# Effect of Spoilage by Lipolytic Fungi Strains on Free Fatty Acids (FFA) Formation in Fermented and Dry Cocoa Beans

Joëlle Stéphanie Tape, Kouakou Richard Houphouët, Koumba Maï Koné, Pauline Mounjouenpou, Benjamin Guibert, Isabelle Piro-Metayer, Didier Montet, Tagro Simplice Guéhi

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## ► To cite this version:

Joëlle Stéphanie Tape, Kouakou Richard Houphouët, Koumba Maï Koné, Pauline Mounjouenpou, Benjamin Guibert, et al.. Effect of Spoilage by Lipolytic Fungi Strains on Free Fatty Acids (FFA) Formation in Fermented and Dry Cocoa Beans. Microbiology Research Journal International, 2023, 33 (4), pp.22-35. 10.9734/mrji/2023/v33i41376. hal-05162282

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# Effect of Spoilage by Lipolytic Fungi Strains on Free Fatty Acids (FFA) Formation in Fermented and Dry Cocoa Beans

Tapé Joëlle Stéphanie<sup>a</sup>, Houphouët Kouakou Richard<sup>a</sup>, Koné Koumba Mai<sup>b</sup>, Mounjouenpou Pauline<sup>c</sup>, Guibert Benjamin<sup>d,e</sup>, Métayer-Piro Isabelle<sup>d,e</sup>, Montet Didier<sup>d,e</sup> and Guéhi Tagro Simplice<sup>a*</sup>

## Authors' contributions

This work was carried out in collaboration among all authors. Author TJS has made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data. Author HKR has contributed to conception and design, or acquisition of data, or analysis and interpretation of data. Author KKM carried out the conceptualization and developed the methodology. Author MP wrote and reviewed the original manuscript. Author MPI has contributed to the isolation and the polyphasic characterization of molds strains. Author GB has carried out and supervised molecular identification of molds strains. Author MD has contributed to the correction of this manuscript as the co-supervisor of the Author TJS. Author GTS as the project coordinator, has reviewed, edited and given final approval of the version to be published. All authors read and approved the final manuscript.

## Article Information

DOI: 10.9734/MRJI/2023/v33i41376

### Open Peer Review History:

This journal follows the Advanced Open Peer Review policy. Identity of the Reviewers, Editor(s) and additional Reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/101630

<footnote><sup>a</sup> Laboratoire de Biotechnologie et Microbiologie des Aliments, UFR Sciences et Technologies des Aliments, Université Nangui Abrogoua, 02 BP 801 Abidjan 02, Côte d'Ivoire.</footnote>

<footnote><sup>b</sup> Institut National Polytechnique Félix Houphouët-Boigny, Bp 1093, Yamoussoukro, Côte d'Ivoire.</footnote>

<footnote><sup>c</sup> Laboratoire de Technologie Alimentaire, IRAD. BP 2067 Yaoundé, Cameroon.</footnote>

<footnote><sup>d</sup> CIRAD, UMR Qualisud, TA B 96/16, 75 Av JF Breton, 34398 Montpellier cedex 5, France.</footnote>

<footnote><sup>e</sup> Qualisud, Univ. Montpellier, Avignon, CIRAD, Institut Agro, La Réunion University, Montpellier, France.</footnote>
## ABSTRACT

**Aim:** High free fatty acids (FFA) content of cocoa beans is high due to alter quality of butter fraction and chocolate manufactured. During primary post-harvest processing, cocoa beans harbor lipase producing mold species could trigger the accumulation of FFA. This study investigated the improvement of the quality of raw cocoa material sourced from Côte d'Ivoire regarding its high FFA concentration.

**Methods:** Fermented and dry cocoa were sampled on-farm level in some main cocoa producing regions of Côte d'Ivoire. Contaminating mold strains were isolated using Sabouraud chloramphenicol agar incubated at 25°C for 7 days. Morphological identification of mold isolates was performed from younger fungal culture. Biochemical study about lipolytic properties of mold isolates was carried out using Cazpeck-Dox Agar medium enriched with olive oil as carbon source and rhodamine B as indicator of lipase activity. Extraction, PCR amplification using a specific transcribed spacer primer (ITS4/ITS5) and sequencing of DNA were performed for fungal species molecular identification.

**Results:** Approximately 154 mold isolates belonging to 8 genera were found in tested cocoa beans. Only *Rhizopus oryzae*, *Absidia corymbifera*, *Mucor heimalis*, *Aspergillus niger*, *A. tubingensis*, *A. aculeatus*, *A. flavus*, *A. tamari*, *A. fumigatus*, *Nigrospora sphaerica*, *Curvularia geniculate* and *Penicillium chrysogenum* species produced greater lipolytic properties. *A. corymbifera*, *A. niger*, *A. fumigatus* and *R. oryzae* were the most frequent and predominant species. Cocoa beans inoculated with *R. oryzae*, *A. corymbifera* or *A. niger* recorded higher FFA content above 1.75%.

**Conclusion:** Reducing of the mold species growth during cocoa primary post-harvest processing could sustainably produce raw cocoa material with less FFA.

**Keywords:** Cocoa beans; lipolytic mold species, free fatty acids; primary postharvest processing; Côte d'Ivoire.

## 1. INTRODUCTION

Cocoa (*Theobroma cacao* L.) is an important food source in many homes due to the frequent consumption of its processed/finished forms such as biscuits, chocolate, chocolate drinks, cocoa powder and sweets [1]. Primary industrial processing of cocoa beans leads to cocoa butter (CB) as the main by-product from cocoa beans beside cocoa liquor and powder [2]. The components of cocoa butter are constituted of 97% of triacylglycerols, di- and mono-glycerides, phospholipids and glycolipids and unsaponifiable matter in the low proportion [3]. According to Zzaman et al. [4], cocoa butter is reported to be the continuous phase that the variable and interesting rheological, physical and chemical characteristics could be affected during both primary post-harvest and industrial processing. Its hardness including crystallization and melting properties depends on the content of saturated and unsaturated fatty acids bound to triglycerides, and on the of free fatty acids (FFA) content. FFA are carboxylic acids with a long carbon chain that are naturally present in small quantities in cocoa butter but released from

triacylglycerides or di- and mono-glycerides under activity of lipase or by oxidation reactions, and accumulated in high concentration inside various agricultural commodities including cocoa beans [5]. However, oxidation risks are negligible in cocoa butter due to its low unsaturated fatty acid content [6]. The Directive 2000/36/EC of European Union [7] set the legal maximum limit for FFA content of cocoa butter at 1.75%. FFA content above this limit may be considered as a factor reducing the commercial value of cocoa beans [8] and altering the technological properties of cocoa butter fraction and chocolate manufacture [9]. In addition, according to Briones and Aguilera [10], FFA contents exceed international standard of 1.75% causes financial losses for both cocoa producers and chocolate manufacture industry. Côte d'Ivoire is the first world producer of cocoa beans with up 2.2 million tons in 2020-2021 [11]. Unfortunately, primary post-harvest processing of cocoa beans vary considerably from a producing region to another cocoa producing region [12] in this country. Consequently, Ivorian cocoa beans are currently subject to various quality defects including high FFA content for several years [6].
The resurgence of the problem of free fatty acid content in Ivorian cocoa since 2010-2011 has become alarming that it is urgent to find a sustainable solution while European Commission is considering reducing FFA content of cocoa and by-products to 1%. In Côte d'Ivoire, various abiotic and biotic factors could trigger the increase of FFA content inside fermented and dried cocoa beans. In this country like all tropical and humid regions, climatic and agronomic conditions are conducive to mold growth and food quality deterioration [11]. In addition, many research activities highlighted the effect of inappropriate primary post-harvest processing of cocoa mediated by a dynamic of biochemical reactions catalyzed by a microbial succession contaminated spontaneously cocoa bean mucilaginous pulp including the considerable growth of specific filamentous fungi [13]. Chaves-López et al. [14] have concluded that cocoa fermentation and drying are two major and essential steps of primary post-harvest processing that depended on various factors such as genotype of cocoa plant, microclimate, local practices of fermentation process, level of maturity and sanitary status of pods, mass of cocoa beans submitted to fermentation, etc. Management of these operations is crucial for the quality of the final product before export [15]. During these post-harvest operations, filamentous fungi can also develop especially if cocoa beans are from damaged pods [16] or if fermentation process is performed without regular turning [17]. In addition, storage of fermented and dried cocoa under high relative humidity values promotes growth of molds [18]. Some of these molds could produce lipase enzyme [19], which could hydrolyzed triacylglycerides of cocoa butter and increase FFA content inside cocoa beans [8]. To date, no

complete and comprehensive research integrating both the identification of lipolytic molds and the real role of fungal microflora contaminated fermented and dried cocoa beans by producing lipase activity for promotion of the FFA formation was carried out. In the previous studies, seven lipolytic mold species are investigated in cocoa beans from Côte d'Ivoire [8]. This study aimed to improve the quality of fermented and dried cocoa beans regarding the FFA content by the identification of lipolytic molds frequently contaminated this agricultural commodity and could promoted the FFA formation.

## 2. MATERIALS AND METHODS

### 2.1. Materials

Fifty fermented and dried cocoa beans samples were randomly collected during October 2018 from Akoupé (6° 23' N, 3° 54' W), Aniasué (6°38'9" N, 3°40'52" W) and Yakassé-Attobrou (6°10'51" N, 3°39'26" W), big cocoa producing regions located in Southeast of Côte d'Ivoire (Fig. 1). Among these dry cocoa beans samples, twenty samples weighing 2000g each were collected at farm's level from twenty diiferent cocoa's farmer and 30 samples weighing 3000g each were collected from controlled primary post-harvest processing. The controlled primary post-harvest processing consisted in application of 3-days pod opening delay, 7-days fermentation time in wooden boxes (45×40×35 cm³) with 2 turnings at 48 and 96 hours and sun-dried on rack for 7-8% moisture on fresh cocoa beans . Cocoa beans of each sample were separately packaged in a sterile biodegradable bag for transportation to the laboratory until analyses.

<figure><img src="image_4.png" /><figcaption>Fig. 1. Geographical location map of cocoa producing regions (Akoupé, Aniassué, Yakassé-Attpbrou) in Cote d'Ivoire</figcaption></figure>
## 2.2. Methods

### 2.2.1 Isolation of molds from cocoa beans

In order to isolate the molds present in the cocoa beans, five fermented and dried cocoa beans from each sample were disinfected by soaking in sodium hypochlorite solution (0.4%) for 2 min [20]. Then, they were aseptically cultured by direct contact technique on the surface of Sabouraud chloramphenicol agar medium in Petri dishes [21]. Cultures were incubated at 25°C for 3-5 days. Molds' isolates were purified by successive sub-culturing onto other Petri dishes containing the same medium.

## 2.3 Identification of Molds' Isolates

### 2.3.1 Polyphasic characterization

The mold isolates from the cocoa bean samples were characterized by cultural and morphological characterization. For morphological identification, molds grown on Sabouraud chloramphenicol medium were checked for macroscopic and microscopic characters as described in appropriate keys [20]. For this purpose, fragment of the colony of each mold isolate was carefully taken from the 7-days old culture and placed fresh state in drop of sterile water between slide and coverslip. Macroscopic characteristics related to shape, appearance, colonies growth rate, production soluble pigments, hyphae colour, spores colour and shape of the colony of each mold isolate were determined by naked eye observation of its cultures on chloramphenicol Sabouraud medium as previously described [22]. Microscopic characteristics related to hyphae structure, spore-forming organs, vesicle shape, presence or absence metula, shape and mode conidia aggregation were determined [23]. A selection of phenotypically similar isolates were subjected to further identification by a molecular approach [1].

### 2.3.2 Molecular identification

*   **DNA extraction and PCR reaction**

For the extraction and purification of genomic DNA of mold, a portion from the 7-day old culture of each isolate grown on Sabouraud chloramphenicol was used [1]. Total genomic DNA was extracted from this portion and sequenced after the amplification of parts of the ITS, β-tubulin and/or calmodulin genes as to modified previous protocol described [24,20].

Genomic DNA quality was checked using electrophoresis and quantification using an ND-1000 spectrophotometer (Thermo Fisher, Waltham, MA, USA). Polymerase Chain Reaction (PCR) were carried out with pairs of primers ITS4-ITS5 [25] targeting the ITS (Internal Transcription Spacer) region of ribosomal DNA. The PCR reactions were carried out in 50 µL of reaction mixes containing 25 µL of PCR master mix X2 (biotechrabbit™) comprising DNA polymerase (0.625 U per sample), Tris/HCl (30 mM, pH 8.05), KCl (100 mM), each dNTP (400 µM) and MgCl₂ (5 mM), 1 µL forward (0.2 µM) and reverse (0.2 µM) primers, 18 µL molecular biology water (Molecular Biology Grade Water, Corning) and 5 µL of 1:10 diluted DNA from different fungal strains. The PCR was carried out with a T100TM thermocycler (Bio-RAD) according to program comprising 10 min of initial denaturation at 94°C followed by 35 cycles consisting of 30 sec of denaturation at 94°C, 30 sec of hybridization at 56° C and 1 min elongation at 72°C followed by final elongation at 72°C for 7 min [22].

*   **PCR products sequencing**

The PCR products were sequenced in forward and reverse direction on a Sanger sequencer (Applied biosystems). Obtained sequences were processed using UGene Unipro version 38.1 software via visualization and cleaning of chromatograms and assembly of direct and indirect sequences to obtain a consensus sequence. For the confirmation of species identity, sequences obtained in this study were compared with sequences in the NCBI database and the internal database of the Westerdijk Fungal Biodiversity Institute (WI) [26].

### 2.3.3 Screening of lipolytic mold isolates

Specific medium contained 10 g of peptone, 3g of yeast extract and 5g of NaCl and 26 g of bacto agar dissolved in 890 mL of distilled water was prepared. Olive oil emulsion (0.1 g. mL-1) prepared as only source of carbon in sterile 2% polyvinyl alcohol (PVA) solution using ultra turax and 10 mL of rhodamine B solution (1 mg.mL⁻¹) filtered using a cellulose membrane syringe 0.22 µm were added to sterilized medium (121°C, 15 min) supercooling. Mixture was carefully homogenized and then poured into Petri dishes. Each mold isolate was cultured on this medium enriched in lipids, as organic nutrients, and incubated at 25°C for 48 to 96 hours. In event of fungal lipase production, there is a
complexation of rhodamine B with fatty acids from olive oil. Final complex is materialized by appearance of orange fluorescent halo around of fungal colony once exposed to UV light at 365 nm [8].

## 2.4 Effect of lipolytic mold inoculation to cocoa beans on FFA formation.

* **Preparation of fungal conidia suspension**

Fungal conidia were collected from each lipolytic mold strain grown on chloramphenicol Sabouraud by scraping in sterile water solution. After counting conidia with Thomas slide, final suspensions were calibrated to 10⁵ conidia.mL⁻¹ for inocula preparation [22].

* **Inoculation of fungal conidia to beans inside cocoa pods**

Cocoa beans were inoculated by the injection of 1 mL of 10⁵ conidia.mL⁻¹ inside healthy pods, as previously described by Kedjebo et al. [22]. Negative pod control was treated with sterile distilled water. The inoculated pods were incubated in climate chamber at 25°C, 80% relative humidity for 7 days.

## 2.5 Determination of FFA contents of cocoa beans

Cocoa beans were dried in oven at 40°C for 5 days to 7-8% moisture. Dried cocoa beans were frozen in liquid nitrogen to be ground (< 500 µm). Ten grams of cocoa powder were placed in Whatman cartridge and then soaked in beaker containing 65 mL of petroleum [22]. The cocoa butter was extracted using Soxtec apparatus at 130°C for 4 h. Approximately 5 g of cocoa butter without solvent was weighed (W) and dissolved in 50 mL of mixture of petroleum ether/absolute ethanol (50:50, v/v) containing phenolphthalein. FFA content of cocoa butter was quantified by ISO 660:2020 method using 0.1 N KOH alcohol solution (V). FFA (% oleic acid) percentage was calculated as previsouly described by Asimah and Sebastian [27]:

$$FFA (\% oleic acid) = \frac{28.2 \times V \times N}{w}$$

## 2.6 Statistical Analysis

Data analysis was performed using SPSS for windows v.17.0 (SPSS® Inc., IL, USA). Means for the FFA content measurements data were

calculated and tested for significance by the Students t-test at p = 0.05.

# 3. RESULTS

## 3.1 Cultural and Morphological Identification of the Genera of Mold Isolates

One hundred fifty four (154) molds were isolated from all fermented and dried cocoa beans samples whatever their post-harvest processing. The examination of these molds showed various characteristics related to the colony and to some specific vegetative organs. Morphological characteristics of vegetative colonies and conidia of representative mold isolates from fermented and dry cocoa beans from Côte d'Ivoire were indicated (Fig. 2). Based on these keys of both macroscopic and microscopic characteristics 13 mold species belonging to 7 genera including Rhizopus Absidia and Mucor Aspergillus, Penicillium, Cuvularia were Nigrospora and Botrytis identified. Main cultural characteristics of all colonies on Sabouraud-chloramphenicol medium showed that they were extensive with fast or moderate growth speed. Microscopic observations revealed the presence of branched or unbranched hyphes as sporangiophores that constitue mycelia. Aspergillus isolates were rouping into 3 sections. Molds' isolates were belonging to the Nigri section presented white or yellow mycelium, black or brown conidia that appeared grainy appearance. Another isolates were grouping into the Fumigati section showed white mycelium with blue conidia. The last mold strains were classifying into Flavi section and were characterized by white or yellow mycelium and green spores. Microscopic examiations showed that molds of Aspergillus genus were characterized by their uniseriate or biseriate conidial head as a smooth hyaline conidiophore which bear spherical or hemispherical vesicle bearing smooth or echinulate globose spores at their end. Mold strain belonging to Penicillium genus was distinguished by blue or often green colonies with moderate or slow growth speed. Table 1 recaps the description of some main cultural and morphological characteristics of the colonies of the mold strains found in tested fermented and dry cocoa beans samples.

## 3.2 Lipolytic Mold Isolates from Cocoa Beans

Table 2 shows the ability of mold strain for production of lipases enzyme. Twelve mold
strains belonging to of 9 genera have produced
lipolytic activity: 3 strains of Mucorales order, 6
*Aspergilli*, *Nigrospora*, *Curvularia*, and
*Penicilium*. Members of Mucorales order

appeared to be greatest lipolytic followed
by the members of *Aspergillus* while
*Botrytis* sp. was not able to produce extracellular
lipase.

<figure><img src="image_5.png" /><figcaption>Fig. 2. Morphological characteristics colonies and conidia of mold isolates from fermented and dry cocoa beans produced in Côte d'Ivoire on Sabouraud-chloramphenicol agar (culture incubation 25°C for 5 days)</figcaption></figure>
<table><caption>Table 1. Description of some main morphological characteristics of some mold isolates from fermented and dry cocoa beans sampled from Côte d'Ivoire</caption><thead><tr><th rowspan="2">Genera</th><th colspan="5">Macroscopic characteristics</th><th colspan="3">Microscopic characteristics</th></tr><tr><th>Growth speed</th><th>Aspect</th><th>Hyphae colours</th><th>Conidia colour</th><th>Reverse</th><th>Hyphae structure</th><th>Conidia-forming organs</th><th>Conidia morphology</th></tr></thead><tbody><tr><td>Rhyzopus</td><td>Fast</td><td>Cottony</td><td>White and grey</td><td>Black</td><td>Yellow</td><td>Unpartitioned / unbranched</td><td>Sporange</td><td>Spherical</td></tr><tr><td>Absidia</td><td>Fast</td><td>Cottony</td><td>Grey</td><td>White</td><td>Colourless</td><td>Unpartitioned / and branched</td><td>Sporange</td><td>Spherical</td></tr><tr><td>Mucor</td><td>Fast</td><td>Cottony</td><td>Whitish</td><td>White</td><td>Colourless</td><td>Partitioned / branched</td><td>Sporange</td><td>Spherical</td></tr><tr><td>Nigrospora</td><td>Slow</td><td>Woolly</td><td>Dew</td><td>White</td><td>Orange</td><td>Partitioned / branched</td><td>-</td><td>Ellipsoidal</td></tr><tr><td>Botrytis</td><td>Fast</td><td>Furry</td><td>Blackish grey</td><td>Black</td><td>Brown</td><td>Partitioned / branched</td><td>-</td><td>Ovoid</td></tr><tr><td>Cuvularia</td><td>Slow</td><td>Compact</td><td>Grey</td><td>White</td><td>Black</td><td>Unpartitioned</td><td>-</td><td>Ellipsoidal</td></tr><tr><td>Aspergillus (section Flavi)</td><td>Moderate</td><td>Granular</td><td>Beige</td><td>Green and brown</td><td>Yellow</td><td>Partitioned / unpartitioned</td><td>Vesicle with phialides</td><td>Spherical</td></tr><tr><td>Aspergillus (section Nigri)</td><td>Moderate</td><td>Granular</td><td>Beige</td><td>Brown</td><td>Yellow</td><td>Partitioned / unpartitioned</td><td>Vesicle with metules and phialides</td><td>Spherical</td></tr><tr><td>Aspergillus (section Fumigati)</td><td>Moderate</td><td>Powdery</td><td>White</td><td>Blue</td><td>Yellow</td><td>Unpartitioned</td><td>Hemispheric vesicle</td><td>Spherical</td></tr><tr><td>Penicillium</td><td>Very slow</td><td>Powdery</td><td>White</td><td>Blue-green</td><td>Yellow</td><td>Partitioned</td><td>Brush shape whorled</td><td>Spherical</td></tr></tbody></table>
<table><caption>**Table 2. Highlighting of lipolytic properties of some mold strains isolated from fermented and dry cocoa beans sampled in Côte d'Ivoire**</caption><thead><tr><th>Genus/order of mold isolates</th><th>Lipolytic enzyme production</th><th>Estimated intensity of lipolytic activity level</th></tr></thead><tbody><tr><td>Mucorales</td><td>yes</td><td>++++</td></tr><tr><td>Aspergillus</td><td>yes</td><td>+++</td></tr><tr><td>Nigrospora</td><td>yes</td><td>++</td></tr><tr><td>Curvularia</td><td>yes</td><td>+</td></tr><tr><td>Penicillium</td><td>yes</td><td>+</td></tr><tr><td>Botrytis</td><td>not</td><td>-</td></tr></tbody></table>

<table><caption>**Table 3. Results of molecular identification of lipase producing mold isolates from fermented and dry raw cocoa material collected in Côte d'Ivoire**</caption><thead><tr><th>Code of molds' isolates</th><th>Nc</th><th>% of homology</th><th>Matching species</th></tr></thead><tbody><tr><td>I</td><td>565</td><td>100</td><td>Rhyzopus oryzae</td></tr><tr><td>II</td><td>561</td><td>100</td><td>Absidia Corymbifera</td></tr><tr><td>III</td><td>551</td><td>100</td><td>Mucor hiemalis</td></tr><tr><td>IV</td><td>485</td><td>100</td><td>Aspergillus flavus</td></tr><tr><td>V</td><td>492</td><td>99.9</td><td>A. tamarii</td></tr><tr><td>VI</td><td>506</td><td>100</td><td>A. niger</td></tr><tr><td>VII</td><td>504</td><td>100</td><td>A. s aculeatus</td></tr><tr><td>VIII</td><td>504</td><td>99.8</td><td>A. tubingensis</td></tr><tr><td>IX</td><td>503</td><td>100</td><td>A. fumigatus</td></tr><tr><td>X</td><td>406</td><td>100</td><td>Nigrospora sphaerica</td></tr><tr><td>XI</td><td>517</td><td>99.9</td><td>Cuvularia geniculata</td></tr><tr><td>XII</td><td>521</td><td>100</td><td>Penicillium chrysogenum</td></tr></tbody></table>

*number of nucleotides compared
**percentage of sequence homology*

<table><caption>**Table 4. Frequency and dominance of lipase producing mold isolates from faw cocoa material sourced from Côte d'Ivoire**</caption><thead><tr><th>Lipolytic mold isolates</th><th>Frequency (%)</th><th>Dominance (%)</th></tr></thead><tbody><tr><td>R. oryzae</td><td>24</td><td>9.38</td></tr><tr><td>A. corymbifera</td><td>50</td><td>19.53</td></tr><tr><td>M. heimalis</td><td>10</td><td>3.91</td></tr><tr><td>N. sphaerica</td><td>12</td><td>4.69</td></tr><tr><td>C. geniculata</td><td>8</td><td>3.13</td></tr><tr><td>Aspergillus flavus</td><td>14</td><td>5.47</td></tr><tr><td>A. tamari</td><td>10</td><td>3.91</td></tr><tr><td>A. niger</td><td>46</td><td>17.97</td></tr><tr><td>A. aculeatus</td><td>20</td><td>7.81</td></tr><tr><td>A. tubingensis</td><td>14</td><td>5.47</td></tr><tr><td>A. fumigatus</td><td>34</td><td>13.28</td></tr><tr><td>P. chrysogenum</td><td>12</td><td>4.69</td></tr></tbody></table>

### 3.3 Identification of Mold Species

Only mold isolates exhibited ability for production of lipase activity were identified by use molecular tools. Table 3 indicates 12 mold species identified including *Rhizopus oryzae*, *A. corymbifera*, *Mucor hiemalis*, *Aspergillus flavus*, *A. tamari*, *A. niger*, *A. aculeatus*, *A. tubingensis*, *A. fumigatus*, *Nigrospora sphaerica*, *Cuvularia geniculata*, *Penicillium chrysogenum* with

significant alignments of 99 to 100% with the isolated fungal species. Table 4 shows the incidence of each strain among these 12 lipolytic molds isolates: *Absidia sp.* was the most predominantly and frequently strain detected in cocoa beans samples, followed by *Aspergilli* and *Rhizopus* species whereas the species of *Curvularia*, *Penicillium* and *Nigrospora* were the least frequent and abundant molds isolates found in our cocoa beans samples.
<figure><img src="image_6.png" /><figcaption>Fig. 3. Overall influence of inoculation of lipolytic mold species conidia to fresh beans inside cocoa pods on the FFA occurrence during incubation at ambient temperature for 7 days</figcaption></figure>

**3.4 FFA Content of Inoculated Cocoa Beans with Lipolytic Mold Isolates**

Fig. 3 illustrates the changes in FFA content of cocoa beans samples inoculated with lipolytic molds according each isolate incubated at 25 °C for 7-days. Cocoa beans inoculated with *R. oryzae* recorded the highest FFA content of 2.05%. FFA content found in those inoculated with *A. corymbifera* showed FFA content of 1.57% while cocoa beans inoculated with *A. niger* exhibited FFA level of 1.41%. Inoculation of cocoa beans with *A. fumigatus* led to FFA content of 1.27%. The inoculation of *A. flavus* or *A. tubingensis* to cocoa beans did not promote the FFA formation due to the lowest levels of FFA content below 1%.

**4. DISCUSSION**

A total of 154 fungal isolates spanning 6 genera were recovered from the 50 samples collected during the cocoa processing and the sampling of peasant cocoa beans. These genera are reported to be described elsewhere. Indeed, the diversity of molds detected in this research were in agreement with those found by previous studies related to the mycobiota contaminated fermented cocoa beans samples and other

agricultural commodities [28,29,30], except for three genera such as Nigrospora, Curvularia and Botrytiswhich. Among these isolates, 12 lipolytic species spanning 5 genera were identified : *R. oryzae*, *A. corymbifera*, *M. heimalis*, *A. niger*, *A. tubingensis*, *A. aculeatus*; *A. fumigatus*, *A. flavus*, *A. tamari*, *P. chrysogenum*, *C. geniculata* and *N. sphaerica*. Our results are in agreement with those found previously by Serra et al. [31]. Regarding to the incidence these isolates could be classified in to 2 groups: one of the isolates with high incidence and the second group was constituted by those with low incidence. The incidence of the species were *Mucorales* such *Absidia* and *Rhizopus* (50% <) followed by the members of *Aspergilli*. The second group was constituted of members of *Curvularia*, *Nigrosporia* and *Penicilium* genera that hace a least incidence. In both fermented and dried cocoa, more than one half of the molds isolates were from peasant cocoa beans while controlled fresh fermented and dried cocoa beans are less contaminated by fungi. Mold species of Mucorales order were predominant than other mold species as previously shown [8]. However, our results disagreed with those previously found in other cocoa producing countries such as Sierra Leone, Equatorial Guinea and Ecuador where the predominance of molds of *Aspergillus*
genera was reported [32]. The absence of A. ochraceus or other OTAproducing species from the tested fermented and dried cocoa beans samples and aflatoxin-producing species from dried agricultural commobities was in agreement with the results founded [1,28] However, these results differed from many other previous studies [30,32]. These disparities in the occurrence of mold may be ascribed to various abiotic factors such as primary post-harvest processing, moisture level which was currently above the recommended values (7-8%) for the peasant cocoa beans samples [6], local microclimat in each cocoa producing region during the sampling of fermented and dried cocoa beans. The influence of biotic factors including interactions between filamentous fungi strains due to the resilience of certain mold species growing in minimal conditions [33] and to the competitiveness for the space [34,35], for the access to the nutrients such as lipid matrix of cocoa beans [8] play essential role in the presence of certain strains. The filamentous fungi species isolated in present work sourced from environment (air, soil, shelling tools and fermentation) and could contaminate cocoa beans at different steps of primary post-harvest processing [11,36]. The contamination of fermented and dry cocoa beans by molds, as it reduces the quality and may produce mycotoxins which consumption that lead poisoning and death of consumers of cocoa end-products such as chocolate [37]. Among mold isolates found in tested fermented and dry cocoa beans samples, some fungal species such as Aspergillus niger contamination could produce mycotoxins, such as ochratoxin A [38,39]. The presence of A. niger could increased the microbita of the fungal strains and the OTA contents of fermented and dry cocoa beans [37]. Fortunately, use of some yeasts, lactic and acetic bacteria strains as starter culture could inhibit both the mycelial growth of fungu species and OTA production during cocoa fermentation process [37,40]. Otherwiew, according to Copetti et al. [41], the accumulation OTA in fermented and dry cocoa beans could also be suppressed by the increasing of organic acids contents during fermentation. And the finale dry cocoa beans produced from such as batches are of a good quality and are safe [37]. The results of the tests related tot the ability of the molds isolates from cocoa beans highlighted the extracellular lipase production by all species belonging to all genera found except Botrytis. Among these molds, all species belonging to Mucorales order such Rhizopus, Absidia and Mucor genera exhibited

the strongest abilties for lipase production as previously described by various studies [42,43]. Our results related to the capacity of various mold species belonged to Aspergillus for the extracellular lipase production are in agreement with those previously founded [44]. A. niger [45] and A. flavus [46] were reported to be greater producer of the extracellular lipase. The accumulation and the increase of FFA content inside inoculated cocoa beans are due to the production of extracellular lipase by these mold isolates. Most excessive FFA concentration of inoculated fermented and dry cocoa beans with Rhizopus oryzae, Absidia corombifera, Aspergillus tubingensis or A. fumigatus confirm that these molds could strongly produce lipase as previously demonstrated [8]. So the presence of these fungi species among microbiota contaminated cocoa beans considerably promote the FFA's occurrence. The combination of high mold contamination level favoured by fungal diseases [19], inadequate primary post-harvest processing such long storage time of the pods prior opening [2] and sanitary condition of beans [6] could promote the formation of FFA and cause excessive FFA content in cocoa beans. In addition, some research have concluded that the duration of cocoa beans fermentation could be considered to critically promote the FFA formation due to both the growth and activity of molds [11,47].

## 5. CONCLUSION

The study investigated the improvement of cocoa material quality regarding the identification of lipase producing molds and the FFA content. We found 13 mold species of 8 genera such as Rhizopus, Absidia, Mucor, Nigrospora, Curvularia, Aspergillus, Penicillium and Botrytis. Mucorales order and Aspergillus species from cocoa beans are more frequent and dominant than other mold species in cocoa mycoflora. Mold species such as A. corymbifera, R. oryzae, A. niger and A. tubingensis were greater lipase producer. Inoculation of R. oryzae, A. corymbifera or A. niger to cocoa beans promotes highest FFA contents. This study clearly indicated that reducing of lipolytic mold species growth in cocoa beans could decrease FFA content and sustainably improve the quality of cocoa beans sourced from Côte d'Ivoire.

## AKNOWLEDGEMENTS

This project is funded by the Education and Research Ministry of Côte d'Ivoire, as the part of
the Debt Reduction-Development Contracts managed by IRD. The research activities were supported by both the "Research Team on Cocoa Post-harvest Technology and Quality from beans to chocolate bar" of Université Nangui ABROGOUA, Côte d'Ivoire and the UMR Qualisud of Cirad, France. The research activities of TAPE Joelle Stéphanie are totally supported by a grant for PhD Student (N° 2018 / BE_D / C2D_AMRUGE-CI) from Debt Reduction-Development Contracts awarded by "Appui à la Modernisation et à la Réforme des Universités et Grandes Ecoles de Côte d'Ivoire (AMRUGE-CI)." The authors thank also a lot all cocoa farmers family living at Akoupé, and Yakkasé-Attobrou locations, cocoa producing regions located in the Southeast of Côte d'Ivoire for providing the cocoa pods and for their agreement to carry out the cocoa primary post-harvest processing on their farms.

COMPETING INTERESTS

Authors have declared that no competing interests exist.

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