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

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.

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 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.

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. Composition of cocoa pulp similar medium (CPSM).

Components	Concentration (g·L−1)
Yeast nitrogen base (YNB)	2.0
Fructose	30.0
Glucose	20.0
Pectin	10.0
Sucrose	6.7
Acetic acid	1.4
Lactic acid	1.0
Citric acid	1.0
Ammonium sulfate (NH4)2SO4	2.8
Monobasic potassium phosphate (KH2PO4)	1.0
Dibasic potassium phosphate (K2HPO4)	6.0
Magnesium sulfate heptahydrate (MgSO4·7H2O)	0.5
Iron (II) sulfate heptahydrate (FeSO4·7H2O)	0.01
Analytic Ethanol	2.0
Polyphenols (catechin 1136 mg·L−1, epicatechin 1441 mg·L−1, isoquercetin 539 mg·L−1)	0.051%

[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:

$$qi (\mathrm{\mu }\mathrm{g}·{\mathrm{m}\mathrm{L}}^{-1})={\displaystyle \frac{A_i\times 0.818}{A_e\times V_i}}$$

• $qi$: Amount of compound i released into the headspace, expressed in µg·mL⁻¹;
• $A_i$: Peak area of compound I;
• $V_i$: Volume of sample suspension introduced into the vial (mL);
• $A_e$: Peak area of the internal standard (3-heptanol);
• 0.818: Mass of the internal standard in µg.

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:

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

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. Distribution of yeast species isolated from cocoa fermentation carried out in 8 main producing areas in Côte d’Ivoire.

Yeasts Isolated		Cocoa Fermentations Areas								Frequence (%)	Dominance (%)	% Identity	GenBank Accession Number
Genera	Species	Adzopé	Agnibilékrou	Biankouma	Bonon	Divo	Guiberoua	Maféré	Meagui
Candida	berthetii	1				1				25	0.72	99	NR_077118.1
	boidinii		3							13	1.08	100	KY445950.1
	incommunis		1							13	0.36	100	MZ506857.1
	tropicalis	1	3	5		3	1	2		75	5.38	100	LC317537.1
Clavispora	lusitaniae			8						13	2.87	100	KP674846.1
Cyberlindnera	sp.		1							13	0.36	100	PP112176.1
Geotrichum	candidum			3						13	1.08	99	MT071788.1
Hanseniaspora	opuntiae		5	5	2	8	2	2	2	88	9.32	100	FM199955.1
	pseudoguilliermondii					2				13	0.72	100	MW990151.1
Kluyveromyces	marxianus								2	13	0.72	100	MT187614.1
Kurtzmaniella	quercitrusa		4			2				25	2.15	100	MF574303.1
Meyerozyma	caribbica		1							13	0.36	100	MT312818.1
Nakaseomyces	glabratus		1		1		1			38	1.08	100	MK998697.1
Pichia	bruneiensis		1			1			1	38	1.08	98	LC431631.2
	ethanolica		1				5	1		38	2.51	100	KM368822.1
	kluyveri	1	2	1	1	1				63	2.15	100	KM982973.1
	kudriavzevii	5	16	18	7	8	13	15	11	100	33.33	100	JQ808004.1
	manshurica			4	2				2	38	2.87	100	OR554031.1
	pseudolambica					1				13	0.36	100	OP418367.1
	sporocuriosa			1						13	0.36	100	FJ153179.1
	terricola		1	1						25	0.72	100	KY495764.1
Rhodotorula	mucilaginosa			2						13	0.72	100	MT465994.1
	paludigena			1						13	0.36	100	MN515011.1
Saccharomyces	cerevisiae	4	9	12	1	3	6	6	3	100	15.77	100	KY816904.1
Saccharomycodes	ludwigii		1						2	25	1.08	100	OR554100.1
Saccharomycopsis	amapae		1							13	0.36	97	HG939418.1
Saturnispora	diversa		3	2						25	1.79	96	KT175190.1
Starmera	stellimalicola		1							13	0.36	100	KM982969.1
Starmerella	bacillaris								1	13	0.36	100	KT029752.1
Torulaspora	delbrueckii				1					13	0.36	100	MK267708.1
Wickerhamomyces	anomalus		4	7		4				38	5.38	100	HM044864.1
Unidentified yeast isolates			3	1	1		2	2	2	75	3.94	N/I	N/A
Total of yeast ioslates		12	62	71	16	34	30	28	26
Total of identified yeast species		5	20	14	7	11	6	5	8

). 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. Hydrolytic enzyme activity produced by each yeast species isolated from cocoa fermentation carried out in 8 main producing areas in Côte d’Ivoire.

Yeasts Isolated		Hydrolytic Enzymes Activity										Total of Hydrolytic Enzyme Activity Produced
Genera	Species	Chitinase	β-Glucanase	Xylanase	Pectinase	Invertase	Esterase	Protease	β-Glucosidase	Polygalac- turonase	Polyphenol Oxidase
Candida	berthetii	−	−	+	−	+	−	−	+	−	+	4
	boidinii	+	−	+	−	+	+	−	+	−	+	6
	incommunis	+	−	+	+	+	+	+	+	−	+	8
	tropicalis	+	−	+	−	+	−	−	+	−	+	5
Clavispora	lusitaniae	+	−	+	+	+	−	+	+	+	−	7
Cyberlindnera	sp.	Not tested
Geotrichum	candidum	Not tested
Hanseniaspora	opuntiae	+	−	−	+	−	−	−	+	+	−	4
	pseudoguilliermondii	+	−	−	−	+	−	−	+	−	−	3
Kluyveromyces	marxianus	Not tested
Kurtzmaniella	quercitrusa	+	−	+	+	+	−	+	+	−	+	7
Meyerozyma	caribbica	Not tested
Nakaseomyces	glabratus	−	−	+	−	−	−	−	−	+	−	2
Pichia	bruneiensis	+	−	+	+	−	+	+	+	−	+	7
	ethanolica	Not tested
	kluyveri	+	−	+	+	+	−	+	+	−	+	7
	kudriavzevii	+	−	+	+	+	−	+	+	−	+	7
	manshurica	+	−	+	+	+	−	+	+	+	+	8
	pseudolambica	Not tested
	sporocuriosa	+	−	+	+	+	−	+	+	−	+	7
	terricola	+	−	−	−	+	+	+	+	−	+	6
Rhodotorula	mucilaginosa	Not tested
	paludigena	Not tested
Saccharomyces	cerevisiae	+	−	+	+	+	−	+	+	+	+	8
Saccharomycodes	ludwigii	−	−	+	−	−	−	−	+	−	-	2
Saccharomycopsis	amapae	−	−	+	+	+	−	−	+	+	+	6
Saturnispora	diversa	−	−	+	−	−	−	−	+	−	+	3
Starmera	stellimalicola	−	+	+	+	+	−	−	−	+	-	5
Starmerella	bacillaris	+	−	+	−	+	+	−	+	−	-	5
Torulaspora	delbrueckii	+	−	+	−	−	−	−	−	+	-	3
Wickerhamomyces	anomalus	+	−	−	+	+	−	−	+	−	-	4
Unidentified yeast isolate		-	−	−	+	+	−	−	+	+	+	5
Total number of each enzyme activity producing yeast species		16	1	18	12	16	5	8	21	8	15	24
Number of each enzyme activity producing yeast species (%)		66.66	3.16	75.0	50.0	66.66	20.83	33.33	87.5	33.33	62.5	100

). Candida incommunis, Pichia manshurica, and Saccahromyces cerevisiae were the greater hydrolytic enzyme-producing yeast species with 8 activities, while Nakaseomyces glabratus and Saccharomycodes 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.

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. Chemical families of volatile compounds produced by yeast species isolated from spontaneous cocoa fermentation carried out in different producing areas of Côte d’Ivoire.

	Average of Concentration of Volatile Compounds per Chemical Family (µg·mL−1) Produced by Yeast Species
Yeasts Species	Alcohols	Aldehydes	Ketones	Esters	Acids
Candida berthetii	6.33 ± 0.27 j	0.06 ± 0.01 nop	7.78 ± 1.21 b	23.16 ± 2.21 de	1.2 ± 0.11 gh
Candida boidinii	7.42 ± 0.47 hij	0.09 ± 0.01 mno	0.48 ± 0.02 nopq	2.64 ± 0.13 mno	1.64 ± 0.09 cd
Candida incommunis	2.13 ± 0.21 lm	0.61 ± 0.11 a	0.72 ± 0 mno	2.57 ± 0.46 mno	0.55 ± 0.03 m
Candida tropicalis	15.47 ± 1.27 c	0.21 ± 0.03 efg	1.19 ± 0.1 lm	12.31 ± 0.79 gh	1.51 ± 0.09 def
Clavispora lusitaniae	10.43 ± 0.46 defg	0.12 ± 0.01 jklm	5.97 ± 0.55 d	23.10 ± 2.08 de	1.91 ± 0.02 b
Cyberlindnera sp.	9.88 ± 0.59 efg	0.13 ± 0.01 ijklm	2.20 ± 0.19 hi	11.67 ± 0.67 h	1.05 ± 0.04 hijk
Geotrichum candidum	3.62 ± 0.32 kl	0.21 ± 0.01 ef	1.45 ± 0.21 kl	37.31 ± 2.67 c	0.07 ± 0.01 n
Hanseniaspora opuntiae	8.97 ± 1.07 fgh	0.29 ± 0.01 d	15.60 ± 0.85 a	36.45 ± 4.17 c	2.70 ± 0.27 a
Hanseniaspora pseudoguilliermondii	9.03 ± 0.87 fgh	0.11 ± 0.0l mn	1.94 ± 0.21 ijk	9.89 ± 0.47 hi	0.96 ± 0.03 ijkl
Kluyveromyces marxianus	14.48 ± 1.11 c	0.22 ± 0.02 ef	2.97 ± 0.06 g	21.42 ± 1.26 e	0.97 ± 0.15 hijk
Kurtzmaniella quercitrusa	3.88 ± 0.13 k	0.05 ± 0 op	1.77 ± 0.05 ijk	3.50 ± 0.39 lmn	1.04 ± 0.04 hijk
Meyerozyma caribbica	8.76 ± 0.6 ghi	0.24 ± 0.02 e	1.66 ± 0.07 ijkl	3.68 ± 0.43 klm	1.29 ± 0.09 fg
Nakaseomyces glabratus	25.62 ± 4.38 a	0.37 ± 0.02 c	1.58 ± 0.13 jkl	6.19 ± 0.65 jk	1.01 ± 0.2 hijk
Pichia bruneinsis	10.60 ± 0.8 def	0.29 ± 0.02 d	0.32 ± 0.01 nopq	3.51 ± 0.42 lmn	1.02 ± 0.23 hijk
Pichia ethanolica	0.23 ± 0.01 c	0.47 ± 0.1 b	0.09 ± 0 q	0.94 ± 0.13 no	0.53 ± 0.07 m
Pichia kudriavzevii	12.04 ± 0.37 d	0.16 ± 0.01 hijk	4.08 ± 0.52 f	14.81 ± 4.79 fg	1.33 ± 0.06 efg
Pichia kluyveri	7.12 ± 0.84 ij	0.11 ± 0.01 lmn	5.07 ± 0.1 e	48.33 ± 0.41 a	0.92 ± 0.06 jkl
Pichia manshurica	0.84 ± 0.04 mno	0.04 ± 0 p	0.40 ± 0.04 nopq	0.38 ± 0.03 o	0.07 ± 0.01 n
Pichia pseudolambica	2.18 ± 0.11 lm	0.05 ± 0 p	0.51 ± 0.05 nopq	2.41 ± 0.23 mno	1.02 ±0.07 hijk
Pichia sporocuriosa	2.03 ± 0.21 lmn	0.10 ± 0 lmn	0.11 ± 0.01 pq	0.46 ± 0.03 o	1.83 ± 0.22 bc
Pichia terricola	11.61 ± 1.19 d	0.11 ± 0.01 lmn	3.73 ± 0.19 f	14.99 ± 0.6 f	1.14 ± 0.18 ghij
Rhodotorula mucilaginosa	0.43 ± 0.04 no	0.05 ± 0.01 op	0.42 ± 0 nopq	0.78 ± 0.07 o	0.12 ± 0.01 n
Rhodotorula paludigena	0.65 ± 0.09 mno	0.07 ± 0.01 nop	0.52 ± 0.02 nopq	1.18 ± 0.22 mno	0.90 ± 0.1 kl
Saccharomyces cerevisiae	14.27 ± 1.49 c	0.21 ± 0.03 efg	0.82 ± 0.1 mn	6.69 ± 0.55 j	1.45 ± 0.36 def
Saccharomycopsis amapae	10.10 ± 0.53 de	0.18 ± 0.01 fgh	2.65 ± 0.03 gh	24.95 ± 0.45 d	1.29 ± 0.25 fg
Saturnispora diversa	4.28 ± 1.20 k	0.14 ± 0.01 hijkl	0.19 ± 0.11 opq	1.95 ± 3.81 mno	0.73 ±0.01 lm
Saccharomycodes ludwigii	8.19 ± 0.21 hi	0.14 ± 0.02 hijkl	0.63 ± 0.01 nop	7.35 ± 0.24 ij	1.18 ± 0.02 ghi
Starmerella bacillaris	4.39 ± 0.14 k	0.12 ± 0.01 klm	2.12 ± 0.28 hij	5.67 ± 0.91 jkl	1.78 ± 0.07 bc
Starmera stellimalicola	1.06 ± 0.2 mno	0.17 ± 0.01 fghi	7.09 ± 0.12 c	17.26 ± 0.14 f	0.60 ± 0.35 m
Torulaspora delbrueckii	14.79 ± 0.55 c	0.17 ± 0 ghij	0.61 ± 0.05 nopq	6.37 ± 0.34 j	1.53 ± 0.04 de
Wickerhamomyces anomalus	21.18 ± 0.44 b	0.11 ± 0.0l mn	16.00 ± 0.56 a	43.69 ± 2.15 b	2.76 ± 0.14 a

The assigned values of the same alphabetic letter do not show any significant difference at the threshold of α = 0.05.

). 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 ethanolica 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 producer 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.

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-1propanol, 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-pentanoe, 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.

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. 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.

Mycotoxinogenic Mold Species	Mycelial Growth of Mycotoxinogenic Molds on YPDA		Yeast Species
	Controls	Antagonistic Effect	By Direct Confrontation	By Aroma Compounds
Aspergillus carbonarius			All 32 tested yeast species	Torulaspora delbrueckii Saccharomyces cerevisiae Candida incommunis Pichia terricola Candida boidinii Hanseniaspora pseudoguillermondii
Aspergillus niger				Torulaspora delbrueckii Candida incommunis Saccharomyces cerevisiae Pichia bruneiensis Candida éthanolica Starmerella davenportii Starmerella stellimalicola
Aspergillus ochraceus				All yeast species were isolated except Pichia kudriavzevii, Candida tropicalis, Wickerhamomyces anomalus, and Saccharomycodes ludwigii

). 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.

4. Discussion

Morphological and molecular characterization of the yeast isolates led to the identification of 31 species belonging to 19 genera, notably Pichia, Candida, Hanseniaspora, Rhodotorula, Wickerhamomyces, Kurtzmaniella, Clavispora, Starmerella, Nakaseomyces, Meyerozyma, Torulaspora, 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 fermentation 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 suggest 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 fermentation [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 hydrolytic enzymes, which drive pigment degradation and aroma precursor formation [40,41]. P. kudriavzevii and Saccharomyces cerevisiae [39], C. tropicalis [17], and Hanseniaspora opuntiae [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 [46,47,48], while pectinase reduces cocoa pulp viscosity, enhancing fermentation efficiency and releases trapped aromatic compounds [49,50,51]. 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 consistent 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, pectinase, and polyphenol oxidase, β-glucanase chitinase, xylanase, protease, β-glucosidase, polyphenol oxidase, and pectinase produced by yeast species involved in the cocoa fermentation 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 [69,70,71].

Volatile compounds profiles revealed the presence of various chemical families, including 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 stellimalicola, 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 [89,90,91], 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 [92,93,94,95].

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.