The Microbiology of Cocoa Fermentation

THE MICROBIOLOGY OF COCOA FERMENTATION

13

Ionela Sarbu, Ortansa Csutak Faculty of Biology, Department of Genetics, University of Bucharest, Bucharest, Romania

13.1 Introduction Cocoa has a long history being related with the evolution of the human society and economy. The Latin name of the genus, Theobroma, meaning the “food of the goods,” reveals the importance given along the history, to the cocoa and to its most known end product, the chocolate. The cocoa was widely cultivated in the pre-Columbian Mesoamerica, its original Olmec name, kakaw (cacao), diffused around 2000 years ago into lower Central America languages, suffering various modifications of the phonologies (Kaufman and Justeson, 2007). The drink prepared from cocoa beans, the chocolatl, introduced in Europe by the Spanish, spread first in France, Italy, and Holland then in England and Austria. According to the World Cocoa Foundation (WCF), a nonprofit international organization including cocoa and chocolate manufacturers, processors, and other companies, the United States (Brazil and Ecuador) accounts for 13% of the global production, Africa (Cameroon, Ivory Coast, Ghana, and Nigeria) assures 73% of the global cocoa production, and Southeast Asia (Indonesia, Malaysia, and Papua New Guinea) the rest of 14% (http://www.worldcocoafoundation.org/). The International Cocoa Organization (ICCO) estimated the 2016/2017 world cocoa production at 4,733,000 ton (https://www.icco.org/). While the chocolate manufacturing is a powerful industry, the primary fermentation of the cocoa beans is still a traditional process. The first studies on cocoa fermentation were done in the late 1890s, revised by Roelofsen in 1958 and described from the perspective of microbiology and chemistry by numerous articles and book chapters from the 2000s until present (Ozturk and Young, 2017). The yeasts are involved in the first steps of the fermentation, followed by the lactic acid bacteria (LAB) and acetic acid bacteria (AAB). During fermentation, internal enzymes along with various compounds released by Caffeinated and Cocoa Based Beverages. https://doi.org/10.1016/B978-0-12-815864-7.00013-1 © 2019 Elsevier Inc. All rights reserved.

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424  Chapter 13  The Microbiology of Cocoa Fermentation

microorganisms contribute to the formation of flavors, aroma, and color of cocoa beans (Ganeswari et al., 2015; Moreira et al., 2013). The cocoa beans are then processed by drying, roasting, and conching for obtaining the chocolate. This chapter presents the main aspects concerning the microbiology, biochemistry, and genetics of cocoa fermentation, the biodiversity of the microorganisms involved in fermentation, and some of the modern approaches used for the improvement of the process.

13.2  Cocoa Fermentation Throughout the fermentation process, the cocoa beans suffer a series of transformations that include: pulp degradation by microbial strains, cotyledon death as a result of pH and temperature changes, astringency and bitterness reduction due to loss of flavonoids, and color transformation due to phlobaphenes formation. All these transformations occurring inside and outside the cocoa beans lead to the accumulation of flavor precursors (Aikpokpodion and Dongo, 2010; Aprotosoaie et al., 2016; Krysiak et al., 2013). The spontaneous fermentation of cocoa beans involves microbial strains originating from workers' hands, fruits surface, tools used in postharvesting processes, insects, banana leaves, trays, or boxes used previous fermentation. But not all these microorganisms will be involved in the fermentation process (Camu et al., 2007). Inside the Theobroma cacao fruits, the seeds are cover by a mucilaginous pulp which contains water 80%–90%, sugars 10%–15% mainly sucrose, pectin 1%–1.5%, citric acid 1%–3%, proteins 0.5%–0.7%, minerals Ca, K, Na, Mg, Fe, and Zn 8%–10%, and vitamin C (Lefeber et al., 2010; Schwan and Wheals, 2004). The pulp compounds represent the basis for the development of microbial strains. The sugars and polysaccharides are fermented mainly by the yeasts. During this time, ethanol and week organic acids are produced in order to reduce the pH values fluctuations. Also, pectinolytic enzymes are synthesized for the maceration of the cocoa pulp and synthesis of flavor and aroma precursors (alcohols, fatty acids, and fatty esters) (Illeghems et al., 2012; Zhao and Fleet, 2014). This step, determine physical and biochemical changes in the cotyledon. Thus, the endogenous enzymes (invertase, glycosidases, proteases, and polyphenol oxidase) are active producing precursor molecules that assure the development of characteristic flavors and color of well-fermented cocoa beans (Afoakwa et al., 2008). After 24–48 h, the yeast population declines mainly due to the high ethanol concentration and the rise of temperature. The yeasts are eclipsed by LAB, which ferment sugar and citric acid to lactic acid, acetic acid, and mannitol. The rise of oxygen level, temperature, and pulp drainage assures the growth of AAB which oxidize the ethanol

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and the lactic acid into acetic acid, then into carbon dioxide and water, having also an important role in formation of chocolate flavor precursors (Illeghems et al., 2015b). In the end of fermentation, spore forming bacterial strains along with filamentous fungi may grow causing damages (Schwan, 1998).

13.2.1  Microbial Biodiversity 13.2.1.1 Yeasts A study focused on the analysis of the genetic diversity of Saccharomyces cerevisiae strains associated with cocoa beans, showed a strong country-level clustering of the strains which form multiple populations reflecting probably a complex pattern of migration events (Ludlow et al., 2016). Moreover, the yeast strains associated with the fermentation of cocoa beans appear to be different from those found in the region from which the plants originate. This suggests a combination of alleles from Europe, Asia, and North America due to the human activity related to the fermentation styles of cocoa. Therefore, some of the yeast species might appear as predominant in fermentations from various countries, while studies lead in the same country but in different regions, seldom identify a different composition of the yeast population (Table 13.1).

13.2.1.2  Lactic Acid Bacteria and Acetic Acid Bacteria The microbial communities involved in cocoa fermentation from different regions have been analyzed through classic microbiological techniques (cultivation, isolation, and biochemical identifications) or by applying various culture-independent techniques (denaturing gradient gel electrophoresis (DGGE), rDNA sequencing, and pyrosequencing) in order to overcome the limitations of classical methods. The succession of microbial groups is the same in fermentation of cocoa beans worldwide. The only variations are represented by the number and diversity of microbial the species involved in the process which may present variation depending on the country, region, and the fermentation technique applied. The oxygen level, temperature, and metabolic end products (lactic acid, acetic acid, and ethanol) are key factors that modulate the microbial dynamics of cocoa beans fermentation process. Thus, the beans mass is characterized by a nonuniform distribution of microorganisms along the fermentation process (Meersman et al., 2013). Species belong to Lactobacillus genus seems to dominate the fermentation process around the world, while other species from genus Leuconostoc, Pediococcus, Weisella, and Lactococcus are occasionally reported. In many regions, the predominant LAB species in cocoa

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Table 13.1  Some of the Yeast Species Identified During Cocoa Fermentation in Various Countries Country

Yeast Species

References

Brazil

S. cerevisiae, P. kluyveri, H. uvarum, I. orientalis, D. etchellsii, K. ohmeri, C. orthopsilosis, P. kundriavzevii S. cerevisiae, C. magnoliae, P. kluyveri P. manshurica, H. opuntiae, P. kundriavzevii, P. kluyveri, C. tropicalis, P. occidentalis, C. ortopsilosis, P. terricola, T. delbruekii C. inconspicua, H. guillermondii, Y. lipolytica, C. zeylanoides, P. fermentans, C. krusei, C. glabrata, H. valbyensis C. krusei, C. tropicalis, Sacch. fibuligera, Kloeckera sp., S. cerevisiae Kl. apis, S. cerevisiae, C. tropicalis P. kundriavzevii, P. kluyveri, S. cerevisiae S. cerevisiae, C. tropicalis, P. kudriavzevii, P. galeiforms, G. geotrichum, W. anomalus H. opuntiae, P. manshurica, P. sporocuriosa, I. hanoiensis, P. kudriavezii, C. insectorum, Candida sp., P. klyvera, P. fermentans S. chevalieri, P. membranaefaciens, C. krusei, T. holmii, T. candida P. kundriavzevii, S. Cerevisiae, H. opuntiae, C. carpophila, C. orthopsilosis, K. ohmeri, M.(P.) carribica, P. manshurica P. kundriavzevii, S. cerevisiae, Sacch. crataegensis, H. guilliermondii

de Melo Pereira et al. (2012)

Cuba

Dominican Republic Indonesia

Ivory Coast

Ghana Mexic

Miguel et al. (2017) Maura et al. (2016)

Gálvez et al. (2007) Jamili et al. (2016) Ardhana and Fleet (2003) Koffi et al. (2017) Koné et al. (2016) Hamdouche et al. (2015)

Ravelomanana et al. (1985) Daniel et al. (2009) Hernández-Hernández et al. (2016)

C., Candida; D., Debaryomyces; G., Galactomyces; H., Hanseniaspora; I., Issatchenkia; K., Kodamaea; Kl., Kloeckera; M., Meyerozyma; P., Pichia; S., Saccharomyces; Sacch., Saccharomycopsis; T., Torulaspora; W., Wickerhamomyces; Y., Yarrowia.

beans fermentation are heterofermentative Lactobacillus plantarum and Lactobacillus fermentum (Table 13.2). According to different researches, the AAB species dominating the cocoa fermentation process are Acetobacter pasteurianus, A. aceti, A. ghanensi, A. tropicalis, and Gluconobacter oxydans (Table 13.2).

13.2.1.3  Factors Influencing the Microbial Diversity and Dynamics During Cocoa Fermentation The microbial diversity and community dynamics also depend on the fermentation method, the characteristics of the microorganisms involved, the location of the fermentation process, and on the cocoa bean genotype.

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Table 13.2  Bacterial Species Identified During Cocoa Fermentation in Various Countries Country

Bacterial Species

References

Indonesia

L. cellobiosus, L. plantarum A. pasteurianus B. pumilus, B. licheniformis L. fermentum, L. plantarum, L. brevis, Lc. mesenteroides, Lc. pseudomesenteroides A. pasteurianus L. plantarum, L. fermentum, L. brevis, L. mali, Leuc. pseudomesenteroides, Leuc. Mesenteroides, W. ghanensis, Ent. casseliflavus, Ent. faecium A. pasteurianus, A. senegalensis, A. senegalensis A. syzygii-like, A. tropicalis, A. tropicalis-like L. plantarum, L. fermentum, L. brevis, P. acidilactici L. plantarum, L. pentosus, L. paracasei subsp. paracasei, L. brevis A. lovaniensis L. plantarum, Leuc. mesenteroides, L. curieae, Ent. faecium, F. pseudoficulneus, L. casei, W. paramesenteroide, W. cibaria L. plantarum, L. fermentum, L. casei, L. rhamnosus, L. lactis, L. delbrueckii, L. acidophilus, L. brevis, P. dextrinicus, L. amylovorus, L. reuteri, Lc. lactis, Lc. mesenteroides, F. pseudoficulneus, S. salivarius, P. acidilactici, Oenococcus oeni A. aceti, A. pasteurianus, A. ghanensis, A. senegalensis, A. lovaniensis, A. fabarum, A. indonesiensis, A. malorum, A. cerevisiae, A. peroxydans, Ga. oxydans, G. xylinus, G. saccharivorans B. licheniformis, B. firmus L. fermentum, Leuc. pseudomesenteroides F. tropaeoli-like, L. fabifermentans, Lc. lactis subsp. lactis, L. nagelii, L. cacaonum, L. amylovorus, E. saccharolyticus, F. ficulneus, L. satsumensis, W. cibaria, W. fabaria A. pasteurianus, A. senegalensis, A. ghanensis, A. fabarum, A. cibinongensis, A. malorum/cerevisiae, A. indonesiensis, A. syzygii, A. orientalis, A. lovaniensis/fabarum, Frateuia aurantia, G. oxydans L. fermentum, L. plantarum, L. mesenteroides, Lactococcus lactis

Ardhana and Fleet (2003)

Malaysia

Ghana

Nigeria Dominican Republic Ivory Coast Brazil

Ecuador

Trinidad

Meersman et al. (2013)

de Bruyne et al. (2010)

Camu et al. (2007) Kostinek et al. (2008) Gálvez et al. (2007)

Ouattara et al. (2017) Moreira et al. (2013), de Melo Pereira et al. (2013), Passos et al. (1984), Papalexandratou et al. (2011a), Illeghems et al. (2012)

Papalexandratou et al. (2011b)

Ostovar and Keeney (1973)

A., Acetobacter; B., Bacillus; E., Enterococcus; F., Fructobacillus; G., Gluconobacter; Ga., Gluconacetobacter; L., Lactobacillus; Leuc., Leuconostoc; Lc., Lactococcus; O., Oenococcus; P., Pediococcus; S., Streptococcus.

428  Chapter 13  The Microbiology of Cocoa Fermentation

Fermentation can be performed in heaps of beans, in baskets, wooden boxes, or trays. The fermentation methods used present different advantages: the heap method assures a uniform process, the box method influences the pH values, the tannins, and sugars contents, while the tray method is mostly adequate for short fermentation processes (Aprotosoaie et al., 2016). According to Romano et  al. (2006), Candida krusei is dominant during heap beans fermentation followed by Pichia membranefaciens, Pichia kluyveri, Hanseniaspora guilliermondii, and Trichosporon asahii. In tray fermentation, the dominant species are S. cerevisiae and Pichia membranefaciens, smaller populations of C. krusei, Pichia kluyveri, and H. guilliermondii being detected. The DGGE analysis of 26S rRNA fragments of yeasts from 12 cocoa bean fermentations from Brazil, Ecuador, Ivory Coast, and Malaysia, revealed that the most frequent species were Hanseniaspora sp., followed by Pichia kundriavzevii (formerly Issatchenkia orientalis, anamorph C. krusei) and S. cerevisiae (Papalexandratou and De Vuyst, 2011). Hanseniaspora sp. could be found even after 4 days due to its resistance to acids and high temperature. P. kundriavzevii and C. nitrativorans (found in Ivorian cocoa fermentation) are well adapted to high temperature (40°C) and ethanol concentration (20%) and exhibited pectinolytic activities involved in the degradation of cocoa bean pulp (Samagaci et al., 2016). Interesting, although C. nitrativorans had killer activity against P. kundriavzevii, it was not dominant, suggesting that the killer phenotype might not be determinant in yeast colonization during fermentation. On the other hand, S. cerevisiae can be found in all stages of fermentation due to its adaptation to the ecosystem: a rapid growth, ethanol tolerance, and enhanced pectinolytic activity (Daniel et al., 2009; Maura et al., 2016). In the Ivorian heap fermentation, P. kundriavzevii was found at the beginning of the process followed by Hanseniaspora sp., while in the Ivorian box fermentation the yeast diversity was augmented, Hyphopichia burtonii and M. (P.) carribica being also present (Papalexandratou and De Vuyst, 2011). In Ghanaian cocoa bean heap fermentations, the most frequent species were P. kundriavzevii, S. cerevisiae, and Hanseniaspora opuntiae followed by C. carpophila, C. orthopsilosis, Kodamaea ohmeri, M. (P.) carribica, Peronospora manshurica, Saccaromycodes ludwigii, and Yamadazyma (Pichia) mexicana (Daniel et al., 2009). Another study conducted on Ghanaian heap and tray fermentations showed that H. guilliermondii was prelevant, followed by P. membranefaciens and P. kundriavzevii (Papalexandratou and De Vuyst, 2011). Jespersen et al. (2005) found that the dominant species in Ghanaian heap bean fermentation was C. krusei followed by P. membranifaciens, P. kluyveri, H. guilliermondii, and T. asahii. In the tray fermentation, S. cerevisiae

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and P. membranifaciens were found in great ammount, while C. krusei, P. kluyveri, and H. guilliermondii were less numerous. Similar experiments (Nielsen et al., 2005) showed that H. guilliermondii, C. krusei, and P. membranifaciens were dominant in Ghanian cocoa in heap as well as tray fermentations, while S. cerevisiae and C. zemplinina were observed almost exclusively in tray fermentations. Experiments lead in Brazil in stainless steel tanks and wooden boxes showed that S. cerevisiae, H. uvarum, and P. kluyveri were dominant in both cases (de Melo Pereira et al., 2013). However, the diversity of non-Saccharomyces yeast species (Pichia, Candida, Debaryomyces, and Schizosaccharomyces) was greater in the wooden boxes. On the contrary, the ethanol yield was higher in the stainless steel tanks fermentation. In order to understand the effects of breeding on cocoa beans fermentation process in wooden boxes, da Veiga Moreira et al. (2013) analyzed the microbial diversity, substrate consumption, and metabolic end products accumulation of three different hybrids of T. cacao (PH 9, PH 15, and PH 16) from Brazil. The molecular identification analysis, revealed that S. cerevisiae was predominant during fermentation in all experiments, followed by H. uvarum and Pichia sp. However, PH 9 had a greater biodiversity of the microbial population than the other two hybrids, Rhodotorula mucilaginosa and Issatchenkia terricola being detected through polymerase chain reaction (PCR)-DGGE fingerprinting. Substrates consumption (glucose, fructose, sucrose, and citric acid) was rather similar during fermentation, while the concentration of acetic acid, lactic acid, and ethanol reached maximum values at different times. However, the PH 16 hybrid presented particular biochemical characteristics with high concentrations of sugars, acids, and ethanol. A study conducted by Gu et al. (2013) in which cocoa beans from China, Indonesia, and Papua New Guinea have been used, revealed that genetic variety, climatic conditions, and fermentation process influence cocoa bean weight, butter content, concentration of polyphenols, and amino acid.

13.2.2 General Aspects of Cocoa Fermentation Biochemistry and Genetics The desired characteristic cocoa flavor, color, and taste are obtained after fermenting, drying, and roasting of the raw cocoa beans (Illeghems et al., 2012). The fermentation processes have significant impact on the final quality of the cocoa beans. Lack of the fermentation or a sub-fermentation process will increase bitterness and astringency of cocoa beans. On the other hand, extending fermentation beyond 5  days could lead to accumulation of off-flavor compounds released by some fungal species.

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Besides the cocoa bean type, cocoa flavor and color are influenced by: (1) the method applied for fermentation process—boxes, baskets, containers, and trays covered with banana leaves; (2) the duration of fermentation—if the process is accelerated, flavor precursors do not have time to accumulate in the cocoa beans; (3) the diversity of the microorganisms involved in fermentation process; (4) the weather conditions during fermentation and drying processes—during the wet season, the beans can contain more water which influences the aeration during fermentation, the high humidity also increasing the risk to mold contamination of beans while drying; and (5) the drying and storage conditions (Camu et al., 2008a; Ganeswari et al., 2015).

13.2.2.1  Yeast Fermentation Cocoa fermentation process begins after harvesting and opening the pods and takes place in anaerobic conditions, at low pH (3.5–4.0), during 24–48 h. In this phase, the yeasts are fermenting the sugars (sucrose, glucose, and fructose) generating ethanol, carbon dioxide, organic acids, and flavor precursors (Kloekera apiculata, S. cerevisiae var. chevalieri, S. cerevisiae, Hanseniaspora sp., P. kluyveri). Some of the yeast species can breakdown the citric acid present in the pulp (Candida sp., Pichia sp.) or ferment the rest of sugars in later fermentation stages due to an augmented resistance to high ethanol concentrations or temperatures (Kluyveromyces marxianus). Other species synthesize pectinolytic enzymes (K. marxianus, Kluyveromyces thermotolerans, S. cerevisiae var. chevalieri) or produce killer toxins with antimicrobial activity (Candida sp.). The fermentation process is influenced by the age of the pod, the cocoa type, or the method used. Hernández-Hernández et al. (2016) determined the pH, temperature, total soluble solids, and acidity during a study comprising fermentations using wooden boxes of 1000, 300, and 100 kg and a wooden rotary drum of 500 kg. Although the parameters were similar for all treatments, the degree of fermentation was close only for 300 and 100 boxes and the rotary drum, while the 1000 kg boxes showed a lower quality of fermentation. Camu et  al. (2008a) performed seven independent cocoa heap fermentations and observed that the concentrations of metabolites (ethanol, citric acid, lacic, and acetic acid) as well as microbial population dynamics were heap dependent. The sugars are metabolized in the yeast cells through glycolysis, pentose phosphate pathway, and the citric acid or tricarboxylic acid cycle (TCA). The fermentations in which S. cerevisiae is a dominant species are more likely to have a higher ethanol concentration than those in which are present Hanseniaspora, Pichia, or Kluyveromyces species (Ho et al., 2014). If the genes involved in the metabolic pathways, such

Chapter 13  The Microbiology of Cocoa Fermentation   431

as the hexokinase and glucokinase genes (HXK1, XK2, and GLK1) are rather well described for S. cerevisiae, progress is still done for other species. Thus, Zhang et al. (2017) functionally identified and characterized two genes: KmHXK1 and KmGLK1 from K. marxianus. Also, Giorello et al. (2014) reported 128 genes associated with fermentation in Hanseniaspora vinae, 87% from them being similar to those from S. cerevisiae. The citric acid is present in unfermented cocoa beans and, although it is metabolized mainly by the LAB, several yeast species are involved in depletion of citric acid. This induces a general response to stress using the high-osmolarity glycerol (HOG) pathway and the mitogen-activated protein kinase (MAPK), in this case the Hog1 phosphorilation depending only on the Sln1 branch (Bermejo et al., 2008). The pectinolytic enzymes, or pectinases, belong to the polysaccharidases family and act on the homogalacturonan and rhamnogalacturonan regions of the pectins, heteropolysaccharides constituting major components of the higher plants cell wall and lamella. The pectinases are classified in de-esterifying and depolymerizing enzymes. The enzymes produced by yeast species are hydrolases (endo and exo-polygalacturonosidases). Yeasts involved in cocoa fermentation produce, in general, endopolygalacturonosidases (endoPG) with optimal activity at and various temperatures and pH values: S. cerevisiae var. chevalieri at 25°C and pH between 3.0 and 5.5 (Gainvors et al., 2000; Sanchez et al., 1984), K. marxianus and K. thermotolerans at 40°C and pH 5.0 (Alimardani-Theuil et al., 2011; Schwan and Wheals, 2004). In S. cerevisiae, the PGU1 (PGL1) gene from chromosome X, encoding the Pgu1 protein (361 amino acids) has been sequenced (Blanco et al., 1999). According to Gognies et al. (2001), the PGU1 activity requires components of MAPK pathway, is enhanced under respiratory conditions, related to filamentous growth, repressed by glucose, and upregulated by pectins. Until present, two genes (EPG1 and EPG1-2) from two K. marxianus strains have been sequenced. They encode for two endoPG of 361, respectively, 362 amino acids with 80%–90% sequence identity (Alimardani-Theuil et al., 2011). The cocoa aroma is the result of complex reactions appeared during the processing of the beans and depends on the cocoa genotype as well as on the environmental conditions, the microbial diversity during fermentation and the subsequent manufacturing stages (drying, roasting, alkalinization, and conching). Although in present there are known more than 14,000 varieties of T. cacao, the main varieties commercially exploited are: Forastero with a strong basic chocolate flavor; Criollo which is highly aromatic; Trinitario, a hybrid between Forastero and Criollo, with a higher resistance to diseases, presenting strong chocolate taste and specific wine-like flavor; the Nacional variety (only in Ecuador) with caramel and fruity notes. The cocoa flavor

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is a combination of nonvolatile components (alkaloids, polyphenols, proteins, and carbohydrates) and volatile components (alcohols, acids, aldehydes, ketones, esters, pyrazines, acids, phenols, phuranones, and pyrones) (Aprotosoaie et al., 2016). Yeasts are involved in synthesis of volatile compounds that are further important in the development of fruity, candy or perfume-like chocolate flavors, and aromas. During a study in Ivory Coast, Koné et al. (2016) identified 33 aroma compounds synthesized by yeasts. The species Pichia kudriavzevii, S. cerevisiae, C. tropicalis, G. candidum, and Wickerhamomyces anomalus produced: higher alcohols (isobutanol and isoamyl alcohol), acids (acetic acid and isovaleric acid), and esters (ethyl acetate, isobutyl acetate, and isoamyl acetate). According to Ho et  al. (2014) no higher alcohols or esters were determined in beans or pulps fermented in the absence of the yeasts, indicating that yeasts are most probably the main producers of these compounds. In Saccharomyces, there are three genes, ATF1, Lg-ATF1, and ATF2, encoding for acohol acetyltransferases (AATases) of 525, 545, and respectively, 525 amino acids. These AATases catalyze acetate ester synthesis with various aromas: ethyl acetate—pineapple and isoamyl acetate—banan and pear like, but also isobutyl acetate—fruit (apple, banana, and cherry), propyl acetate—pear or 2-phenylethyl acetate—raspberry (Saerens et al., 2010). The gene ATF1, located on chromosome XV, is the main responsible for synthesis of volatile acetate esters during fermentation, being also involved in lipid and sterol metabolism. Using gas chromatography combined with mass spectroscopy (GC-MS), Verstrepen et al. (2003a) showed that the expression level of ATF1 is an important limiting factor for ester synthesis. Its close homolog, Lg-ATF1 (lager acohol acetyltransferases), is found only in Saccharomyces bayanus and has a less important role. The transcription of the ATF1 and LgATF1 genes is co-regulated, is induced by the addition of glucose in anaerobic conditions under the control of Ras/cAMP/PKA signaling pathway, is repressed by aeration and the presence of unsaturated fatty acids (Verstrepen et al., 2003b; Yoshimoto et al., 1998). The gene ATF2 is located on chromosome VII and, besides volatile esters, is involved in steroid detoxification. Strains belonging to Hanseniaspora and Pichia genera, were also able to produce 2-phenylethyl acetate and isoamyl acetate but under aerobic conditions (Rojas et al., 2001). In K. marxianus, the production of acetate esters strongly depends on the nitrogen and carbon source and some esterases (e.g., Iah 1p) could play a potential role in the metabolic pathway. Also, bioinformatic and phylogenetic studies suggested that the ATF2 encoded AATase (Atf2) may not be involved in synthesis of volatile acetate esters in K. marxianus (Gethins et al., 2015). Pyrazines contribute to the chocolate flavor and are formed during roasting by the reaction between the reducing sugars and

Chapter 13  The Microbiology of Cocoa Fermentation   433

amino acids. Therefore, the presence during frementation of yeast species with high fermentation rates, such as P. kudriavzevii and S. cerevisiae, is very important. Moreover, cocoa-substitute compounds (powder extenders and replacers) can be produced using various yeast species also found in cocoa fermentation (S. cerevisiae and C. tropicalis) which are grown on hop or non-hop-based media, combined with reducing sugars in an aqueous slurry, then heated under pressure (Abbas, 2006).

13.2.2.2  Bacterial Fermentation There are three types of bacteria involved in the fermentation process: LAB, acid acetic bacteria (AAB), and Bacillus sp. Lactic Acid Bacteria The biochemical roles of LAB in cocoa fermentations have been described by many researchers in the recent years. The most common LAB involved in cacao fermentation includes species belonging to the genera: Lactobacillus (L.), Lactococcus (Lc.), Leuconostoc (Leuc.), Pediococcus (P.), and Weisella (W.). First studies on cocoa fermentation, considered the role of LAB strains as insignificant due to their short presence during the process and to the fact that the residual lactic acid produced could be responsible for the off-flavor of cocoa beans. Further studies have highlighted the important role LAB strains in pulp degradation, flavors accumulation, and inhibitions of spoilage microorganisms. After the first day of fermentation in which yeasts dominate the process and ethanol accumulates, the conditions become more favorable for development of microaerophilic LAB, which start to multiply for the next 2 days by consuming the rest of cocoa pulp sugars (60% sucrose and 39% a mixture of glucose and fructose) and releasing lactic acid in the environment (Ardhana and Fleet, 2003). Studies have shown that the dynamics and LAB diversity is high during the fermentation process. Heterofermentative LAB dominate cocoa beans fermentations, they utilize glucose via the pentose phosphate pathway, forming along with lactic acid, acetic acid, ethanol, mannitol, and carbon dioxide. Homofermentative LAB use glucose via the Embden-Meyerhof-Parnas pathway, the lactic acid being the main final end product of fermentation (Schwan, 1998). Both heterofermentative and homofermantative LAB strains seem thus to be involved in cocoa beans fermentation. Cocoa pulps represent an ideal substrate for strictly heterofermentative and fructophilic LAB species due to the high concentration of fructose and citric acids (Lefeber et al., 2011). The most frequent LAB strains isolated from cocoa beans fermentation are heterofermentative L. fermentum and L. plantarum.

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Citrate metabolism is considered as an important step in the development of flavor compounds in cocoa beans. Yeasts along with LAB are the main groups of microorganisms responsible for the breakdown of citric acid in cocoa fermentation (Ouattara et al., 2014). Thus, an important feature of LAB strains is their ability to metabolize citric acid via oxaloacetate pathway transforming it into either succinic acid or pyruvic acid, which is then converted into lactic acid, acetic acid, and ethanol (Axelsson, 2004). Using citrate metabolism LAB convert citric acid also into acetoin and 2,3-butandiol which are known to be part of the flavor profile of certain cocoa beans products (Lefeber et al., 2011). The assimilation of citric acid by LAB strains causes the increase pH allowing for the growth of AAB (Camu et al., 2007; Moens et al., 2014). Using the pyrosequencing technique, Illeghems et al. (2015a) sequenced two LAB strains isolated from spontaneous cocoa beans fermentation in Ghana. Their results revealed that the two LAB strains possess an extended metabolic system and contain various genes involved in niche adaptations which explain their domination during the fermentation process. L. fermentum 222 contains genes encoding for accumulation of flavors compounds like α-acetolactate decarboxylase (aldB) and 2,3-butanediol dehydrogenase (butC) which are necessary for citrate and aspartate metabolism or a hydroxymethyltransferase (shm) involved in conversion of serine into acetaldehyde. Besides the genes involved in the heterofermentative pathway, genes necessary for adaptation to stress conditions were discovered, such as those encoding for exopolysaccharides and ornithine cyclodeaminase (ocd) with important role in acid tolerance and osmotic protection. L. plantarum 80 seems to have just the aldB gene, acetoin being therefore the only flavor compound that could be produced by this strain. Instead, the strain harbored several genes for citrate catabolism, oxidative stress (cydABCD and katA), and protein degradation, necessary for cocoa pulp assimilation (Illeghems et al., 2015a). Acid Acetic Bacteria AAB are a group of microorganisms with multiple applications in food industry and are used for obtaining vinegar, kombucha, cocoa, ascorbic acid, and tartaric acid. The AAB are also involved in spoilage of beer, wine, and cider among fermentation (Illeghems et al., 2013). In recent years, the AAB have been extensively analyzed due to their major contribution to cocoa beans fermentation. They are assumed to be some of the most valuable microorganisms in cocoa fermentation producing oxidation of ethanol and lactic acid to acetic acid. The last one diffuses into the cotyledons which suffer a series of biochemical modifications leading to synthesis of cocoa color and flavor precursors (Adler et al., 2014; Soumahoro et al., 2015).

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After the third day of fermentation when the pulp of cocoa beans is reduced, the temperature and the level of air increased inside the fermenting mass, the environmental conditions become favorable for the growth of AAB. Since glucose and fructose are no more available due to their consumption by yeast and LAB strains, the metabolism of AAB is shifted toward the utilization of ethanol released by yeasts as a main carbon source. The AAB use an alcohol dehydrogenase to transform ethanol into acetaldehyde which is than reduced by an aldehyde dehydrogenase into acetate (Illeghems et al., 2013). AAB oxidize mainly ethanol produced by yeasts and the lactic acid produced by LAB into acetic acid, a part of which is reduced in the end via acetyl-CoA to carbon dioxide and water by a modified TCA. The acetic acid fermentation is an extremely exothermic process enhancing the temperature to over 50°C (Peláez et al., 2016). Only a small amount of lactate 2%–4% is converted into acetate by AAB species. Lactate is oxidized by A. pasteurianus via TCA cycle and another part is transformed into acetoin. The presence of AAB is important since they are responsible for the degradation of lactate, which in high amount, may lead to a decrease of the organoleptic qualities of the cocoa final products (Adler et al., 2014). During the fermentation, the acetic acid is found in a higher concentration than the lactic acid. Toward the end of the process, the levels of acetic and lactic acids are comparable due to the high volatility of the acetic acid. The most prevalent AAB species isolated from spontaneous cocoa bean fermentation are A. pasteurianus, A. ghanensis, and Gluconoacetobacter (Yao et al., 2014). All the modifications that take place during acetic acid fermentation, increases of temperature and acetic acids concentration followed by the diffusion of acetic acid into the beans, trigger the death of cotyledons which cause the activation of endogenous hydrolytic enzymes. These enzymes associated with accumulation of flavors precursor are active for a short period of time, most of them being subsequently inactivated (aminopeptidase, invertase, and polypenol oxidase) or partly inactivated (carboxypeptidase). Endoproteases and glycosidases remain active throughout the fermentation process (Camu et al., 2008b). The most important compounds involved in flavors are: alcohols, carboxylic acids, aldehydes, ketones, esters, and pyrazines. Some of the compounds are released by the microbial strains involved in the fermentation process, while others are released following the action of endogenous enzymes from cotyledons (Aprotosoaie et al., 2016). It seems that AAB have a crucial role in the formation of the chocolate flavor precursors. On the other hand, lactic and acetic acids released by bacterial strains during fermentation inhibit ochratoxigenic fungal growth and other food spoilage microorganisms.

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Genome analysis of the strain A. pasteurianus 386B isolated from a cocoa bean fermentation (Illeghems et al., 2013) showed that it owns genes that can be associated with fermentation of cocoa beans: endopolygalacturonase, alcohol and lactate dehydrogenase (adh, ldh), pyruvate decarboxylase (pdc), genes encoding for synthesis of trehalose and for mechanosensitive channels (MscL) which are associated with tolerance to high osmolarity. Since AAB are exposed to high concentration of acid, they developed mechanisms of protection: consuming acetate from citosol (acetyl-CoA synthetase—acn; acetate kinase— ackA; and phosphate acetyltransferase—pta); efflux pumps (aatA); exopolysccharide formation (polABCDE); and conversion of urea into ammonia. Bacillus sp. Spore forming bacteria from the genus Bacillus as well as molds are occasionally developed during cacao beans fermentation. Even if Bacillus species were detected throughout the whole fermentation process, during the third day their number starts to grow. The genus Bacillus includes species that are able to use various carbon sources offering them the possibility to cover large ecological niches and to proliferate under extreme environmental conditions. The increase of temperature (40–50°C) and pH values in the bean mass favors the development of these spore forming bacteria which can also be found in the final cocoa products (Binh et al., 2017). The ability of some Bacillus spp. to metabolized lactic acid, acetic acid, and mannitol could also explain their presence in the later stages of fermentation process. Nevertheless, the role of Bacillus spp. during cocoa bean fermentation is still not very well understood. Some researchers demonstrated the ability of Bacillus spp. to release polygalacturonase and pectin lyases which could indicate an important functional role of this microbial group during cocoa beans fermentation (Ouattara et al., 2008).

13.2.2.3  Filamentous Fungi Filamentous fungi can also be present in small numbers throughout the fermentation process most of them being present in the aerated and cooler areas of the fermenting mass. For example, in Indonesia, Penicillium citrinum was detected during a natural fermentation of cocoa beans but its role in fermentation could not be explained (Mounjouenpou et al., 2008). Some researchers sustain that filamentous fungi have an important role in cocoa beans fermentation by releasing pectinolytic, amylolytic, and lipolyctic enzymes which could be involved in pulp degradation. Usually, fungal strains start to grow after the second day of fermentation when temperature and ethanol concentration

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decrease. The fungal species involved in fermentation differ from those associated with cocoa bean spoilage which appear during the drying process. The ability of some fungal strains to degrade lipids by releasing fatty acids could have a big impact on cocoa products flavor (Schillinger et  al., 2010). More investigations are required in order to establish the exact role of the fungal strains in cocoa beans fermentation process. Since fresh cocoa beans contain: fats 45%–55%, polysaccharides 14%–20%, pectin 2%, sugar 0.5%–2%, protein 1.5%, polyphenols 7%–10%, alkaloids 3.5%, etc., which could easily be degraded by enzymatic equipment of some fungal species, there is the possibility that some of these might have a beneficial effect on cocoa bean fermentation (Ardhana and Fleet, 2003; Schillinger et al., 2010). In general, the presence of fungi in cocoa is associated with spoilages and mycotoxin contamination. The most common ochratoxigenic species isolated from cocoa beans are Aspergillus carbonarius, Aspergillus niger, Penicillium verrucosum, and Penicillium nordicum (Amézqueta et al., 2008).

13.3  Approaches for Improvement of Cocoa Fermentation Due to the great importance of cocoa and chocolate industry, researchers and manufacturers are searching for new approaches meant to increase and improve the quality of fermentation. Most studies from the domain deal with the addition of starter cultures: yeasts, yeastsLAB, or yeasts-LAB-AAB. In order to establish the most appropriate starter culture is essential to know which microbial species are dominant in a specific fermentation process, the microorganism/strains selected must present good fermentation rates, high pectinolytic activity, augmented ability to synthesize flavor and aroma precursors, and present antimicrobial activity (de Melo Pereira et al., 2016).

13.3.1 Yeast Starter Cultures The studies for development of starter cultures showed that, in general, S. cerevisiae, the species with most incidence in cocoa fermentation, is used for its high ability to synthesize acetate esters, pectinases, and for obtaining thermotolerant hybrids, P. kundriavzevii is a good ethanol producer enhancing the fermentation efficiency, Kluyveromyces sp. have high pectinolytic activity and are thermotolerant, P. kluyveri increases the flavor of the cocoa beans and cocoa pulp degradation, Torulaspora delbrueckii has a positive influence on the flavor and aroma of the final product, and Candida sp. inhibits the development of potential pathogenic microorganisms.

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Cempaka et al. (2014) used a laboratory strain of S. cerevisiae var. chevalieri as starter for fermentation of Forastero cocoa beans from Indonesia and observed a better use of glucose, a shift of ethanol peak (16.1 mg/g pulp after 96 h when the starter was used, compared to 5.4 mg/g pulp after 48 h in natural fermentation) and an improvement of mucilagous pulp removal. Meersman et al. (2015) used mass mating assay between a natural S. cerevisiae strain (Y927) and a strain from the bioethanol industry (Y115) for obtaining hybrids with high fermentation efficiency and temperature tolerance. Other experiments were also lead in Indonesia with Forastero cocoa beans fermented in plastic boxes using seven starter cultures represented by: one strain of each C. krusei, Saccharomycopsis fibuligera, Kloeckera sp., and S. cerevisiae and three strains of C. tropicalis. The inoculated fermentations showed clear advantages compared to the natural/control fermentations: a shortened time of fermentation from 5 to 3 days, an increased fat content of cocoa and a rapid development of chocolate characteristics (Jamili et al., 2016). An yeast starter culture containing the strains S. cerevisiae UFLA CA11, P. kluyveri UFLA YCH194, and H. uvarum UFLA YCH203 was spread over cocoa beans in fermentation experiments in Brazil (Batista et al., 2015). The qPCR revealed that in the inoculated fermentation H. uvarum was inhibited by the other two species. The liquid chromatography analyses showed that the carbohydrates were consumed faster when the starter culture was used than in the control experiment, the citric acid reached less than 1 g/kg only after 72 h and the ethanol had a higher concentration (8 g/kg from 48 to 72 h) compared with the control (4.6 g/kg after 96 h). During the sensory analyses of the chocolates (70% cocoa) obtained from the inoculated and the control fermented beans, the consumers noticed stronger coffee and sour taste for the inoculated assay. The strain S. cerevisiae UFLA CA11 has been also used in a mixed culture with P. kluyveri CCMA0237 and H. uvarum CCMA0236 to inoculate cocoa beans for fermentation in wooden boxes in Brazil (Batista et al., 2016). The impact of the inoculation on the volatile compounds was evaluated. Thus, aldehydes and ketones with desired aromas were found mainly in the inoculated fermentation and the esters were found in a higher concentration. The tests for the sensory profile of the chocolates showed that the fruity notes were more intense and lasted longer in the case of chocolates obtained from inoculated fermentation compared with those from the chocolates obtained from the control fermentation. Two strains of P. kudriavzevii (LPB06 and LPB07) with superior aroma producing abilities were used as starters for laboratory experiments at bench scale, in Brazil (Pereira et  al., 2017). During the fermentation assays high yields of acetaldehyde, ethyl acetate and

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ethanol were obtained. Also, an efficient polyphenol oxidase activity was observed, indicating a good fermentative process. The hybrid strain K. marxianus MMIII-41 with high extracellular inulinase activity was used for inoculation of cocoa beans fermentated in plastic laundry baskets in Brazil (Leal et al., 2008). The result was a higher liquid drainage (sweatings) from the fermentation mass, 66.7%, in the inoculated fermentation compared with 59.4% in control fermentation after only 24 h which contributes to a superior quality of the fermented seeds, cocoa liquor, and chocolate. Moreover, most of the yeast species could be detected even after 120 h. Teng-Sin et  al. (2016) used 16 yeast species as starters in laboratory cocoa fermentation experiments and obtained augmented metabolism of total soluble solids from the pulp. C. ethanolica and C. tropicalis showed the strongest correlations with the fermentation index of nibs, while C. jaroonii, C. quercitrusa, C. xylopsoci, H. opuntiae, Hanseniaspora sp., P. kudriavzevii, and W. anomalus showed the highest degree of correlation with total soluble solids of pulps. The effect of the yeast starter culture might depend on various factors. Thus, Visintin et al. (2017) used a mixed culture of strains S. cerevisiae IC67 and T. delbrueckii IC103 with high tolerance to stress, for inoculating cocoa pods from two different hybrids (PS1319 and SJ02). While the inoculum influenced positively the end product (chocolate) quality of the PS1319 hybrid, no significant changes have been recorded in the case of the SJ02 hybrid.

13.3.2  The Yeasts Can Be Used as Starters in Combination With Bacteria Strains of L. fermentum and A. pasteurianus were mixed with a highly aromatic P. kluyveri strain, respectively, with a K. marxianus strain with high pectinolytic activity (Crafack et  al., 2013). Although only slight differences were observed between the inoculated and control fermentations in terms of microbial diversity and chemical composition, the mixed starter culture of L. fermentum, A. pasteurianus, and P. kluyveri had a positive impact on the flavor of the chocolate. The same bacteria species, L. fermentum and A. pasteurianus, were used in combination with S. cerevisiae on cocoa pods from the PH15 cocoa hybrid from Brazil (da Veiga Moreira et al., 2017). The experiments showed that glucose and fructose were consumed during the first day in the inoculated assay. The analysis of volatile compounds revealed the presence of 2,3-butanediol and 2,3-dimethylpyrazine that influenced positively the sensory characteristics of the chocolate. A starter consortium of S. cerevisiae, L. plantarum, and A. aceti was used in various proportions (10% up to 60%) for inoculation of cocoa beans fermented in boxes (Sandhya et  al., 2016). The experiments

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indicated that using a 10% concentration of the inoculum reduced the time of the fermentation process, improved alcohol production, and lead to appearance of intense cocoa flavor. Kresnowati and Febriami (2016) obtained a high fermentation index (1.38) for lab experiments on cocoa beans inoculated with a combination of S. cerevisiae var. chevalieri and L. plantarum. The yeast population increased within 24 h, the sugars (glucose and fructose) were rapidly degraded and the fermentation was completed in 4 days. A microbial cocktail with S. cerevisiae var. chevalieri, Lactococcus lactis, L. plantarum, A. aceti, and G. oxydans subsp. suboxydans have been used to inoculate 200 kg boxes of cocoa beans in Brazil. Although there were no major differences between spontaneous and controlled fermentation regarding the speed, microbial succession, metabolites and flavors accumulation, the sensorial analysis showed that using a defined starter culture leads to a better taste chocolate (Schwan, 1998).

13.3.3  Using Bacteria for Cocoa Fermentation Improvement Lately, consumers request regarding good quality chocolates have increased, therefore, obtaining high-quality cocoa became a new challenge for many farmers. Since cocoa flavor precursors are accumulated during fermentation, the possibility to control the process can have beneficial effect on the end products. The researchers proposed various combinations of starter cultures including bacterial strains isolated from cocoa fermentation or from other natural sources. A part of these starter cultures has been already successfully implemented. However, until now there are no starter cultures for cocoa fermentation available in the market. Different studies showed that addition of bacterial strains to a natural or artificial fermentation process has a positive effect on the cocoa beans quality by accelerating and, in the same time, stabilizing the process. Besides developing flavor precursors, LAB strains are also able to release antimicrobial compounds (bacteriocins, organic acids, hydrogen peroxide, etc.) that inhibit the growth of microorganisms associated with cocoa spoilage: Escherichia coli, Salmonella sp., and Listeria sp. (Saltini et al., 2013). Genome analysis of L. fermentum 222 and L. plantarum 80 strains conducted by Lefeber et  al. (2010) revealed that these two strains could be used as starter culture for cocoa beans fermentation due to a complex enzymatic equipment adapted to cocoa pulp ecosystem and to the existence of multiple mechanisms developed for resistance to stress conditions. Yao et  al. (2017) studied stress resistance of some Bacillus sp. strains presenting pectinolytic activity, citrate metabolism, and acids production, in order to use them as starter culture

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for cocoa beans fermentation. Five Bacillus strains were able to grow under stress conditions similar with those from cocoa fermentation: high temperature (50°C), ethanol concentration 4%, pH variation (4.0–8.0). As a conclusion, Bacillus sp. can be used as starter culture since they have a complex enzymatic profile necessary for cocoa pulp degradation.

13.4 Conclusions The process of cocoa beans fermentation, the first step of the worldwide industry of cocoa and chocolate products, is complex and depends on numerous factors that have a major influence on the fermentation process parameters and the quality and sensory characteristics of the final products. The scientific community working in the domain is constantly preoccupied with the impact of various factors (the type of cocoa cultivars, the geographical location where the fermentation is performed, and the method used for fermentation) on the microbial diversity and dynamics and the biochemical changes triggered during the process. Therefore, a deeper knowledge of cocoa process technology will bring a multiple benefits: reduce the risk of contamination, increase the nutritional value of the end products, and add new flavors. In general, cocoa fermentation takes place by traditional methods that are uncontrollable and involve certain risks. The microbial strains originating from a location are responsible for certain flavors which confer cocoa products unique organoleptic characteristics. Thus, an accurate identification and metabolic characterization of the whole microbial community involved in the fermentation process, would allow the isolation of new strains able of delivering a specific flavor to cocoa beans. At the same time, the growing demand for cocoa and chocolate products of high variety and the need to generate a standardized fermentation process, is constantly generating an increasing number of studies dealing with improvement of the fermentation process mainly by using starter cultures based on specific physiological characteristics of microorganisms involved in fermentation.

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