Annals of Microbiology (2019) 69:975–987 https://doi.org/10.1007/s13213-019-01488-1 ORIGINAL ARTICLE Filamentous fungi diversity in the natural fermentation of Amazonian cocoa beans and the microbial enzyme activities Jean Aquino de Araújo1 & Nelson Rosa Ferreira1 & Silvia Helena Marques da Silva2 & Guilherme Oliveira 3 & Ruan Campos Monteiro4 & Yamila Fernandes Mota Alves1 & Alessandra Santos Lopes1 Received: 18 September 2018 /Revised: 13 May 2019 /Accepted: 29 May 2019 /Published online: 20 June 2019 # Università degli studi di Milano 2019 Abstract Purpose The purpose of this study was to investigate the diversity of filamentous fungi and the hydrolytic potential of their enzymes for a future understanding of the influence of these factors on the sensory characteristics of the cocoa beans used to obtain chocolate. Methods Filamentous fungi were isolated from the natural cocoa fermentation boxes in the municipality of Tucuman, Pará, Brazil, and evaluated for the potential production of amylases, cellulases, pectinases, and xylanases. The fermentation was monitored by analyzing the pH and temperature. The strains were identified by sequencing the ITS1/ITS4 section of the 5.8S rDNA and partially sequencing the 18S and 28S regions, and the molecular identification was confirmed by phylogenetic reconstruction. Result The fungi isolated were comprised of three classes from the Ascomycota phylum and one class from the Basidiomycota phylum. There were found 19 different species, of this amount 16 had never been previously reported in cocoa fermentation. This fact characterizes the fermentation occurring in this municipality as having wide fungal diversity. Most of the strains isolated had the ability to secrete enzymes of interest. Cladosporium cladosporioides, Fomitopsis subtropical, Aspergillus versicolor, Penicillium pimiteouiense, Phanerochaete australis, Neonothopanus nambi,andAspergillus parasiticus were the strains that excelled in the secretion of the following enzymes: amylase, pectinase, cellulase, and xylanase. Conclusion The presence of 16 species not yet reported in cocoa seed fermentations and their potential hydrolytic activities show a diversity of filamentous fungi in this microbial biome that needs to be better understood. Keywords Amylase . Cellulase . Filamentous fungi . Pectinase . Theobroma cocoa . Xylanase Introduction Fermentation is one of the steps in cocoa processing. It occurs * Nelson Rosa Ferreira spontaneously and starts soon after harvesting and breaking of [email protected] the fruits, which is carried out by the cocoa cultivators to * Alessandra Santos Lopes develop the characteristic chocolate flavor (Nielsen et al. [email protected] 2007). In this step, the overlay of the seed pulp is degraded by the action of microorganisms (fungi and bacteria) and this 1 Laboratory of Biotechnological Processes. Program of process causes an elevation in temperature which can reach Post-Graduation in Food Science and Technology (PPGCTA), Federal University of Pará (UFPA), Belém, Pará 66075-110, Brazil values near 50 °C (Abdullahi et al. 2018). The microorganisms present in the cocoa fermentation pro- 2 Mycology Laboratory, Section of Bacteriology and Mycology, Evandro Chagas Institute (IEC/SVS/MS), BR 316, km 7, cess hydrolyze the sugars and organic acids of the pulp into Ananindeua, Pará 67.030-000, Brazil simple sugars, which are processed into ethanol, lactic acid, 3 Instituto Tecnológico Vale (ITV), Belém, Pará 66.055-090, Brazil and acetic acid (Schwan and Wheals, 2004). The acids gener- ated penetrate the seeds, and together with the increase in 4 Graduate Program of Biology of Infectious Agents and Parasitological, Institute of Biological Sciences, Federal University temperature, cause the death of the embryo, a desirable action of Pará (UFPA), Belém, Pará 66075-110, Brazil in the cocoa fermentation process (Macedo et al. 2013). 976 Ann Microbiol (2019) 69:975–987 Of the microorganisms present in the fermentation of co- required for the identification of microorganisms, such as coa, the filamentous fungi are highly important, since they DNA extraction, the polymerase chain reaction (PCR), aga- grow abundantly in habitats where polymeric compounds rose gel electrophoresis, and genetic sequencing (Justé et al. are present, such as those found in the cocoa pulp (pectin, 2008; Godet and Munaut 2010). starch, and cellulose). The fungi possess advantages over oth- It is important to note that the filamentous fungi are present er microorganisms due to their ability to assimilate a variety of during the cocoa fermentation stage regardless of the geo- substrates to produce enzymes (Chan et al. 2018). graphic location and are potential producers of extracellular The filamentous fungi are found during all the primary enzymes of biotechnological interest, but detailed work on stages of cocoa bean processing (fermentation, drying, and this class of microorganisms during cocoa fermentation has storage), but their activity during cocoa fermentation is not not yet been carried out. Thus, the present study aimed to yet fully known (Copetti et al. 2011a). It is therefore of utmost identify the species involved in the fermentation of cocoa in importance to identify and characterize the compounds gener- the State of Pará, Brazil, and evaluate their potential to pro- ated by the strains, especially the enzymes that act during duce hydrolytic enzymes of interest both in cocoa fermenta- cocoa fermentation. tion and for other biotechnological applications. A study carried out in Cameroon reported the occurrence of filamentous fungi during the cocoa fermentation process and identified Aspergillus and Penicillium species Material and methods (Mounjouenpou et al. 2012). A study carried out in Brazil on the presence of filamentous fungi during cocoa fermenta- Cocoa seed collection tion identified the species Absidia corymbifera, Aspergillus candidus, A. carbonarius, A. flavus, A. fumigatus, A. niger, The cocoa seeds were collected in the first week of July in A. parasiticus, A. sydowii, A. versicolor, Eurotium Tucuman, a municipality in the Brazilian Amazon, located in amstelodami, Geotrichum candidum, Monascus ruber, thesouthernregionoftheStateofPará(latitude:06°51′ 44″S Mucor sp., Paecilomyces variotii, Penicillium paneum, and longitude: 51° 09′ 40″ W). The seeds were maintained in Rhizopus,andSyncephalastrum sp. (Copetti et al. 2011a). three wooden trough fermenters (A1, A2, and A3), each con- The production of extracellular enzymes by filamentous fungi taining approximately 500 kg of seeds, and were covered by is diverse, but some are more technologically relevant, such as banana leaves for 7 days of natural fermentation under the amylase, cellulase, xylanase, and the pectinases, since these are conditions laid down by local cocoa farmers. Samples were used to hydrolyze complex polysaccharides into simple sugars. taken at zero time and at 24-h intervals from different points in These enzymes are useful in food processing, breweries, and the the trough fermenters, for analysis (pH) and the isolation of biofuel industries (Reddy and Sreeramulu 2012). It is therefore microorganisms. Samples (200 g per day for 8 sampling pe- important to search for new sources of extracellular enzymes in riods) were taken from seeds located at the surface, in the their natural habitats, which have not yet been reported and middle, and at the bottom of the troughs, giving a total weight which might constitute a real technological potential with desir- of 4.8 kg. The samples were placed in polyethylene bags, but able characteristics (high activity, high temperature stability, and one part (50%) of each sample was immediately placed at − wide pH ranges) (Damaso et al. 2012). 18 °C and subsequently maintained at this temperature, while Different methods can be used to select microorganisms by the other part was stored at a temperature of 4 °C for the determining the presence of extracellular enzymes. These microbiological analyses. methods include the use of solid culture medium containing substrate inductors which allow for visualization of the hydro- lysis reaction (Larone, 2002). This technique is based on the Monitoring of the fermentation process use of a solid medium consisting of agar, mineral salts, and the substrate inductor (a single carbon source available in the The temperatures and pH values were determined after 0, 24, environment), and coloring agents that visualize the hydroly- 48, 72, 96, 120, 144, and 168 h of fermentation. The temper- sis reaction. These coloring agents bind to systems between ature was measured in situ by immersing the electrode directly the polymeric molecule structures, such that when the mole- into the pulp at random points on the surface, middle, and cules are hydrolyzed, a halo of hydrolysis is formed. bottom of the fermenter troughs using a digital thermometer Taxonomic classification methodologies by way of molec- (Instruthemp ®, Mod. HT-600). ular biology are increasingly common and necessary as The hydrogen potential (pH) was measured in the labora- against classical taxonomy, since they use specific molecular tory using a digital pH meter (Bel ®, Mod. W3B) verifying markers, which, in the case of fungi, is the 18S rDNA gene the pH of samples corresponding to the same fermentation (ribosomal DNA) (Cocolin and Ercolini, 2008; Watanabe, periods cited above according to the AOAC 970.21 method 2010). These methodologies incorporate various techniques (APHA 2001). All samples were analyzed in triplicate.