located in Southern Italy. *Front. Microbiol.* **2024**, *15*, 1399968. [CrossRef]

2. Asiegbu, F.O. Biotechnology: History, processes, and prospects in forestry. In *Forest Microbiology*; Academic Press: Cambridge, MA, USA, 2025; pp. 3–14. [CrossRef]

3. Kitessa, D.A. Review on effect of fermentation on physicochemical properties, anti-nutritional factors and sensory properties of cereal-based fermented foods and beverages. *Ann. Microbiol.* **2024**, *74*, 32. [CrossRef]

4. Rai, A.K.; Jeyaram, K. Role of Yeasts in Food Fermentation. In *Yeast Diversity in Human Welfare*; Satyanarayana, T., Kunze, G., Eds.; Springer: Singapore, 2017; pp. 83–113. [CrossRef]

5. Craparo, V.; Viola, E.; Vella, A.; Prestianni, R.; Pirrone, A.; Naselli, V.; Amato, F.; Oliva, D.; Notarbartolo, G.; Guzzon, R.; et al. Oenological capabilities of yeasts isolated from high-sugar matrices (Manna and Honey) as potential starters and Co-starters for winemaking. *Beverages* **2024**, *10*, 48. [CrossRef]

6. Johnson, E.A.; Echavarri-Erasun, C. Yeast biotechnology. In *The Yeasts*; Elsevier: Amsterdam, The Netherlands, 2011; pp. 21–44. [CrossRef]

7. Kadjo, A.C.; Houphouët, K.R.; Koné, K.M.; Fontana, A.; Durand, N.; Montet, D.; Guéhi, T.S. Link between the Reduction of Ochratoxin A and Free Fatty Acids Production in Cocoa Beans Using Bacillus sp. and the Sensory Perception of Chocolate Produced Thereof. *Acta Sci. Nutr. Health* **2024**, *7*, 11–20. [CrossRef]

8. Kouassi, A.D.D.; Koné, K.M.; Assi-Clair, B.J.; Lebrun, M.; Maraval, I.; Boulanger, R.; Guéhi, T.S. Effect of spontaneous fermentation location on the fingerprint of volatile compound precursors of cocoa and the sensory perceptions of the end-chocolate. *J. Food Sci. Technol.* **2022**, *59*, 4466–4478. [CrossRef]

9. Kédjébo, K.B.D.; Guéhi, T.S.; Brou, K.; Durand, N.; Aguilar, P.; Fontana-Tachon, A.; Montet, D. Effect of post-harvest treatments on the occurrence of ochratoxin A in cocoa beans. *Food Addit. Contam. Part A* **2015**, *33*, 157–166.

10. Kouakou, B.J.; Irie, B.Z.; Dick, E.; Nemlin, G.; Bomisso, L.E. Caractérisation des techniques de séchage du cacao dans les principales zones de production en Côte d'Ivoire et détermination de leur influence sur la qualité des fèves commercialisées. *J. Appl. Biosci.* **2023**, *64*, 4797–4812. [CrossRef]

11. Wen, L.; Yang, L.; Chen, C.; Li, J.; Fu, J.; Liu, G.; Kan, Q.; Ho, C.-T.; Huang, O.; Cao, Y. Applications of multi-omics techniques to unravel the fermentation process and the flavor formation mechanism in fermented foods. *Crit. Rev. Food Sci. Nutr.* **2024**, *64*, 8367–8383. [CrossRef] [PubMed]

12. Ragab, W.S.; Gomah, N.H.; Abdein, M.A. Biological Control of Mold and Mycotoxin Contaminations in Food and Dairy Products. *Int. J. Biol. Pharm. Allied Sci.* **2020**, *9*, 1128–1145. [CrossRef]

13. Nguyen, P.A.; Strub, C.; Fontana, A.; Schorr-Galindo, S. Crop molds and mycotoxins: Alternative management using biocontrol. *Biol. Control* **2017**, *104*, 10–27. [CrossRef]

14. Sandoval-Lozano, C.J.; Caballero-Torres, D.; López-Giraldo, L.J. Screening wild yeast isolated from cocoa bean fermentation using volatile compounds profile. *Molecules* **2022**, *27*, 902. [CrossRef]

15. Gutiérrez-Ríos, H.G.; Suárez-Quiroz, M.L.; Hernández-Estrada, Z.J.; Castellanos-Onorio, O.P.; Alonso-Villegas, R.; Rayas-Duarte, P.; Cano-Sarmiento, C.; Figuero-Hermandez, C.Y.; González-Rios, O. Yeasts as producers of flavor precursors during cocoa bean fermentation and their relevance as starter cultures: A review. *Fermentation* **2022**, *8*, 331. [CrossRef]

16. Assi-Clair, B.J.; Koné, M.K.; Kouamé, K.; Lahon, M.C.; Berthiot, L.; Durand, N.; Lebrun, M.; Julien-Ortiz, A.; Maraval, I.; Boulanger, R.; et al. Effect of aroma potential of *Saccharomyces cerevisiae* fermentation on the volatile profile of raw cocoa and sensory attributes of chocolate produced thereof. *Eur. Food Res. Technol.* **2019**, *245*, 1459–1471. [CrossRef]

17. Koné, M.K.; Guéhi, S.T.; Durand, N.; Ban-Koffi, L.; Berthiot, L.; Tachon, A.F.; Brou, K.; Boulanger, R.; Montet, D. Contribution of predominant yeasts to the occurrence of aroma compounds during cocoa bean fermentation. *Food Res. Int.* **2016**, *89*, 910–917. [CrossRef]

18. Csutak, O.; Csutak, T. Phylogenetic Analysis and Characterization of an Alkane-Degrading Yeast Strain Isolated from Oil-Polluted Soil. *Turk. J. Biol.* **2014**, *38*, 601–610. [CrossRef]

19. Tigrero-Vaca, J.; Maridueña-Zavala, M.G.; Liao, H.L.; Prado-Lince, M.; Zambrano-Vera, C.S.; Monserrate-Maggi, B.; Cevallos-Cevallos, J.M. Microbial diversity and contribution to the formation of volatile compounds during fine-flavor cacao bean fermentation. *Foods* **2022**, *11*, 915. [CrossRef] [PubMed]

20. Khodadadi, H.; Karimi, L.; Jalalizand, N.; Adin, H.; Mirhendi, H. Utilization of size polymorphism in ITS1 and ITS2 regions for identification of pathogenic yeast species. *J. Med. Microbiol.* **2017**, *66*, 126–133. [CrossRef] [PubMed]

21. Saoudi, B.; Habbeche, A.; Kerouaz, B.; Haberra, S.; Romdhane, Z.B.; Tichati, L.; Boudelaa, M.; Belghith, H.; Gargouri, A.; Ladjama, A. Purification and characterization of a new thermoalkaliphilic pectate lyase from *Actinomadura keratinilytica* Cpt20. *Process Biochem.* **2015**, *50*, 2259–2266. [CrossRef]
22. Moubasher, H.; Wahsh, S.S.; El-Kassem, N.A. Purification of pullulanase from *Aureobasidium pullulans*. *Microbiology* **2010**, *79*, 759–766. [CrossRef]

23. Bavaro, S.L.; Susca, A.; Frisvad, J.C.; Tufariello, M.; Chytiri, A.; Perrone, G.; Mita, G.; Logrieco, A.F.; Bleve, G. Isolation, characterization, and selection of molds associated to fermented black table olives. *Front. Microbiol.* **2017**, *8*, 1356. [CrossRef]

24. Strauss, M.L.A.; Jolly, N.P.; Lambrechts, M.G.; Van Rensburg, P. Screening for the production of extracellular hydrolytic enzymes by non-*Saccharomyces* wine yeasts. *J. Appl. Microbiol.* **2001**, *91*, 182–190. [CrossRef]

25. Delgado-Ospina, J.; Acquaticci, L.; Molina-Hernandez, J.B.; Rantsiou, K.; Martuscelli, M.; Kamgang-Nzekoue, A.F.; Vittori, S.; Paparella, A.; Chaves-López, C. Exploring the capability of yeasts isolated from Colombian fermented cocoa beans to form and degrade biogenic amines in a lab-scale model system for cocoa fermentation. *Microorganisms* **2020**, *9*, 28. [CrossRef]

26. Hamdouche, Y.; Guehi, T.; Durand, N.; Kedjebo, K.B.D.; Montet, D.; Meile, J.C. Dynamics of microbial ecology during cocoa fermentation and drying: Towards the identification of molecular markers. *Food Control* **2015**, *48*, 117–122. [CrossRef]

27. Waehrens, S.; Zhang, S.; Hedelund, P.; Petersen, M.; Byrne, D. Application of the fast sensory method 'Rate-All-That-Apply' in chocolate quality control compared with DHS-GC-MS. *Int. J. Food Sci. Technol.* **2016**, *51*, 1877–1887. [CrossRef]

28. Cruz-Rojas, L.L.; Oliva-Cruz, S.M.; Rondinel-Mendoza, N.V.; Villegas-Vilchez, L.F.; Caetano, A.C. Identification, semi-quantification, and dynamics of volatile organic compounds during spontaneous fermentation of two cocoa varieties from northern Peru, using HS-SPME/GC-MS techniques. *Food Sci. Technol.* **2023**, *43*, e0000072. [CrossRef]

29. Owusu, M.; Petersen, M.; Heimdal, H. Effect of fermentation method, roasting and conching conditions on the aroma volatiles of dark chocolate. *J. Food Process. Preserv.* **2012**, *36*, 446–456. [CrossRef]

30. Llano, S.; Vaillant, F.; Santander, M.; Zorro-González, A.; González-Orozco, C.E.; Maraval, I.; Boulanger, R.; Escobar, S. Exploring the Impact of Fermentation Time and Climate on Quality of Cocoa Bean-Derived Chocolate: Sensorial Profile and Volatilome Analysis. *Foods* **2024**, *13*, 2614. [CrossRef]

31. Kadjo, A.C.; Beugre, G.C.; Sess-Tchotch, D.A.; Kedjebo, K.B.D.; Mounjouenpou, P.; Durand, N.; Fontana, A.; Guehi, S.T. Screening of anti-fungal Bacillus strains and influence of their application on cocoa beans fermentation and final bean quality. *J. Adv. Microbiol.* **2023**, *23*, 8–17. [CrossRef]

32. Verce, M.; Schoonejans, J.; Hernandez Aguirre, C.; Molina-Bravo, R.; De Vuyst, L.; Weckx, S. A combined metagenomics and metatranscriptomics approach to unravel Costa Rican cocoa box fermentation processes reveals yet unreported microbial species and functionalities. *Front. Microbiol.* **2021**, *12*, 641185. [CrossRef]

33. Ouattara, H.G.; Niamké, S.L. Mapping the functional and strain diversity of the main microbiota involved in cocoa fermentation from Cote d'Ivoire. *Food Microbiol.* **2021**, *98*, 103767. [CrossRef]

34. Hamdouche, Y.; Meile, J.C.; Lebrun, M.; Guehi, T.; Boulanger, R.; Teyssier, C.; Montet, D. Impact of turning, pod storage and fermentation time on microbial ecology and volatile composition of cocoa beans. *Food Res. Int.* **2019**, *119*, 477–491. [CrossRef]

35. Batista, N.N.; Ramos, C.L.; Ribeiro, D.D.; Pinheiro, A.C.M.; Schwan, R.F. Dynamic behavior of *Saccharomyces cerevisiae*, *Pichia kluyveri* and *Hanseniaspora uvarum* during spontaneous and inoculated cocoa fermentations and their effect on sensory characteristics of chocolate. *LWT-Food Sci. Technol.* **2015**, *63*, 221–227. [CrossRef]

36. Jespersen, L.; Nielsen, D.S.; Hønholt, S.; Jakobsen, M. Occurrence and diversity of yeasts involved in fermentation of West African cocoa beans. *FEMS Yeast Res.* **2005**, *5*, 441–453. [CrossRef]

37. Díaz-Muñoz, C.; De Vuyst, L. Functional yeast starter cultures for cocoa fermentation. *J. Appl. Microbiol.* **2022**, *133*, 39–66. [CrossRef]

38. De Vuyst, L.; Leroy, F. Functional role of yeasts, lactic acid bacteria and acetic acid bacteria in cocoa fermentation processes. *Microbiol. Rev.* **2020**, *44*, 432–453. [CrossRef]

39. Chagas Junior, G.C.A.; de Espírito-Santo, J.C.A.; Ferreira, N.R.; Marques-da-Silva, S.H.; Oliveira, G.; Vasconcelos, S.; de Almeida, S.F.O.; Silva, L.R.C.; Gobira, R.M.; de Figueiredo, H.M.; et al. Yeast isolation and identification during On-Farm Cocoa Natural Fermentation in a Highly Producer Region in Northern Brazil. *Sci. Plena* **2021**, *16*, 121502. [CrossRef]

40. Hansen, C.E.; del Olmo, M.; Burri, C. Enzyme activities in cocoa beans during fermentation. *J. Sci. Food Agric.* **1998**, *77*, 273–281. [CrossRef]

41. Zhao, J.; Fleet, G. Yeasts are essential for cocoa bean fermentation. *Int. J. Food Microbiol.* **2014**, *174*, 72–87. [CrossRef] [PubMed]

42. Maura, Y.F.; Balzarini, T.; Borges, P.C.; Evrard, P.; De Vuyst, L.; Daniel, H.M. The environmental and intrinsic yeast diversity of Cuban cocoa bean heap fermentations. *Int. J. Food Microbiol.* **2016**, *233*, 34–43. [CrossRef] [PubMed]

43. Samagaci, L.; Ouattara, H.G.; Goualié, B.G.; Niamke, S.L. Growth capacity of yeasts potential starter strains under cocoa fermentation stress conditions in Ivory Coast. *Emir. J. Food Agric. (EJFA)* **2014**, *26*, 861–870. [CrossRef]

44. Mager, W.H.; Ferreira, P.M. Stress response of yeast. *Biochem. J.* **1993**, *290* Pt 1, 1–13. [CrossRef]

45. Stanley, D.; Bandara, A.; Fraser, S.; Chambers, P.J.; Stanley, G.A. The ethanol stress response and ethanol tolerance of *Saccharomyces cerevisiae*. *J. Appl. Microbiol.* **2010**, *109*, 13–24. [CrossRef] [PubMed]
46. Carvalho, J.K.; Panatta, A.A.S.; Silveira, M.A.D.; Tav, C.; Johann, S.; Rodrigues, M.L.F.; Martins, C.V.B. Yeasts isolated from a lotic continental environment in Brazil show potential to produce amylase, cellulase and protease. *Biotechnol. Rep.* **2021**, *30*, e00630. [CrossRef] [PubMed]

47. Koffi, O.; Samagaci, L.; Goualie, B.; Niamke, S. Screening of potential yeast starters with high ethanol production for a small-scale cocoa fermentation in Ivory Coast. *Food Environ. Saf.* **J. 2018**, *17*, 113–130.

48. Sousa, L.S.; Rocha, F.D.S.; Silveira, P.T.D.S.; Bispo, E.D.S.; Soares, S.E. Enzymatic activity of proteases and its isoenzymes in fermentation process in cultivars of cocoa (*Theobroma cacao* L.) produced in southern Bahia, Brazil. *Food Sci. Technol.* **2016**, *36*, 656–663. [CrossRef]

49. Freimüller Leischtfeld, S.; Hämmerli, A.; Lehmann, A.; Tönz, A.; Beck, B.M.; Wild, J.; Weis, S.; Neutsch, L.; Miescher Schwenninger, S. *Wickerhamomyces pijperi*: An Up-And-Coming Yeast with Pectinolytic Activity Suitable for Cocoa Bean Fermentation. *Appl. Microbiol.* **2025**, *5*, 43. [CrossRef]

50. Mulindwa, J.; Atukwase, A.; Bitalo, D.N.; Archileo, N.K. On-farm cocoa fermentation practices and their effect on the quality of cocoa beans in three agroecological zones of uganda. *Eur. J. Nutr. Andamp Food Saf.* **2025**, *17*, 1–15. [CrossRef]

51. Samagaci, L.; Ouattara, H.G.; Goualié, B.; Niamké, S. Polyphasic analysis of pectinolytic and stress-resistant yeast strains isolated from Ivorian cocoa fermentation. *J. Food Res.* **2014**, *4*, 124. [CrossRef]

52. Santos, M.P.F.; Bezerra, C.O.; Casteliano, G.A.; Uetanabaro, A.P.T.; da Silva, E.G.P.; da Costa, A.M. Production of enzyme for *Pleurotus pulmonarius* by solid-state fermentation on peach-palm and cocoa waste. *Fine Chem. Eng.* **2025**, *6*, 102–111. [CrossRef]

53. Silva, A.B.D.C.; e Silva, M.B.D.C.; de Souza Silveira, P.T.; Marques, E.D.L.S.; Soares, S.E. Extraction and determination of invertase and polyphenol oxidase activities during cocoa fermentation Extração e determinação da atividade de invertase e polifenoloxidase durante fermentação de cacao. *Braz. J. Dev.* **2021**, *7*, 80316–80330. [CrossRef]

54. Meersman, E.; Struyf, N.; Kyomugasho, C.; Jamsazzadeh Kermani, Z.; Santiago, J.S.; Baert, E.; Hemdane, S.; Vrancken, G.; Courtin, C.M.; Hendrickx, M.; et al. Characterization and degradation of pectic polysaccharides in cocoa pulp. *J. Agric. Food Chem.* **2017**, *65*, 9726–9734. [CrossRef]

55. Ouattara, H.G.; Koffi, B.L.; Karou, G.T.; Sangaré, A.; Niamke, S.L.; Diopoh, J.K. Implication of Bacillus sp. in the production of pectinolytic enzymes during cocoa fermentation. *World J. Microbiol. Biotechnol.* **2008**, *24*, 1753–1760. [CrossRef]

56. Delgado-Ospina, J.; Molina-Hernández, J.B.; Chaves-López, C.; Romanazzi, G.; Paparella, A. The role of fungi in the cocoa production chain and the challenge of climate change. *J. Fungi* **2021**, *7*, 202. [CrossRef] [PubMed]

57. Visintin, S.; Ramos, L.; Batista, N.; Dolci, P.; Schwan, F.; Cocolin, L. Impact of *Saccharomyces cerevisiae* and *Torulaspora delbrueckii* starter cultures on cocoa beans fermentation. *Int. J. Food Microbiol.* **2017**, *257*, 31–40. [CrossRef] [PubMed]

58. Ooi, T.S.; Sepiah, M.; Khairul Bariah, S. Effect of yeast species on total soluble solids, total polyphenol content and fermentation index during simulation study of cocoa fermentation. *Int. J. Eng. Technol. Sci. Innov.* **2016**, *3*, 477–483.

59. Macedo, A.S.L.; Rocha, F.D.S.; Ribeiro, M.D.S.; Soares, S.E.; Bispo, E.D.S. Characterization of polyphenol oxidase in two cocoa (*Theobroma cacao* L.) cultivars produced in the south of Bahia, Brazil. *Food Sci. Technol.* **2016**, *36*, 56–63. [CrossRef]

60. Daniel, H.M.; Vrancken, G.; Takrama, J.F.; Camu, N.; De Vos, P.; De Vuyst, L. Yeast diversity of Ghanaian cocoa bean heap fermentations. *FEMS Yeast Res.* **2009**, *9*, 774–783. [CrossRef]

61. Hirko, B.; Mitiku, H.; Getu, A. Role of fermentation and microbes in cacao fermentation and their impact on cacao quality. *Syst. Microbiol. Biomanuf.* **2023**, *3*, 509–520. [CrossRef]

62. Schwan, R.F.; Wheals, A.E. The microbiology of cocoa fermentation and its role in chocolate quality. *Crit. Rev. Food Sci. Nutr.* **2004**, *44*, 205–221. [CrossRef]

63. Campos, S.D.M.; Martínez-Burgos, W.J.; dos Reis, G.A.; Ocán-Torres, D.Y.; dos Santos Costa, G.; Rosas Vega, F.; Badel, A.B.; Coronado, L.S.; Serra, J.L.; Soccol, C.R. The Role of Microbial Dynamics, Sensorial Compounds, and Producing Regions in Cocoa Fermentation. *Microbiol. Res.* **2025**, *16*, 75. [CrossRef]

64. Aprotosoaie, A.C.; Luca, S.V.; Miron, A. Flavor chemistry of cocoa and cocoa products—An overview. *Compr. Rev. Food Sci. Food Saf.* **2016**, *15*, 73–91. [CrossRef]

65. Haase, T.B.; Babat, R.H.; Zorn, H.; Gola, S.; Schweiggert-Weisz, U. Enzyme-assisted hydrolysis of *Theobroma cacao* L. pulp. *J. Agric. Food Res.* **2024**, *18*, 101466. [CrossRef]

66. Almeida, S.D.F.O.D.; Silva, L.R.C.; Junior, G.C.A.C.; Oliveira, G.; Silva, S.H.M.D.; Vasconcelos, S.; Lopes, A.S. Diversity of yeasts during fermentation of cocoa from two sites in the Brazilian Amazon. *Acta Amaz.* **2019**, *49*, 64–70. [CrossRef]

67. Ferreira, O.D.S.; Chagas-Junior, G.C.; Chisté, R.C.; Martins, L.H.D.S.; Andrade, E.H.D.A.; Nascimento, L.D.D.; Lopes, A.S. *Saccharomyces cerevisiae* and *Pichia manshurica* from Amazonian biome affect the parameters of quality and aromatic profile of fermented and dried cocoa beans. *J. Food Sci.* **2022**, *87*, 4148–4161. [CrossRef]

68. López-Enríquez, L.; Vila-Crespo, J.; Rodríguez-Nogales, J.M.; Fernández-Fernández, E.; Ruipérez, V. Non-*Saccharomyces* yeasts from organic vineyards as spontaneous fermentation agents. *Foods* **2023**, *12*, 3644. [CrossRef] [PubMed]

69. Purbaningrum, K.; Hidayat, C.; Witasari, L.D.; Utami, T. Flavor precursors and volatile compounds improvement of unfermented cocoa beans by hydrolysis using bromelain. *Foods* **2023**, *12*, 820. [CrossRef]
70. Santander Muñoz, M.; Rodríguez Cortina, J.; Vaillant, F.E.; Escobar Parra, S. An overview of the physical and biochemical transformation of cocoa seeds to beans and to chocolate: Flavor formation. *Crit. Rev. Food Sci. Nutr.* **2019**, *60*, 1593–1613. [CrossRef]

71. Castro-Alayo, E.M.; Idrogo-Vásquez, G.; Siche, R.; Cardenas-Toro, F.P. Formation of aromatic compounds precursors during fermentation of Criollo and Forastero cocoa. *Heliyon* **2019**, *5*, e01157. [CrossRef] [PubMed]

72. Figueroa-Hernández, C.; Mota-Gutierrez, J.; Ferrocino, I.; Hernández-Estrada, Z.J.; González-Ríos, O.; Cocolin, L.; Suárez-Quiroz, M.L. The challenges and perspectives of the selection of starter cultures for fermented cocoa beans. *Int. J. Food Microbiol.* **2019**, *301*, 41–50. [CrossRef]

73. Mota-Gutierrez, J.; Barbosa-Pereira, L.; Ferrocino, I.; Cocolin, L. Traceability of functional volatile compounds generated on inoculated cocoa fermentation and its potential health benefits. *Nutrients* **2019**, *11*, 884. [CrossRef]

74. Koné, K.M.; Assi-Clair, B.J.; Kouassi, A.D.D.; Yao, A.K.; Ban-Koffi, L.; Durand, N.; Guéhi, T.S. Pod storage time and spontaneous fermentation treatments and their impact on the generation of cocoa flavour precursor compounds. *Int. J. Food Sci. Technol.* **2021**, *56*, 2516–2529. [CrossRef]

75. Ordoñez-Araque, R.H.; Landines-Vera, E.F.; Urresto-Villegas, J.C.; Caicedo-Jaramillo, C.F. Microorganisms during cocoa fermentation: Systematic review. *Foods Raw Mater.* **2020**, *8*, 155–162. [CrossRef]

76. Dulce, V.R.; Anne, G.; Manuel, K.; Carlos, A.A.; Jacobo, R.C.; de Jesús, C.E.S.; Eugenia, L.C. Cocoa bean turning as a method for redirecting the aroma compound profile in artisanal cocoa fermentation. *Heliyon* **2021**, *7*, e07694. [CrossRef]

77. Cai, W.; Wan, Y.; Chen, Y.; Fan, H.; Li, M.; Wu, S.; Lin, P.; Zeng, T.; Luo, H.; Huang, D.; et al. Transcriptomics to evaluate the influence mechanisms of ethanol on the ester production of Wickerhamomyces anomalus with the induction of lactic acid. *Food Microbiol.* **2024**, *122*, 104556. [CrossRef]

78. Kadow, D.; Bohlmann, J.; Phillips, W.; Lieberei, R. Identification of main fine flavour components in two genotypes of the cocoa tree (*Theobroma cacao* L.). *J. Appl. Bot. Food Qual.* **2013**, *86*, 90–98. [CrossRef]

79. Criollo-Nuñez, J.; Sandoval-Aldana, A.P.; Bolívar, G.; Ramírez Toro, C. Selection of promising yeasts for starter culture development promoter of cocoa fermentation. *Ing. Compet.* **2024**, *26*, e20413296.

80. Frauendorfer, F.; Schieberle, P. Key aroma compounds in fermented Forastero cocoa beans and changes induced by roasting. *Eur. Food Res. Technol.* **2019**, *245*, 1907–1915. [CrossRef]

81. Mendoza Salazar, M.M.; Martínez Álvarez, O.L.; Ardila Castañeda, M.P.; Lizarazo and Medina, P.X. Bioprospecting of indigenous yeasts involved in cocoa fermentation using sensory and chemical strategies for selecting a starter inoculum. *Food Microbiol.* **2022**, *101*, 103896. [CrossRef]

82. Coria-Hinojosa, L.M.; Velásquez-Reyes, D.; Alcazar-Valle, M.; Kirchmayr, M.R.; Calva-Estrada, S.; Gschaedler, A.; Gschaedler, A.; Lugo, E. Exploring volatile compounds and microbial dynamics: *Kluyveromyces marxianus* and *Hanseniaspora opuntiae* reduce Forastero cocoa fermentation time. *Food Res. Int.* **2024**, *193*, 114821. [CrossRef] [PubMed]

83. Choińska, R.; Piasecka-Jóźwiak, K.; Chabłowska, B.; Dumka, J.; Łukaszewicz, A. Biocontrol ability and volatile organic compounds production as a putative mode of action of yeast strains isolated from organic grapes and rye grains. *Antonie Van Leeuwenhoek* **2020**, *113*, 1135–1146. [CrossRef]

84. Ruiz-Moyano, S.; Hernández, A.; Galvan, A.I.; Córdoba, M.G.; Casquete, R.; Serradilla, M.J.; Martín, A. Selection and application of antifungal VOCs-producing yeasts as biocontrol agents of grey mould in fruits. *Food Microbiol.* **2020**, *92*, 103556. [CrossRef]

85. Olana, R.C.; Haituk, S.; Karunarathna, A.; Cumagun, C.J.R. Elucidating the Efficacy of Antagonistic Yeasts by Using Multiple Mechanisms in Controlling Fungal Phytopathogens. *J. Phytopathol.* **2025**, *173*, e70097. [CrossRef]

86. Ruggirello, M.; Nucera, D.; Cannoni, M.; Peraino, A.; Rosso, F.; Fontana, M.; Cocolin, L.; Dolci, P. Antifungal activity of yeasts and lactic acid bacteria isolated from cocoa bean fermentations. *Food Res. Int.* **2019**, *115*, 519–525. [CrossRef]

87. Mewa-Ngongang, M.; du Plessis, H.W.; Ntwampe, S.K.O.; Chidi, B.S.; Hutchinson, U.F.; Mekuto, L.; Jolly, N.P. The use of *Candida pyralidae* and *Pichia kluyveri* to control spoilage microorganisms of raw fruits used for beverage production. *Foods* **2019**, *8*, 454. [CrossRef]

88. Hathout, A.; Abdel-Nasser, A. The efficiency of *Saccharomyces cerevisiae* as an antifungal and antimycotoxigenic agent. *Biointerface Res. Appl. Chem.* **2023**, *13*, 354. [CrossRef]

89. Chen, L.; Zhao, C.; Yan, T.; Li, B.; Wang, S.; Gong, D.; Long, D. Antifungal potentiality of non-volatile compounds produced from *Hanseniaspora uvarum* against postharvest decay of table grape fruit caused by Botrytis cinerea and Penicillium expansum. *Postharvest Biol. Technol.* **2025**, *222*, 113364. [CrossRef]

90. Cabañas, C.M.; Hernández León, A.; Ruiz-Moyano, S.; Merchán, A.V.; Martínez Torres, J.M.; Martín, A. Biocontrol of Cheese Spoilage Moulds Using Native Yeasts. *Foods* **2025**, *14*, 2446. [CrossRef]

91. Maluleke, E.; Jolly, N.P.; Patterton, H.G.; Setati, M.E. Antifungal activity of non-conventional yeasts against Botrytis cinerea and non-Botrytis grape bunch rot fungi. *Front. Microbiol.* **2022**, *13*, 986229. [CrossRef] [PubMed]

92. Yang, T.; Wang, C.; Li, C.; Sun, R.; Yang, M. Antagonistic effects of volatile organic compounds of *Saccharomyces cerevisiae* NJ-1 on the growth and toxicity of *Aspergillus flavus*. *Biol. Control* **2023**, *177*, 105093. [CrossRef]
93. Natarajan, S.; Balachandar, D.; Senthil, N.; Velazhahan, R.; Paranidharan, V. Volatiles of antagonistic soil yeasts inhibit growth and aflatoxin production of *Aspergillus flavus*. *Microbiol. Res.* **2022**, *263*, 127150. [CrossRef]

94. Galvan, A.I.; Hernandez, A.; de Guía Córdoba, M.; Martin, A.; Serradilla, M.J.; Lopez-Corrales, M.; Rodriguez, A. Control of toxigenic *Aspergillus* spp. in dried figs by volatile organic compounds (VOCs) from antagonistic yeasts. *Int. J. Food Microbiol.* **2022**, *376*, 109772. [CrossRef] [PubMed]

95. Vepštaitė-Monstavičė, I.; Lukša-Žebelovič, J.; Apšegaitė, V.; Mozūraitis, R.; Lisicinas, R.; Stanevičienė, R.; Blažytė-Čereškienė, L.; Serva, S.; Servienė, E. Profiles of killer systems and volatile organic compounds of rowanberry and rosehip-inhabiting yeasts substantiate implications for biocontrol. *Foods* **2025**, *14*, 288. [CrossRef] [PubMed]

**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.