INTRODUCTIONThe Latin name for cocoa, Theobroma Cocao,translates as “food for the gods”. The plant has its originin the upper Amazon, where archeologists discoveredtheobromine alkaloid from the oldest organic cocoamatter in history at the Santa Ana archaeological sitein La Florida, in the province of Zamora Chinchipe,Ecuador. From this place, cocoa beans spreadthroughout the rest of the continent. On the YucatanPeninsula, fragments of once-vast cocoa plantationshave been found on the territories that were occupied bythe Mayan civilization. The traces of cultivated cocoawere also discovered in Central America on the territoryof the modern Mexico. Currently, cocoa is cultivatedin many tropical countries of the world. It grows in thearea between 20 degrees latitude north and south of theequator [1, 2].The cocoa beans come from the Theobroma cacaotree. They grow in a pod that contains 30–40 beans156Ordoñez-Araque R H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 155–162wrapped in a jelly-like mucilaginous substance. Rawcocoa has an unpleasant astringent taste, which meansthat its volatile compounds have to be generatedartificially. During treatment, microorganisms modifytheir state and components through various processes,e.g. fermentation. After fermentation, beans are driedand exposed to the sun. Only then do the typical sensoryproperties develop, and the beans acquire the pleasantcharacteristics we associate with chocolate [3, 4].Fermentation is considered the most critical stepin the processing of cocoa, since the beans are themain raw material in chocolate industry. It is at thefermentation stage that they develop their sensoryproperties. These properties come from aromaprecursors generated during chemical changes inthe phenolic content [5]. Fermentation occurs in thepulp of the pod. The pulp is a white carbohydrate-richmucilaginous mass that surrounds and protects thebeans. The process lasts several days and depends onseveral groups of microorganisms [6].The microbial activity during cocoa fermentationhas complex biochemical implications. In fact, cocoa isone of few foods where so many changes and processesoccur during the same process. It includes the successivegrowth of several species of yeasts, lactic acid bacteria,acetic acid bacteria, and, to greater or lesser extent,species of Bacillus and filamentous fungi [7]. Yeastsare active at the earliest stage of fermentation. The mostcommon strains include Saccharomyces spp, Candiddaspp, and Pichia spp. The yeast stage is followed by lacticacid bacteria, which are represented by Lactobacillusplantarum and Lactobacillus fermentum. Acid bacteriaacetics belong to the genus of Acetobacter, Acetobacterpasteurianus being the most common.What exactly determines the quality of the cocoabeans still remains a mystery: groups of microorganismsor individual species? Most likely, all the strains areessential for the fermentation process, since newgenera and new species are constantly being discovered[8, 9]. Therefore, the first objective of this reviewwas to specify the characteristics and reactions ofmicroorganisms during fermentation. The secondobjective was to formulate some recommendations onimproving fermentation conditions or maintaining theoptimal ones to achieve the best transformation in thechemical compounds.STUDY OBJECTS AND METHODSThe bibliographic review was conducted accordingto three databases, namely Science Direct, Scopus,and Medline. The descriptors included the followingkey words: cocoa fermentation, microbiology of cocoafermentation, and phenolic compounds in cocoafermentation. The articles were in English or Spanishand indexed in high impact journals. They wereselected according to their relevance in terms of cocoafermentation. Of 350 initially selected articles, 149 wereexcluded as irrelevant, and 101 did not fit the languagecriterion. Out of 100 English and Spanish articles, only50 were selected as corresponding with all the specifiedrequirements.RESULTS AND DISCUSSIONCocoa fermentation. Cocoa fermentation is apost-harvest process which includes several stages.The first step after cultivation is to open the pods andremove the beans. They are covered with white pulp, ormucilage, which is mainly sugars and water [10]. Theinitial pH of the pulp is 3.6. It is a nutrient-rich mediumthat encourages microbial growth. The pulp has thefollowing composition: about 85% of water, 10–15%of sugars (the concentration of glucose, fructose, andsucrose depending on the age and maturation), 2–3%of pentoses, 1–3% of citric acid, and 1.5% of pectin,proteins, amino acids, vitamins, and minerals. VitaminC and potassium are the most common representativesof vitamins and minerals. They are minor but veryimportant components [10–12].The microbiological changes during fermentationare obvious. First, yeasts ferment pulp carbohydratesand transform them into ethanol and carbon dioxide.The secretions of their pectinolytic enzymes generateanaerobic environment. The yeast stage takesapproximately 36 h. The next stage involves lacticacid bacteria that appear between 16 and 48 h offermentation. They generate lactic and citric acid,increase the acidity of the medium, and change thecomposition of the pulp.As fermentation continues, oxygen begins to comein. As a result, the temperature rises above 37°C,which boosts the growth of acetic acid bacteria. Theirpopulation reaches its peak in 88 h. Between 48 and112 h of fermentation, one can even feel the smellemanating from acetic acid. After alcohol and lacticacid turned to acetic acid, the temperature rises up to50°C. The heat finally inhibits the microorganisms thathave a life span of 120 h. After fermentation, severalfilamentous fungi have been registered in the surfaceareas and in the excess fermentation mass [13–16].Basically, the fermentation of cocoa beans beginswith the initial acidity of the mucilage and the low levelsof oxygen, which are the optimal conditions for yeasts.As these factors decrease, the lactic acid bacteria reachtheir maximum growth point. As their amount graduallydecreases, it is replaced by acetic bacteria, whichprefer ethanol, good aeration, and heat. Aerobic sporeformingbacteria and filamentous fungi often appear atthe final stage of fermentation. They are responsible forunpleasant flavors of fermented cocoa beans [17, 18].On the other hand, prolonged fermentation leads to anincrease in bacilli and filamentous fungi, which can alsocause unpleasant flavors. The physiological functions ofthe predominant microorganisms have been the subjectof countless studies, which established the crucial157Ordoñez-Araque R H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 155–162role of microbial succession in the development of thecharacteristic cocoa aroma [16].The sensory properties of cocoa beans can bedeveloped by basic conditions or by external factors,especially those connected with fermentation. The cocoaflavor increases as the fermentation time elapses, whichmeans a negative correlation with astringency. In otherwords, the astringency of the beans decreases duringfermentation [19].Stages and changes of cocoa beans. Fermentationof raw cocoa beans occurs in two stages, which, inturn, are divided into four steps. The first stage involvesmicrobial reactions that take place in the pulp and on thesurface of the beans. The second phase involves severalhydrolytic reactions that occur within cotyledons [20].The system formed to ferment the mucilage thatcovers the cocoa beans is metabolized by a successionof microorganisms. When cocoa beans are harvestedand extracted from the pod, they are exposed to naturalbiodiverse microflora that comes from the contactwith environment, crop handling personnel, transportcontainers, tools, pod surfaces, etc. [21, 22].Reactions initiated during fermentation continue atthe drying and roasting stage. Thus, oxidation reactionsreduce acidity and the amount of phenolic compoundsresponsible for bitterness and astringency. Cocoa beanscan be dried in the sun or in special dryers. However,the latter method often implies extra high temperatures,which can harden the cotyledons and decrease thequality of the finished product. The quality of cocoabeans directly depends on the genotype, harvest,fermentation, drying, and roasting. For instance, beansof different cocoa genotypes should not be fermentedtogether, as it can spoil their sensory properties [23, 24].Microorganisms present in cocoa fermentation.Traditionally, cocoa fermentation is an uncontrolledprocess initiated by microorganisms that naturallyappear in fermentation sites. These fermentingorganisms use pulp as the main substrate. At the onsetof fermentation, pulp reduces the diffusion of oxygenwithin the mass of the fermented bean, thus creatinganaerobic conditions [25]. As it was already mentioned,there are five main groups of microorganisms thatparticipate in cocoa fermentation: yeasts, lactic acidbacteria, acetic acid bacteria, and various speciesof bacilli and fungi. Unlike other fermented rawmaterials, endogenous enzymes play a crucial role inthe development of the flavor of cocoa beans: withoutfermentation, cocoa beans have no flavor. Duringfermentation, microorganisms eliminate pulp andproduce indispensable metabolites [26, 27].Yeasts. Yeasts are eukaryotic microorganisms with ahigh biotechnological potential for food industry. Theirproperties are completely different from prokaryoticbacteria. Yeasts are resistant to antibiotics, sulfa drugs,and other antibacterial agents. This resistance is genetic,i.e. natural: it cannot be modified or transmitted toother microorganisms [28]. Yeast particles are 5×10 μmin size, which is significantly bigger than the size ofbacteria (0.5×5 μm) [29].The yeast species that have been identified asthe main colonizers during cocoa fermentation arethe Saccharomyces cerevisiae, Candida pelliculosa,Candida tropicalis, Candida zeylanoides, Torulopsiscandida, Torulopsis castelli, Torulopsis holmii,Kloeckera apiculata, Kloeckera apis, Schizosaccharomyces,Kluyveromyces marxianus, Pichiamembranifaciens, Pichia kudriavzevii, and Pichiamembranaefaciens. The Saccharomyces cerevisiae is themost common strain reported in all cocoa plantations.The exact reason why a certain strain of yeast entersfermentation process still remains unknown. However,the Kloeckera apiculata does not survive 24 h offermentation as it is inhibited by the concentrationof ethanol produced in the medium. As for theKluyveromyces marxianus, it grows slowly and degradesgradually. Yeasts are active for approximately 48 h andreach the peak of their activity in 24 h. By that time,their activity has changed conditions of the medium, andother microorganisms join in [16, 30, 31].Yeasts play an important role in the pulp degradationprocess. Cocoa pulp can be fermented to produce analcoholic beverage. Yeasts demonstrate pectinolyticactivity. The secondary products of yeast metabolisminvolve organic acids, aldehydes, ketones, higheralcohols, and esters. The production of glycosidasesenzymes is important and affects the quality of beansand, subsequently, that of chocolate [32].Lactic acid bacteria. Lactic acid bacteria comprisea group of microorganisms linked by the formation oflactic acid as the main metabolite. They are a product ofcarbohydrate fermentation. Depending on the amount ofthis product, they can be homo- or heterofermentative.They share similar morphological, physiological,and metabolic characteristics. They are Grampositive,catalase and oxidase negative, not mobile,and they do not form spores. They can be anaerobic,microaerophilic, and airborne [33].Lactic acid bacteria can appear at the onset offermentation. However, they increase their numberand become active only when the pulp with its sugarsbegins to hydrolyze and leave the fermentative system,which is boosts yeast metabolism. The main speciesthat have been isolated so far include Lactobacillusplantarum, Lactobacillus fermentum, Lactobacilluscellobiosus, Leuconostoc mesenteroides, Lactococcus(Streptococcus) lactis, Pediococcus spp, and variousspecies of Bacillus.As for heat-resistant flora, Lactobacillus curieae,Enterococcus faecium, Fructobacillus pseudoficulneus,Lactobacillus casei, Weissella paramesenteroidesand Weissella cibaria have also been registered,but to a lesser extent. They exist during the first72 h of fermentation and reach their peak in 36 h.158Ordoñez-Araque R H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 155–162Their maximum growth period is 16–48 h. Theprevailing species include Lactobacillus plantarumand Lactobacillus fermentum. Lactic acid bacteriamostly produce lactic acid, but they also generate smallamounts of alcohol and acetic acid from fructose andglucose. In addition, they can use citric acid to produceacetaldehyde, diacetyl, mannitol, acetic acid, and lacticacid [34–36].Acetic acid bacteria. Acetic bacteria are Gramnegativeand belong to the Acetobacteraceae family.They are strict aerobics, non-spore-forming, ellipsoidalor bacillus-shaped. They may occur in pairs or in chains.Members of the Acetobacter genus are so common dueto their ability to grow in ethanol environment. Aceticbacteria are known to partially oxidize a variety ofcarbohydrates and to release various metabolites, e.g.aldehydes, ketones, and organic acids, in different media.For a long time, they have been used to perform specificoxidation reactions via processes called “oxidativefermentations” [37]. The first step in the productionof acetic acid is the conversion of ethanol from acarbohydrate by yeasts. The second step is the oxidationof ethanol to acetic acid by acetic acid bacteria [38].During cocoa fermentation, the population of yeastsand lactic acid bacteria decays, thus creating an aerobicenvironment favorable for the growth of acetic bacteria.The temperature reaches approximately 37°C. Theincrease in temperature triggers protein hydrolysis andacidification of the beans. As a result, ethanol dissolvesinto acetic acid, carbon dioxide, and water. Somestrains appear at 24 h and reach their growth peak at88 h. After 120 h, they can no longer be detected. Theybegin to disappear when the mass reaches 50°C. Part ofthe generated acid volatilizes, while the rest enters thebean and is responsible for killing the germ [39]. Thesebacteria play a fundamental role in the generation ofvolatile compounds that affect the quality of chocolate.The Acetobacter and Gluconobacter geni are usuallyobserved during fermentation, the most common beingAcetobacter aceti and Acetobacter pasteurianus, aswell as the recently discovered Acetobacter ascendens,A. rancens, A. xylinum, A. lovaniensis, A. xylinum,A. peroxydans, and Gluconobacter oxydans [16, 40, 41].Fermentation stages.Stage I. During the first stage of fermentation, thevolume of the pulp that surrounds the beans reducesthe diffusion of oxygen within the medium. Thisis where the beans will be fermented in anaerobicconditions. During this stage, first yeasts and thenlactic bacteria consume sugars and organic acidsfrom the pulp, thus producing ethanol, lactic acid,etc. [25].The yeast population starts with 107 CFU/g pulpand reaches a maximum of 108 CFU/g pulp. After that,it starts to decline until it reaches the bottom levelof 10 cells per gram of pulp. Yeasts are prevailingmicroorganisms, and their depectinization activitycauses liquefaction of the pulp with its subsequentdrainage, or “sweating”. The pulp loses its viscosityand lets in air [42]. As a result, the simple sugars of themucilage, namely sucrose, fructose, and glucose, turninto ethanol. The pectin degrades, causing the texture ofthe bean to change, and eliminates citric acid. The yeastswhich are generally responsible for metabolizing thisacid are Candida spp. and Pichia spp., which generatean alkaline pH. This parameter, together with alcoholand oxygen, coincidentally inhibits the yeasts and theiractivity, but contributes to the development of lacticbacteria. Yeasts also form such organic acids as acetic,oxalic, phosphoric, and malic acids. They help reducepH fluctuations [43, 44].Yeasts have become focus of numerous cocoa beanfermentation studies since they release pulp degradationenzymes. Moreover, they are also the main producers ofesters and higher alcohols, which can contribute to thecomplex mix of aromatic volatile compounds that makeup the cocoa aroma. The main yeasts that generate thesevolatile compounds are Candida sp., Kluyveromycesmarxianus, Kloeckera apiculata, S. cerevisiae, andS. cerevisiae var. chevalieri [45].The second phase of this stage involves severalhydrolytic reactions that occur within the cotyledons.As the fermentation continues and the pulp drains, moreoxygen enters the system, thus creating the optimalconditions for the growth of lactic bacteria [42]. Theycolonize the cocoa mass, degrade the glucose of the pulpinto lactic acid, and assimilate the remaining citric acid.Several studies on microbial fermentation indicatethat two most prevalent species in this process areLactobacillus plantarum and Lactobacillus fermentum.They also produce acetate esters from acetic acid, whichgive different tones to cocoa-based products [31]. Lacticbacteria can reach a population of 6.4×107 CFU/g pulp.At first, they increase the acidity by producing citricacid, but then they lower the pH by releasing productsthat are not acidic. Lactic acid bacteria are able tometabolize malic acid. These bacteria have no majorproteolytic activity and can only ferment two typesof amino acids: serine and arginine. After all thesereactions, the environment is totally aerobic, whichallows for the growth of acetic bacteria [7, 34, 46].Stage II. During the second stage, the environmentis oxygenated, and the pH has decreased due to theremoval of some components and variability of theremaining compounds. At last, acetic acid bacteria canconvert the previously obtained ethanol into acetic acidvia the oxidation of alcohol. The optimal temperatureof the acetic fermentation process is between 28°Cand 30°C, and the optimum pH is 4.5. The oxidationof ethanol is carried out in two stages. First, ethanol isoxidized into acetaldehyde. Second, the acetaldehydebecomes acetic acid. Other products include ethylacetate, butanol, isopropanol, intermediate acetaldehydecompounds, and organic acids [47].The formation of acetic acid is very important at thisstage of the process. It occurs due to the activity of acetic159Ordoñez-Araque R H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 155–162bacteria. The exothermic reactions of the bacteria raisethe temperature of the mass. The population reaches itspeak at 1.2×107 CFU/g pulp and falls down after threedays of activity precisely because of the high temperatureit generates. In some cases, the population can reach3.5×103 CFU/g pulp [16, 48].As the volume of oxygen increases, the pH reaches3.5–5.0, and the temperature becomes 45–50°C. Underthese conditions, several aerobic spores of Bacillusbacteria may appear in the fermentation. After the pileof beans has been stirred, one can detect the presenceof Bacillus licheniformis, B. megaterium, B. pumilus,B. pumilus. B. coagulans, B. circulans. B. subtilis.B. cereus, and B. megaterium. Most of them are heattolerantand can survive during drying and roasting.They are capable of producing numerous enzymes,both proteolytic and lipolytic, which catalyze reactions.However, they give cocoa unpleasant taste and smellsince they degrade proteins and fats by producingchemical substances that can distort the flavor [49, 50].CONCLUSIONThe fermentation stage is considered the mostimportant process in the transformation of cocoa tochocolate. The changes that occur in its volatile andaromatic compounds trigger structural changes in thecomposition. The changes are due to the activity ofvarious microorganisms. Their main objective is to killthe germ and thus stop the metabolism of the bean. Theresulting alcohol is broken down into acetic acid andother acids, which produce desirable sensory propertiesthat will be accentuated during drying and roasting.Yeasts, lactic bacteria, and acetic bacteria playa fundamental role in the fermentation process. Byknowing the main strains, their action parameters, mainreactions, and products, food scientists can improve thisprocess to obtain cocoa beans with a better chemicalcomposition.Each strain of microorganisms requires a separateresearch with regard to the variety of cocoa beans.The species vary from zone to zone, and plantations indifferent parts of the world are unlikely to have similarcharacteristics.CONTRIBUTIONRoberto Ordoñez-Araque and Julio Urresto-Villegascompiled the manuscript. Edgar Landines-Vera andCarla Caicedo-Jaramillo collected the data, checked thestructure, and performed the final review.CONFLICT OF INTERESTThe authors declare that there is no conflict ofinterest related to the publication of this article.ACKNOWLEDGEMENTSThe present research is our homage to the indigenouspeoples of Ecuador, who started growing cocoa5.000 years ago and introduced this delicious treat to theworld.