The Microbiology of Cocoa Fermentation and Its Role In

This article was downloaded by: [Universidad Austral De Chile] On: 31 January 2014, At: 16:10 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Critical Reviews in Food Science and Nutrition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/bfsn20 The Microbiology of Cocoa Fermentation and its Role in Chocolate Quality a Rosane F. Schwan & Alan E. Wheals a b Department of Biology , Lavras, Brazil b Department of Biology and Biochemistry , Bath, England Published online: 10 Aug 2010. To cite this article: Rosane F. Schwan & Alan E. Wheals (2004) The Microbiology of Cocoa Fermentation and its Role in Chocolate Quality, Critical Reviews in Food Science and Nutrition, 44:4, 205-221, DOI: 10.1080/10408690490464104 To link to this article: http://dx.doi.org/10.1080/10408690490464104 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions meaning food of the gods. The physiological roles of the predominant micro-organisms are now reasonably well understood and the crucial importance of a well-ordered microbial succession in cocoa aroma has been established. acetic acid bacteria 1. Rosane F. The specific name.000 years before Hernando Cort´es brought it to Europe in 1528. Reduction of the amount of pectin by physical or mechanical means can also lead to an improved fermentation in reduced time and the juice can be used as a high-value byproduct.5M tonnes and the major producers are the Ivory Coast. particularly of “fine” cocoa. Brazil. There has been a long battle in Europe to prevent chocolate products that contain only approximately 20% (w/w) cocoa solids being called chocolate but a compromise has been reached with terms such as “Family milk chocolate” being legally permitted (EU Directive 2000/36/EC). and Address correspondence to Dr. seven-day microbial fermentation of the pectinaceous pulp surrounding beans of the tree Theobroma cacao.2. Federal University of Lavras. is grown in Ecuador. fermenter design. such as ethanol. Copyright  ISSN: 1040-8398 DOI: 10. Department of Biology. Nigeria. but there are many other smaller producers. Over-fermentation leads to a rise in bacilli and filamentous fungi that can cause off-flavors. Schwan. Federal University of Lavras. Malaysia. INTRODUCTION 1. The Arriba type. processors turn this into cocoa products. and as medicine for over 2. Cocoa and Chocolate Probably originating in Mesoamerica.1. This revision of the 1973 Council Directive 205 .3 Its special status in human culture is reflected in its Latin name with genus Theobroma. University of Bath. and several multinationals such as Nestl´e. E-mail: rschwan@ufla. probably originated as an Olmec word from Mexico. Schwan Department of Biology. more research is needed on pectinase production by yeasts. 44:205–221 (2004) C Taylor and Francis Inc. 37 200 000. Jacobs-Suchard. warehouses store beans. and Zareena.000/tonne during the last decade. including members of the 3 principal groups. Bath. Cadbury-Schweppes.000/tonne in 1977 the price of roasted beans has fallen to an average about US $1. a beverage. Lavras. Trinitario. England The first stage of chocolate production consists of a natural. Cameroon.1 chocolate or cacao had already been used as a food. and Rowntree-Mackintosh. There is a microbial succession of a wide range of yeasts. yeasts. better depulping. Forastero from the Amazonas region. It has been possible to use a synthetic microbial cocktail inoculum of just 5 species. Keywords Theobroma cacao.2 The principal varieties are Criollo. lactic-acid. and acetic-acid bacteria during which high temperatures of up to 50◦ C and microbial products. now rarely grown because of its disease susceptibility. traders ship to mainly North America and Europe. To improve the quality of the processed beans. which constitutes about 5% of world trade. and manufacturers convert this into consumable products.1080/10408690490464104 The Microbiology of Cocoa Fermentation and its Role in Chocolate Quality Downloaded by [Universidad Austral De Chile] at 16:10 31 January 2014 Rosane F. and a hybrid.br Ecuador. Brazil Alan E.Critical Reviews in Food Science and Nutrition. and acetic acid. the latter two forming most of the “bulk” market. lactic acid. World annual production is approximately 2. but other important companies are the beverage conglomerate. Wheals Department of Biology and Biochemistry. Unilever. After reaching a peak of well over US$3. Firms that make chocolate almost exclusively are Mars. and the use of starter cultures. Trade in cocoa is complex: farmers produce fermented beans. to mimic the natural fermentation process and yield good quality chocolate. Hershey. with a “fine” flavor. Philip Morris. lactic acid bacteria. The “first” world dominates the commodities market that determines the price of cocoa for the “third” world farmers. kill the beans and cause production of flavor precursors. Ghana. MG. Lavras. Indonesia. Brazil. cacao. A microbial fermentation and drying process is required to initiate the formation of the precursors of cocoa flavor. and polyphenols. WHEALS Figure 1 Schematic of a microbial succession during cocoa bean fermentations. Fermentation Mature fruits (pods) rise directly from the stem of the cocoa tree and are thick walled and contain 30–40 beans (seeds). mucilaginous pulp that comprises approximately 40% of seed fresh weight. At this stage the cut beans show a purple color due to the presence of anthocyanins. The open boxes indicate the periods during the fermentations when a particular microbial group is most abundant and/or important. The beans can then be stored for up to a year but staling will eventually occur.Downloaded by [Universidad Austral De Chile] at 16:10 31 January 2014 206 R. (73/241/EEC) permits up to 5% non-cocoa vegetable fat to be used in the manufacture of chocolate throughout the EU. If there were worldwide adoption. The stars indicate the timing of peaks of metabolites and temperature.2. .5bn. 1. Brazil. The seed embryo is killed and the fruit tissues degrade which makes it much easier to dry the beans. artificial dryers are used but it is important to keep the temperature not exceeding 60◦ C and to dry slowly (at least 48 hours) during which time some excess acids may volatilize and some oxidation will occur. Each bean consists of two cotyledons and an embryo (radicle) surrounded by a seed coat (testa) and is enveloped in a sweet. caffeine. which takes from one to four weeks. A schematic of a microbial succession (Figure 1) summarizes the key events during the process that occurs during cocoa fermentations in Bahia. It would probably result in a loss in demand for cocoa beans exceeding 184. This can be done in the sun (using movable roofs to protect from tropical showers) with regular turning until the water content is less than 8%. Diffusion of these metabolites triggers complex biochemical reactions to occur in the cotyledons. the loss of revenue to cocoa producers could be more than US$1. E. F. white. Alternatively. SCHWAN AND A.4 Harvested seeds are immediately allowed to undergo a natural fermentation during which microbial action on the mu- cilaginous pulp produces ethanol and acids as well as liberating heat. The testa provides a barrier to acid penetration into the bean and diffusion out of undesirable theobromine. both of which are beneficial.000 tonnes. like all fats. and oligopeptides. is composed of a mixture of fatty acids and is typically the saturated fatty acids palmitic acid (25%) and stearic acid (35%). The sugars come from sucrose and its hydrolysis products. Most amino acids and . including catechins and epicatechin. a pale yellow. The source of hundreds of volatiles found in roasted beans (both fermented and unfermented) are the reducing sugars. the fermentation process creates hot. which undergo a further series of chemical reactions leading to the development of full chocolate flavor. Unfermented cocoa seeds do not produce cocoa flavor on roasting so an understanding of the development of cocoa flavor precursors during fermentation is required. and cocoa butter substitutes depending on the requirements of the final product. in addition to being released from glycosides. The antioxidants in cocoa can prevent the oxidation of LDL-cholesterol. flavan-3-ols. including confectionery. During this process the cracked husks are air-separated (winnowing) from the entire separated cotyledons (nibs). Endogenous acids (malic. For the finest chocolate. Bitter and astringent flavors are due to polyhydroxyphenols such as catechins. milk products.15 Theobromine and caffeine and their complexes are major components of cocoa’s bitter taste but they also tend to diffuse out of the bean during fermentation. perhaps because of the inhibitory presence of methylxanthines. Polyphenols tend to diffuse out of the bean during the fermentation and also are oxidized by polyphenol oxidazes to produce mostly insoluble tannins. Possible medicinal/health benefits of chocolate have been reported for many years but it is only recently that some of these claims are being more clearly identified and studied. “conching” is performed in order to get fine crystallization. a dark bitter material with astringent flavors from the polyphenols and tannins.8 The technology of chocolate production effectively limits mycotoxin contamination by moulds that might have occurred in the period at the end of fermentation. Mycotoxins have been found on shells but never in cotyledons. 207 Metabolic and epidemiological studies indicate that regular intake of such products increases the plasma level of antioxidants. Likewise. The nibs are ground several times at elevated temperatures to make a fluid paste (cocoa liquor) that on cooling yields cocoa mass. a dark brown residue (58% of the total). phosphoric. To make finished chocolate products. Thus.5. The basis of its “addictive” properties for chocoholics has not been identified although cannabinoids are found in chocolate at low levels. free amino acids. while lecithin is added followed by repeated milder heating and cooling cycles before filling moulds. and proteins. Processing The next stage is to roast the beans from 5 to 120 minutes and from 120◦ C to 150◦ C depending on nature of the beans and the required product. and cocoa (press) cake. Chemistry Chocolate flavors and aromas have been the subject of extensive research. Therefore it is important to ensure that neither the initial conditions nor fermentation and drying produce excess acid. In turn they depend on the sugars in the pulp and availability of oxygen for their production by bacteria.MICROBIOLOGY OF COCOA FERMENTATION Downloaded by [Universidad Austral De Chile] at 16:10 31 January 2014 1. and the polyunsaturated fatty acid linoleic acid (3%) with some others (2%). a few studies show that ROS associated with carcinogenic processes is also inhibited. citric) are probably less important because it is the diffusion of lactic and acetic acids into the bean that dominate bean acidity. Health and Nutrition Most of the health problems associated with high chocolate consumption stem from the high concentration of carbohydrates in processed chocolate rather than the chocolate itself.3 Research shows that the cocoa bean and its derived products are rich in specific antioxidants. emulsifiers. Some lactic acid is lost during drying but most of the acetic acid remains. sweeteners. The chocolate is typically heated to between 50 and 60◦ C for several hours. although it can be up to 5 days for specialist chocolate.5 In Colombia the nutritional role of chocolate is emphasized because natural cane sugar and chocolate are combined into a nutritious beverage with an excellent balance of carbohydrates. related to the mechanism of protection in heart disease. fatty liquid without any cocoa flavor. acid conditions.3.4. 1. Typically 2/3 of this material is then pressed to separate cocoa butter. 1. oxalic. a desirable attribute as a defense against reactive oxygen species (ROS). and proanthocyanadins. and the beans are roasted at over 100◦ C. contamination in cocoa mass although some bacilli may survive.7 Food poisoning organisms rarely have been reported in the final processed chocolate. The cocoa cake will then be ground to cocoa powder for use by the confectionery and other industries. lipids. during drying or if allowed to get wet during transport and storage. The melting point of cocoa butter is around 35◦ C with softening around 30–32◦ C and it becomes brittle fracture below 20◦ C. There may also be a pre-roast and thermal shock (to loosen the husk). the monounsaturated fatty acid oleic acid (35%). There is also a loss during drying and roasting. glucose and fructose. most of the cocoa butter is mixed back with the cocoa mass (liquor) together with sugar. cocoa butter is less bioavailable and has minimal effect on serum cholesterol. Cocoa butter.6 The fats from cocoa butter are mainly stearic triglycerides (C18:0) that are less well absorbed than other fats and tend to be excreted in the feces. presumably arising from contamination at a late stage in the factory. The roasted beans are then processed into chocolate. anthocyanins. it is not surprising that there has never been a single report of Escherichia coli or Salmonella spp.9 Since there are abundant health claims for polyphenols10−14 efforts are being made to maintain their levels while avoiding taste problems. tartaric. Cocoa cake is a strongly flavored but inedible material that needs further processing to become palatable.6 Since the starting material is sterile. and especially the polymers procyanidins and polyphenols similar to those found in vegetables and tea. 21.6). The initial acidity of the pulp (pH 3. The exothermic reactions of acetic-acid bacteria raise the temperature of the fermenting mass even further up to 50◦ C or more. principally lactic-acid bacteria and acetic-acid bacteria. unwashed baskets used for transport of seeds. due to citric acid. Chocolate shares with wine the distinction of being an ancient fermented product with a combination of nutritional. It consists of 82–87% water.5% pectin. The number of these organisms reaches a peak around 36 hours after the fermentation process begins and the bacterial population reached 6. then remains almost constant for the next 12 h after which there is a dramatic decline of four orders of magnitude over the next day followed by a slower decrease leading to a final population of only 10 viable cells per gram of pulp. These compounds undergo non-enzymatic browning reactions during drying and roasting. several species of yeasts proliferate. lignin. These Maillard reactions are condensations between the α-amino group of amino acids. which is followed by growth of aerobic spore-forming bacteria. beans are manually turned up to once per day. They are quite distinct from caramelization of sugars. Although they can hold up to 2000 kg of beans the depth does not exceed 50 cm to ensure good aeration. acetic acid bacteria became the dominant organisms. lined and covered with leaves. Cocoa Pulp: The Fermentation Substrate Cocoa pulp is a rich medium for microbial growth. The concentration of glucose. This is followed by a phase in which bacteria appear. are also used. than yeasts for a short period of time. favor colonization by yeasts25 that are able to utilize pulp carbohydrates under both aerobic and anaerobic conditions. Some are tiered on slopes that facilitates transfer of beans from one box to a lower one with simultaneous aeration. 2. Baskets.5 and 66. 1–3% citric acid. fermentations are often done in heaps of beans from about 25 kg to 2000 kg enclosed by banana or plantain leaves with some turning to assist aeration. 2–3% pentosans. After 120 hours of fermentation . the pulp becomes contaminated with a variety of microorganisms many of which contribute to the subsequent fermentation.22. perforated wooden boxes allowing pulp to drain away and air to enter. To ensure uniform fermentation and increase aeration.16 Proteins. and the population reached a peak at 88 hours with 1. knives. decreases progressively towards the end of the fermentation.16. 2. Finally. proteins.19 Pectin content. Smoky off-flavors from wood fires used for drying are now less of a problem.22 As aeration of the fermenting mass increases and the temperature rises above 37◦ C. The global improvement in wine quality over the last 25 years has been significantly due to better control of the fermentation process itself. evident from 48 to 112 h. and minerals are also present. sucrose. Organisms come mainly from the hands of workers. The decrease in the number of acetic-acid bacteria from three days onwards is probably due to their inhibition by the high temperature in the cocoa mass. and pectin. F. leading to production of ethanol and secretion of pectinolytic enzymes.22 The amended conditions favor the development of lactic-acid bacteria.2 × 107 CFU/g of pulp.4 × 107 CFU/g of pulp (Figure 2). E. and mystical properties.22 This stage in the microbial succession is reflected in a decline in the concentration of ethanol and lactic acid. Changes in the local climatic conditions influence the sequence of microorganisms involved in cocoa fermentation but a similar succession of groups of organisms has often been reported. or amines and the carbonyl group of reducing sugars.Downloaded by [Universidad Austral De Chile] at 16:10 31 January 2014 208 R.17 More glucose and fructose and a slight increase in total sugar concentration were observed in samples 6 days after harvest than in freshly harvested (ripe) pods. and increase in acetic acid. Microbial Fermentation On small-holdings. and Malaysia. If implemented. THE FERMENTATION PROCESS 2. and 1–1. 10–15% sugar. Plantations usually ferment for a longer period than small-holders and 6 to 7 days is usual. differences were found in the amounts of water. which does not involve amino acids.20−24 Early on in the fermentation. Typical “hammy” off-flavors are produced by over-fermentation when bacilli and filamentous fungi grow on cocoa husks and nibs to produce short chain fatty acids.22 The microbial succession in the fermentation process has been clearly established.20. A comprehensive and representative set of data from both the fermentation process and subsequent sun-drying are shown in Figure 2 (previosuly unpublished data). hemicellulose. The purpose of this review is to describe research over the last 15 years into the fermentation process and discoveries on how cocoa fermentation is involved in production of chocolate flavor precursors. Seeds within the ripe pod are microbiologically sterile. In larger farms fermentations are performed in large.24 The lactic acid bacteria exhibit the fastest growth rate during the 16–48 h period of fermentation and are present in greater numbers. perhaps yield better financial returns for farmers. SCHWAN AND A. and dried mucilage left on the walls of boxes from previous fermentations. together with low oxygen levels. some filamentous fungi may appear on the surface. WHEALS oligopeptides are produced during acid hydrolysis that occurs during fermentation.1 g kg−1 dry weight pulp.1. citrate. Nigeria.18 In a comparative analysis of pulp from beans collected in the Ivory Coast. This period of time is coincident with the decline of the yeast population.2. approximately 1% on a fresh weight basis. The beans are covered with banana leaves or sacking to conserve the heat generated during fermentation. but not necessarily in biomass. was found to 37. The size of the yeast population increases from 107 CFU/g of pulp to 108 CFU/g of pulp during the first 12 h (Figure 2). amino acids. When the pod is opened with a knife. this knowledge will enable high quality natural chocolate to be routinely produced and. medicinal. The strong odor of acetic acid. vitamins (mainly vitamin C). and fructose is a function of fruit age. . Saccharomyces cerevisiae. Filamentous fungi: open triangles. most commonly in the aerated and cooler.. Brettanomyces clausenii. C. Schizosaccharomyces spp. At Table 1 the end of the fermentation the beans are usually transferred to platforms and sun-dried. there is a sharp decrease in the total microbial population.. spore-forming bacteria can be isolated during the first three days of fermentation with populations around 104 CFU/g of pulp but their numbers remain virtually unchanged. Lactic-acid bacteria: open diamonds. Rhodotorula spp. S. C. Kluyveromyces thermotolerans.22 This phase in the succession coincides with increases in oxygen tension.. Hansenula spp.5 × 107 CFU/g of pulp... guilliermondii. Torulopsis spp. cerevisiae var.. Candida pelliculosa. Kloeckera spp. Pichia membranaefaciens. Kloeckera spp. and filamentous fungi can survive. Aerobic. During sun drying cocoa beans are often humidified to help the workers remove the rest of the mucilage with their feet but eventually only microorganisms that are able to form spores.. Thereafter they start to dominate the microbial population to such an extent that they form over 80% of the microflora. Saccharomyces cerevisiae. Saccharomycopsis spp. reukaufii. Saccharomyces spp. C. Candida rugopelliculosa.. Counts are per ml of pulp. Torulaspora pretoriensis Candida spp. Schizosaccharomyces spp. temperature. superficial areas of the fermenting mass. cacoai. Acetic acid bacteria: open squares. Filamentous fungi are found in small numbers throughout the fermentation. Saccharomyces spp.. Other Yeasts isolated from cocoa fermentations in four countries Brazil22 Ghana32 Malaysia32 Belize100 Candida bombi. Debaryomyces spp. Candida spp. There is a minor increase in the number of yeasts to 3.22. commencing after 156 hours. Candida spp. acetic acid bacteria were not found.209 Downloaded by [Universidad Austral De Chile] at 16:10 31 January 2014 MICROBIOLOGY OF COCOA FERMENTATION Figure 2 Cell density in the fermentation box and during sun drying. Schizosaccharomyces malidevorans.. .25. krusei. Saccharomycopsis spp. Pichia spp.3. Spore-forming bacteria: closed circles. Hansenula spp. The cocoa beans were taken to a sun drying platform after 156 hours. During this process. Kloeckera apiculata. chevalieri.26 reaching 5. Torulopsis spp. Saccharomyces chevalieri. intermedia. Candida rugosa. bacilli. Pichia fermentans. Kloeckera apis.. Yeasts Yeasts have been isolated from cocoa fermentations by many groups23 but only four studies have simultaneously identified yeasts and bacteria (Table 1). Hanseniaspora spp... To avoid confusion the names used in the original literature have been retained but current nomenclature is given in the appendix (see page 221).. boidinii. C.5 × 103 CFU/g of pulp22 around 132–160 hours. C. 2. Kluyveromyces marxianus. This is due to growth of thermotolerant yeasts utilizing some of the acids coinciding with an increase in the oxygen content in the fermenting mass22 as well as survivors in the cooler external layers of the fermentation. and pH of the fermenting mass. C.. Yeasts: open circles.. Lodderomyces elongisporus. Delbrueckii. and T. cerevisiae dominated the alcoholic fermentation phase and survived throughout the fermentation process. Leuconostoc mesenteroides. Lb. A. K. which were present in considerable numbers in the Brazilian study. and two species of the genus Pediococcus together with Lactococcus lactis and Leuconostoc mesenteroides were isolated29 (Table 2). were present at the early stages whereas Lactococcus spp. Lb. and Torulospora pretoriensis. A. Lb. and the yeast population was declining. Lb.27 Acetic acid bacteria isolated from cocoa fermentations in four countries Brazil31 Ghana32 Malaysia32 Belize100 Acetobacter aceti subsp. castelli. Lb. In the most comprehensive study22 (Table 1). pasteurianus.210 Downloaded by [Universidad Austral De Chile] at 16:10 31 January 2014 Table 2 R. fermentum Lb.27 and Pichia membranaefaciens. and Schizosaccharomyces in Java. Lactic-Acid Bacteria Lactic-acid bacteria increased in numbers when part of the pulp and “sweatings” had largely drained away. Plantarum Lactococcus lactis.23 Table 3 2. Lb. xylinum. Gluconobacter oxydans subsp. Candida rugosa was present up to the end of fermentation when the temperature was approximately 50◦ C. have not been reported from cocoa bean fermentations in other countries. on average.. Candida pelliculosa. Lactis. has been attributed to the metabolism of these organisms. F. casei. the Lactobacillus spp. mali Lb. These bacteria are responsible for the oxidation of ethanol to acetic acid and further oxidation of the latter to carbon dioxide and water. Pediococcus acidilactici.28 It is not possible to determine whether these differences in the yeast flora were due to geography or to fermentation practices. delbrueckii. fermentum Lb. It was also found in high numbers during the first 24 h of cocoa fermentation in Trinidad. Casei pseudoplantarum.21. Ln. Torulopsis candida. fructivorans.18 All these sugars are fermented by the above species. peroxydans. frequency of species with time was also monitored in detail.30 In general. WHEALS Lactic acid bacteria isolated from cocoa fermentations in four countries Brazil29 Ghana32 Malaysia32 Belize100 Lactobacillus. paramesenteroides studies have identified isolates of the genera Candida. cellobiosus. dextrinicus Lb.21 Kluyveromyces marxianus. the members of genus Acetobacter were found more frequently than those of Gluconobacter (Table 3). and Saccharomyces cerevisiae in Indonesia. gasseri. and Lactococcus (Streptococcus) lactis were the most abundant species in the first 24 h of fermentation. Gluconobacter oxydans Acetobacter spp. Thus the acetic acid bacteria play a key role in the formation of the precursors of chocolate flavor.27 B. Acetic-Acid Bacteria After the decline in the populations of yeasts and lactic-acid bacteria. which causes diffusion and hydrolysis of proteins in the cotyledons. Trichosporon.22 Kluyveromyces marxianus grew slowly at the outset of fermentation and then declined gradually. Candida tropicalis. the fermenting mass becomes more aerated. Candida spp. plantarum. Lb. plantarum Lb. rancens. Small numbers of Pichia fermentans and Lodderomyces ellongisporus were isolated but only during the first few hours of fermentation. plantarum. Candida zeylanoides. A. Lb. Lb. 60% is sucrose and 39% a mixture of glucose and fructose. Lb. xylinum. brevis. Two different strains of S. P. but even so. Lactobacillus fermentum. Gluconobacter oxydans . Lb. casei. A. plantarum Lb. thermotolerans. liquefaciens. Bacteria A. suboxydans Acetobacter ascendens. Glucononbacter oxydans Acetobacter lovaniensis. rancens. The acidulation of cocoa beans and the high temperature in the fermenting mass. Torulospora pretoriensis and Kluyveromyces thermotolerans were found also when the temperature of the fermenting mass was approximately 50◦ C. S. Lb. In general. brevis. occurred during the final stages of fermentation. kandleri. A. 14% of sugars. Leuconostoc mesenteroides. In Bahia (Brazil). Lb. E. Lb. Lb. A. Saccharomyces. lactic-acid bacteria. Yeast metabolism favors the growth of acidoduric. Of the lactic acid bacteria isolated from cocoa fermentations21 (Table 2). Kloeckera. Saccharomyces cerevisiae was the dominant yeast in the cocoa beans taken from boxes immediately after filling.16 Kloeckera apis. Of these. buchneri. Acidophilus. Leuconostoc mesenteroides. oenos. Kloeckera apiculata grew during the early phase of fermentation but declined rapidly such that it could not be isolated after 24 h of fermentation which probably reflects its intolerance of ethanol at concentrations above 4% (v/v). Candida spp. fermentum. Saccharomyces cerevisiae. increased in numbers after 24 h. T. Lb. SCHWAN AND A. holmii in the Ivory Coast. Ln. Pichia. Lactic acid bacteria were isolated in cocoa fermentation in Indonesia and Lactobacillus plantarum and Lactobacillus cellobiosus were the principal species. which is not surprising since cocoa bean pulp contains. cerevisiae was the most common species of yeast identified in the study probably because of its rapid growth and ethanol-tolerance. collinoides Lb. The yeast flora was abundant and varied. Lb. six Lactobacillus spp.4.31 Species of Acetobacter aceti and Acetobacter pasteurianus were isolated in Indonesia but the populations were only approximately 105 to 106 CFU/g. collinoides. This creates conditions suitable for the development of acetic-acid bacteria. Aerobic Spore-Forming Bacteria Increased aeration. implicatus. P. stearothermophilus. Mucor sp. B. including Candida spp. C. it is reputedly the acetic acid that kills the beans. B. B. mycoides. B.30 B. 2.40.26. pumilus. Production of Organic Acids Several of the yeast isolates produce organic acids including acetic. coagulans. phosphoric. This. B.7 Aerobic spore-forming bacteria produce a variety of chemical compounds under fermentative conditions. B. pasteurii.3-butanediol. B. Bacillus cereus. Indeed it has been suggested that C3 –C5 free fatty acids found during the aerobic phase of fermentation and considered to be responsible for off-flavors of chocolate34 are produced by B. Filamentous Fungi Filamentous fungi are not considered to be an important part of the microbial succession of cocoa fermentation. subtilis. subtilis Downloaded by [Universidad Austral De Chile] at 16:10 31 January 2014 C. B. These weak organic acids will have a buffering capacity and will tend to reduce fluctuations in pH.38 3. subtilis B. increased pH value (3. Fusarium moniliforme.37 It is likely that they may cause hydrolysis of some of the pulp and even the testa of the seeds. B. Other substances such as acetic and lactic acids. a great diversity of species was seen in the first 44 h of fermentation.MICROBIOLOGY OF COCOA FERMENTATION Table 4 Aerobic spore-forming bacteria isolated from cocoa fermentations in four countries Brazil26 Trinidad21 Ghana and Malaysia101 Bacillus brevis.32 (Table 4). Bacillus licheniformis. It is not uncommon for yeast species isolated from Brazil to show higher maximum growth temperatures than the corresponding species isolated from temperate sources. B. and Pichia spp. A. Thereafter Aspergillus fumigatus and Mucor racemous dominated the fungal population up to the end of fermentation. metabolize citric acid causing the pH value to increase in the pulp which allows growth of bacteria. licheniformis. cereus. circulans were isolated from cocoa beans that had been subjected to drying and roasting (150◦ C) temperatures. Paecilomyces varioti.0) of cocoa pulp. B. 400) are formed due to biochemical and enzymatic reactions that occur within the cotyledon. and a rise in temperature to about 45◦ C in the cocoa mass in the later stages of fermentation are associated with the development of aerobic spore-forming bacteria of the genus Bacillus21. Lasiodiplodia theobromae. B. 3.5 to 5. Many Bacillus spp. Ethanol Production The sugar-rich.41 Many different species of microorganisms have been characterized and the microbial succession has been defined. The major role of microorganisms is to produce acids and alcohols that will penetrate the testa and start the chemical reactions that will form the precursors of chocolate flavor. are thermotolerant and others grow well at elevated temperatures. however. Trichoderma viridae. B.34. and B. Thielaviopsis ethaceticus. oxysporum. niger. So far the roles of all these microorganisms have not been explicitly described particularly in their relative contribution to the overall quality of the final product.36. stearothermophilus. The first step in understanding this is to determine the physiology of the microorganisms and what they contribute to the dynamics of the fermentation process. glucose. acidic pulp presents ideal conditions for rapid yeast growth. B. Breakdown of Citric Acid Some of the yeasts. The loss of citric acid both in the “sweatings” and by microbial metabolism causes an alkaline drift in pH. cereus. stearothermophilus. Mucor racemosus. macerans. are also produced by Bacillus spp. ROLES OF MICROORGANISMS DURING COCOA FERMENTATION The great majority of flavor compounds (ca. all of which are deleterious to the flavor of chocolate. megaterium. spinosum. B. polymyxa. and tetramethylpyrazine. succinic. they may also produce acids or impart off-flavors to the beans. inhibits the yeasts and their activity wanes. oxalic. subtilis firmus. megaterium. Roles of Yeasts A. in the well-aerated parts of the fermenting mass and during the drying process. B. P. B. B. together with the increasing levels of alcohol and aeration.35 2. However. Measurements of ethanol show clearly how. licheniformis. it penetrates the cotyledons of the beans. B.37 Filamentous fungi isolated from fermenting cocoa in Bahia were Aspergillus fumigatus.39. These may contribute to the acidity and perhaps at times to the off-flavors of fermented cocoa beans. circulans. and fructose to ethanol and CO2 is the primary activity of the fermentative yeasts. pumilus.37 Although the numbers were small. but they have been isolated when the 211 temperature of the fermenting mass was around 50◦ C. . coagulans. and three different isolates of Mycelia sterilia. B. B. Penicillium citrinum. after rising in concentration in the pulp.5. B.1.. cereus var. B. Most of these fungi are reported to be unable to grow at temperatures higher than 45◦ C.16 They have been found quite often.. and B. Conversion of sucrose. F. There is no evidence that enzymes from the microorganisms penetrate the testa and create flavor compounds but hydrolytic enzymes inside the beans are activated by microbial metabolites such as acetic acid. Then it is possible to define the potential ecological roles of these microorganisms. and malic acids. laterosporus. megaterium. coagulans. 3 . glycerol. 3. PG of K. The great majority of lactic-acid bacteria isolated during cocoa fermentation utilize glucose via the Embden-Meyerhof pathway yielding more than 85% lactic acid. principally fusel alcohols. F.51 Dissimilation of these acids leads to an overall drop in acidity and rise in pH value. Although this study was done in pure culture. isoamyl alcohol.43. 2. Aerobic Spore-Forming Bacteria The Bacilllus species that were isolated produced a variety of chemical compounds under fermentative conditions such as 2. S. serine and arginine. chevalieri produced large amounts of aroma compounds suggesting that these strains might be collaborating in the elaboration of aroma and flavor characteristics (Schwan. Studies on wine fermentations have shown the importance of the range of volatiles that can be produced by different strains and different species. Lactic acid bacteria are virtually nonproteolytic and their ability to ferment amino acids is also restricted with only two.” The collapse of the parenchyma cells in the pulp between the beans results in the formation of void spaces into which air percolates. acids. F. These bacteria are responsible for the oxidation of ethanol to acetic acid and further oxidation of the latter to carbon dioxide and water.51 All lactic acid bacteria isolated from cocoa fermentations were able to metabolize malic and citric acids.46. Concentrations of a maximum of 6 g/L of pulp of acetic acid was found in cocoa pulp after 88 hours of fermentation. and K. marxianus was the most abundant pectinolytic yeast. marxianus. some of them could be defined as transients that only show spasmodic appearance and it is possible that the different species found in different countries or in different types of fermentation are not important in respect of the fermentation process per se. cerevisiae. fatty acids. It is clear that excess acid will interfere with chocolate flavor22. cerevisiae var. This enzyme activity is crucial during the first 24 hours because it breaks down the pulp and allows penetration of oxygen into the fermenting cocoa mass enabling aerobic acetic acid bacteria to grow. C.3 butanediol.24 Part of this acid is volatilized and part penetrates the testa (approximately 2%) and is responsible for killing the embryo. marxianus produced large quantities of heat stable endopolygalacturonase (PG). Acetic-Acid Bacteria • Production of acetic acid. chevalieri.43 It is known that volatile compounds are important in the development of full chocolate flavor. Candida sp. and water diffusing into the cotyledon act as solvents so that cellular components are transported to sites of enzyme activity and vice versa. and CO2 . some species utilize glucose via the hexose monophosphate pathway producing 50% lactic acid. F. cerevisiae var.212 R.22. Candida rugopelliculosa.50 None of the bacterial species present in the early stages of the fermentation have been shown to have pectinolytic activity.44. Production of Volatiles Yeasts produce a large array of aroma compounds. Production of Pectinolytic Enzymes Some strains produce pectinolytic enzymes20. Roles of Bacteria A. and 2-phenylethanol. chevalieri.. The exothermic reactions of the acetic-acid bacteria raise the temperature of the fermenting mass.24 However. Indeed.45 The five major yeasts that produce these volatiles (Kloeckera apiculata. and Kluyveromyces marxianus) have been studied individually. thermotolerans).48−50 that break down the cement between the walls of the pulp cells and the resultant juice (or “cacao honey”) drains away as “sweatings. ethyl acetate.29. The acidity of cocoa beans. SCHWAN AND A. Yeast Varieties It is likely that all of these biochemical transformations are necessary for a normal fermentation and species that perform some or all of them are probably essential but the other yeast species are probably unimportant.2. acetic acid.47 both necessary and sufficient for there to be representatives of each physiological/ecological group to provide the appropriate transformations during the fermentation (see section 5). However.52 B. S. diethyl succinate. Kloeckera apiculata and S. it disappeared quickly from the pulp when the mass temperature rose above 50◦ C. R. and fatty acid esters42 and different species produce different aromas. and the diffusion and hydrolysis of protein in the cotyledons has been attributed to the metabolism of these microorganisms. chevalieri were the major producers of volatiles such as isopropyl acetate.28. methanol. marxianus has been studied in more detail. Only 4 out of 12 yeast species showed pectinolytic activity (K. Among the yeasts with high fermentative power S.42.49. Their relative proportion will thus change the composition of the pulp substrate and thus may consequently change the microbial succession. and ethanol. that are extensively attacked by some of these organisms. WHEALS Downloaded by [Universidad Austral De Chile] at 16:10 31 January 2014 D. mannitol. S. unpublished observations). Lactic-acid bacteria first contribute to an increase in acidity by producing citric acid and then lower the pH by metabolizing it and liberating non-acid byproducts. Production of citric acid. E. it does give clues as to which species might be added. Only the first two showed substantial activity and only K. 1-propanol.54 even though most of the acetic acid will eventually be volatilized. It may be Production of lactic acid. sometimes to 50◦ C or more. cerevisiae var. the high temperature in the fermenting mass.53 Ethanol. It had strong maceration activity that reduced cocoa pulp viscosity during the first 36 hr of fermentation when K. Lactic-Acid Bacteria • • E. The detailed levels of chemical reactions inside the bean are still unknown. cerevisiae var. Conclusions Growth in relation to sugar and oxygen are the key parameters that establish and change the microbial succession. and rise in pH value of the cotyledon from 4. Mechanical Removal of Cocoa Pulp Brazilian and Malaysian cocoas tend to be extremely acidic (cotyledon pH about 4. Unfortunately these results could not be reproduced using a commercial depulping machine.67 This value represents . In Brazil.3. it has been used traditionally to make jelly. oxygen becomes more plentiful and then the strictly aerobic acetic acid bacteria develop. Removal of some of the pulp before fermentation reduces acidity and this presents a possible solution to the acidity problem. These bacteria may contribute to the acidity and perhaps at times to off-flavors of fermented cocoa beans.55 Revenue generated by these products exceeds that obtained from selling cocoa beans to processors. pyrazines. The acetic acid bacteria are aerobic and can grow at high concentrations of ethanol and tolerate temperatures around 45◦ C. Today the juice for commercial jelly production is pressed from the seeds before fermentation. There was a more rapid progression in the microbial succession. acetic. This mucilage blocks the void spaces in the cocoa mass. causes under-fermentation.5. It was reported that at least 10% by weight can be removed by pressing the beans prior to fermentation without measurable consequences.66.58 213 4. This liquid is almost transparent and is rich in fermentable sugars. cacao juice. Lactic acid bacteria are the next group in the succession and are microaerophilic.2. Processing of post-harvest residues and by-products of cacao (eg. If this occurs. The viscosity of the pulp still needs to be reduced in addition to reduction of pulp quantity. temperature increase. Novo Nordisk Ferment) sprayed over the seeds and allowing a reaction time of 30 minutes. 4. They produce acetic acid from sugars and also can oxidize ethanol to acetic acid and then to CO2 and water. there is no reduction of acidity of the cocoa compared to traditional box fermentations.65 This also yielded a pulp-depleted bean that when fermented gave rise to fermented cocoa beans that were less acidic. Enzymatic Removal of Cocoa Pulp The addition of pectinolytic enzymes improves the efficacy of mechanical pulp extractors. Ghana and Malaysia are also developing these industries. but little sugar on the seeds. water. and extends the fermentation period. Washing of the seeds has been used to produce a product suitable for wine production.62 Genetic differences in material cultivated in Malaysia and Brazil may be responsible for these differences since cacao cultivars in Malaysia have about three times more pulp sugars than the Brazilian comum cultivar. One liter of a 0.56. increases the quantity of pulp extracted to approximately 23% compared to the batch-type depulper. These conclusions about the physiological roles of the major groups were experimentally tested (see section 5). pectin.8 (as in traditional cocoa fermentation) to 5.60 Using a modified domestic washing machine it was shown63 that partial (20%) removal of cocoa pulp gave an accelerated fermentation.57. and acids. Facultatively anaerobic yeasts are metabolically active at the beginning of fermentation when oxygen is not available because of its occlusion by the mucilaginous pulp surrounding the seed.MICROBIOLOGY OF COCOA FERMENTATION butanediol. Oxygen is one of the factors that determines the microbial succession.59 This produced a less acidic cocoa in Brazil60 although the acidity of beans was not reduced when some of the pulp was removed prior to fermentation in Malaysia.61 A decrease in volume.51.1. Quantity of Pulp Surrounding the Cacao Seed Not all pulp is necessary for a successful fermentation of cocoa beans.3. Such a process does require a very high level of water quality and worker hygiene. Some depulpers leave loose mucilage. 4. impairs aeration. COCOA PULP Cocoa pulp is the raw material on which the fermentation proceeds and this section will describe how it seems to be a key determinant of both quality and financial viability of the process.2) and this has adversely affected the development of their international markets.5 and 4. but they also tend to remove the tightly adhering mucilaginous layer immediately surrounding the bean. Ethanol tolerant yeasts ferment the sugars at low pH (pH 3. 4. cacao jam.64 Such depulpers remove from 17 to 20% of pulp in terms of the fresh weight of the seed.59 A normal bean fermentation occurred when up to 20% of total fresh weight of beans (including pulp) was removed. and lactic acid.59 Pulp extraction on a larger scale for the cacao juice industry has been done with commercially available depulpers. Downloaded by [Universidad Austral De Chile] at 16:10 31 January 2014 3. and sugar content in cocoa pulp occurred when beans were spread out in a thin layer before fermentation in Malaysia and this method produced cocoa with less acidity. but excess pulp can also be sold as a high value commodity.2) and pectinolytic enzymes open the structure of the pulp for the ingress of air. vinegar and liquor of cacao juice) may offer opportunities for diversification on farms. especially where cocoa production is the major enterprise.2% (w/w) solution of pectinase (Ultrazym 100G. When the pulp has been degraded. The economy of cocoa-producing areas in Brazil is very dependent on the acceptance of its products in the market. Not only is the quantity of pulp crucial in affecting the efficiency and nature of the fermentation. This was probably due to differences in the technology: centrifugation is the basis of separation in a washing machine while gentle scraping is the principle of depulpers. Lactic acid bacteria are micro-aerophilic and members of the homolactic group are able to ferment sugars and tolerate this acidity. Loss of pulp occurs naturally during a fermentation because the ‘sweatings’ drain out through the holes in the fermentation box. The carboxyl groups of the D-galactopyranosiduronic acid residues are partially esterified with methanol. Some of the secondary alcohol groups at C-2 and C-3 are acetylated. and (2) chain-splitting enzymes (depolymerases) that split the βb-(1. They found that the yeast enzymes had the same optimum pH value of activity (5. The PG secreted from K. marxianus consisted of PG. holmii produced PME and that S. PG) or by trans-elimination (pectin and pectate lyases. Addition of commercial enzymes is costly and prohibitive on a large scale. and degree of polymerization are the principal elements of heterogeneity in pectic compounds of diverse origins. candida and K. indicating that PG production is constitutive under these conditions but it was unable to grow on pectin or galacturonic acid as the sole carbon source like Rhodotorula spp.4. among isolates from cocoa fermentations. marxianus.stanford/saccharomyces) has revealed a PG gene but not a PME gene suggesting either erroneous assays or mis-identification of species in at least one case.0) but differed from each other in their optimum temperature and thermal stability. with an optimum at pH 5 typical of endoPG secreted by yeasts. Genome sequencing of Saccharomyces cerevisiae (http://genome-www. SCHWAN AND A. E. no monitoring of the microbial population was done. including Kluyveromyces marxianus. High yields of PGs were obtained with selfinduced anaerobic batch fermentations of K. marxianus could macerate potato and cucumber slices and decrease the viscosity of cocoa pulp by 50% within 18 min. WHEALS an increase of about 5% of total weight over the machine. This change also helps pulp processing for pasteurized juice as well as for cacao soft drink production that is bottled and stored at ambient temperatures. Of the 12 yeast species isolated from cocoa fermentations in Brazil. and K. the pulp had a lower viscosity. and Torulopsis candida were the only pectinolytic yeasts isolated from cocoa fermentations in another study. An increasing number of yeast species have been discovered to have pectinolytic activity. and 33 kDa. either by hydrolysis (polygalacturonases.69 The degree of esterification.74 The researchers suggested that K. According to analysis of all bands by densitometry. (1) to increase microbial pectinolytic activity at the onset of fermentation. gel filtration resolved four peaks containing PG activities. Pectinases Produced by Yeasts Pectins give pulp its sticky.74 In trials with pure “starter” cultures of yeasts. and T. and (2) making a source of enzyme obtainable from yeast cultures themselves. 41.55 As the pectin chains were broken by the added enzymes.50 A study of the kinetics of appearance of the enzyme using sub-cellular fractionation showed it was secreted by the classical yeast secretory pathway. 4. Unlike some pectinases. The enzymes of T.78 PG secreted by K. S. thermotolerans produced extracellular endopolygalactoronase (endoPG). marxianus with 100 g l−1 glucose as the sole carbon source75 but production is inhibited by oxygen.69 The neutral sugars. which was also found to be the most active producer of PG. Candida norvegensis. marxianus endoPG activity was not affected by buffers used across the pH range studied. cerevisiae var. norvegensis produced the greatest amount of extracellular enzyme. Since high endoPG activity in early stages of fermentation speeds the fermentation process and leads to better quality of chocolate.50 These data suggest that the anaerobic conditions that rapidly predominate after initiation of natural cocoa fermentations are ideal for the appearance of the enzyme but that its production becomes self-limiting as the pulp drains away and air percolated through the fermenter. 35.77 Addition of pectin or polygalacturonic acid to the growth medium did not increase enzyme secretion. overproducer . marxianus interfered with the development of the wild yeast flora. cerevisiae72. and Zygosaccharomyces were found. fragilis had the highest optimum temperature (60◦ C).67 However. and cohesive properties. The amounts and properties of each PG differed but all were relatively unstable compared with that of K.Downloaded by [Universidad Austral De Chile] at 16:10 31 January 2014 214 R. about 85% of total protein secreted into culture medium by K. marxianus was similar to that reported for PGs from other yeasts. K. Two alternative approaches are being explored to provide a better quality of fermented beans by speeding up this process.48. The relative molecular masses were calculated and the four PG forms had apparent Mr of 47. rugopelliculosa. Torulopsis candida. chevalieri and Candida zeylanoides secreted PG. the proportion of neutral saccharides. C.76. This strain fermented the major pulp sugars as well as degrading pectin. viscous. PL).4)-linked residues of D-galactopyranosiduronic acid. the natural substrates of pectic enzymes. Since good color and flavor are also due to bacterial activity.50 Neither PME nor PL was detected in culture filtrates.49. Pectin and pectic acid. From concentrated culture supernatant of K. Pichia. Among the other strains studied.4)-glycosidic bond. Dgalactose and L-arabinose and sometimes D-xylose and L-fucose. marxianus was characterized and showed activity from pH 4 to 6. Some laboratory experiments have suggested that the yield could be improved by inoculation with pectinolytic yeasts.71 Yeasts from cocoa fermentations produced various pectinolytic enzymes that aided the maceration of cocoa pulp and the drainage of “sweatings. Saccharomyces. marxianus.69 Enzymes that attack pectin can be assigned to two main groups: (1) de-esterifying enzymes (pectinmethylesterases. and the fermented beans were only analyzed by anthocyanin content. chevalieri. total fermentation time was reduced from seven to four days and the acidity of the final product was reduced. PME) that remove the methoxyl groups from the esterified acid. form the side-chains of the pectin molecule. the results could have been partly due to other microbiological activity. marxianus. the experiments are difficult to evaluate since only 1 kg samples of beans were used. F. Based on an assessment of both external and internal (“cut-test”) color of beans. chevalieri. the pH value did not rise during the early stages of fermentation. K.73 ). C.”28 They claimed that Saccharomyces chevalieri (now classified as S. are branched heteropolysaccharides in which the backbone contains L-rhamnose residues and αa-D-(1. S.70 In Java pectinolytic yeasts belonging to the genera Candida. The effect of temperature on endoPG activity from K.68. with special status for quality. Two species of Lactobacillus were selected—L.1. ethanol. marxianus could be used directly on cocoa beans to speed up the process and enhance the quality of the final product. chevalieri produces pectinase. quality will become even more important. The medium in which the cells are grown is thus suitable for direct use without purification or concentration. over-expressing strains are constructed with heterologous DNA sequences and are therefore classified as genetically modified organisms (GMOs). Genetics of EndoPG Production The genes for endoPG have recently been cloned from strains of Saccharomyces cerevisiae. The advantage of these hosts is that (1) the plasmid carrying the endoPG is stable and requires no selection using conventional systems and (2) that the strains can be grown on either cheese whey (an industrial waste product) or on sugar cane juice. A cocktail of these microorganisms was inoculated on cocoa beans immediately after the pod was broken open and left to ferment in sterilized 200 kg wooden boxes for 7 days. The lactic-acid bacteria were selected after observing production of lactic acid at acidic pH. and temperature tolerance. The enzyme output from these sources is at least 50% higher than wild strains and constitutes about 90% of secreted protein. An attempt was made to do this by conventional chemical mutagenesis using nitrosoguanidine. can ferment all pulp sugars at pH 3. oxygen requirement. Even more important is that GMOs are currently under public scrutiny and are often perceived as abnormal and undesirable.85 By isolating the structural gene and putting it on an expression vector it has been shown that it is secreted and functional. Poor practices are widely reported and even led Ecuador. unpublished data). lactis and L.79. The yeast Saccharomyces cerevisiae var. a widely available and cheap commodity in tropical countries where cacao is grown. It consisted of one pectinolytic yeast species.87 Over-expression strains should facilitate the process of producing a pure enzyme. Gluconobacter oxydans was also added to oxidize the ethanol to acetic acid and to CO2 and water. two lactic acid bacterial species. However.000 mutagenized cells only a few strains produced enhanced levels of the enzyme and the best was only 25% above wild type levels. PG from K. marxianus to create new expression systems (Jia and Wheals. unpublished data).84 One of the most interesting observations with respect to Saccharomyces cerevisiae was that the non-pectinolytic laboratory strains used in genetic studies contain a PG homologue. K.86 Only one copy of the gene is present on the genome in both of these yeasts in contrast to filamentous fungi where at least four are present in Aspergillus niger.82 Transcription analysis has shown that the gene can be induced under special conditions of nitrogen starvation with induction of the pseudohyphal development pathway. It therefore seems likely that the best approach would be to screen additional strains for the desired enhanced activity. marxianus has the status of an organism that is generally regarded as safe (GRAS). The three key metabolites in the pulp.2. This is a lengthy and costly process. even better would be the inoculation of fermentations with overproducer strains. site-directed mutagenesis of the active site of endoPG) but without success (Jia and Wheals.5–4. lactic acid. and acetic acid showed similar sequential rises and falls to that found in spontaneous fermentation. Chocolate produced with such a strain would never find a market! Alternative strategies have been tried (increase in chromosomal copy number.5. To this end the endoPG from a number of these strains has been taken from the original cloning strain (S. cerevisiae) and transferred in turn to both K. However. substantial improvements in productivity were never likely and in a screen of 18. to have its rating downgraded in 1994. Starter Cultures From knowledge of the microorganisms responsible for spontaneous cocoa fermentations and their physiological roles during the process. Contamination from extraneous microorganisms was kept to a minimum. Indeed. an attempt was made to manipulate the fermentation.79–82 Saccharomyces bayanus. plantarum. is ethanol tolerant.50 It was concluded that a more directed approach might be more profitable. CHOCOLATE QUALITY One of the reasons that chocolate quality has not been a priority for farmers is that there is no financial incentive to produce high quality fermented cocoa beans. 5. However. Downloaded by [Universidad Austral De Chile] at 16:10 31 January 2014 4.24 From 12 yeast species. and was present at the beginning of natural fermentation. a defined microbial cocktail was selected for use as an inoculum. with a constitutive (deregulated) gene. such as methanol from pectin methylesterase. The beans were then dried and . This section is directed to pointing towards general improvement in practice that can be achieved by using appropriate procedures.82. Although the addition of an enzyme would be useful. and 30 bacterial species that had been identified. the possibility of local production of the enzyme has been investigated. This would have the additional benefit of being suitable for use on any other pectinaceous fruit. and two acetic acid bacterial species. In the absence of contaminating enzymes and undesirable byproducts. particularly if they were stable enough to continually re-infect fresh batches of cocoa beans. As an alternative to over-producing strains.83 and Kluyveromyces marxianus.MICROBIOLOGY OF COCOA FERMENTATION strains might be useful in improving quality. The best producers of acetic acid that were also tolerant to temperatures of 45◦ C were isolates of Acetobacter aceti. lactis and K. 5. as increasing numbers of farmers and countries are attempting to take control of all the processing that occurs in the tropical countries and the global market reaches saturation. To use such a GMO 215 would require satisfying the safety aspects for the regulatory authorities of the country concerned. It would also require disposal of the husks that are currently left to rot in the fields. The natural course of a fermentation takes about seven days and the microbial flora evolves in a more or less predictable manner. and fermentation boxes would all need decontaminating. It is likely that the events of the first 24 hours of the fermentation entrain the subsequent microbial succession (see section 3. If non-GMO yeast strains can be produced that can secrete enhanced amounts of endoPG. Bringing unopened pods close to the boxes for washing would only help if there were a source of washing water that was free of fecal contamination. transport them in unwashed containers. Clearly there is still room for improvement since there was no guarantee that the best species had been selected or that they had been inoculated at the most appropriate rates. It is likely therefore that all cocoa fermentations contained both strongly fermentative and pectinolytic yeasts.Downloaded by [Universidad Austral De Chile] at 16:10 31 January 2014 216 R. This would certainly enable control of the inoculum to be achieved but there could be adverse effects on the chemistry of the pectin and the beans leading to deleterious changes in overall quality. subsequent enzymological work suggested that Kluyveromyces marxianus alone or K. Pulp removal by shearing alone in commercial depulpers was not as effective as a combination of centrifugation and shearing given by a domestic washing machine. and may even outgrow. cerevisiae might be a better choice of yeast(s). Some of these companies also have the capacity to produce batches of special yeasts and other. non-yeast microorganisms to order. This will not be easy without a change in some traditional practices. the starter culture inoculum. On a global scale. It has already shown that supplementing natural endopolygalacturonase (PG) with commercial pectinase led to a faster fermentation and a higher quality product.” However. there is little change between countries and the ones used in the cocktail were always present. Since there is no quick way in which raw materials and tree varieties are going to change. Provided the product is of high quality and the chocolate producers are prepared to pay a premium for this enhanced quality then it will be economic for farmers to purchase and continue to use starter culture strains. containers. natural fermentations are subject to random fluctuations in the inoculum and the fermentation does not always proceed correctly. washing the pods before breakage does help considerably to reduce contamination but at present farmers break open the pods in the fields with contaminated machet´es.65 Redesign of fermenters could be possible and this is being investigated. more likely. 5. a large number of companies already supply fresh pressed. Bacterial spore-formers and filamentous fungi that appear during the latter stages of the fermentation are usually associated with the appearance of off-flavors and spoilage. Once a suitable strain or.2. E. These results show that representatives of the three major groups may be sufficient to complete the complex fermentation and that the basic features of the microbiology of cocoa fermentation are understood. In the only other study where yeast identification went to species level.E) but a detailed study of the lactic acid and acetic acid bacteria may also be useful. A taste panel found the product as good as a “natural fermentation. and pour them straight into wooden fermentation boxes containing the residues of the previous batch. or “instant” yeast cultures for baking. both of which include pectinolytic yeasts. Education and training for the farmers may also encourage them to let the fermentation take its full course. consortium of strains has been defined it will be possible to create cultures for direct addition to initiate fermentations. marxianus together with S. Furthermore it implies bringing the pods to a more centrally located “factory” with appropriate facilities and economies of scale. these should have the same effect as adding pectinase. Mechanical turning is not a realistic alternative and forced aeration requires careful control. or wine making. Further ahead we can envisage the need for proper pasteurization of the materials. perhaps with a disinfectant. One way to explore the generality of this result is to examine the species found in other countries (Tables 1–3). Aeration is known to be important in the fermentation but adequate supplies of oxygen are dependent on reduced viscosity of the pulp and regular turning of the bean mass. Field trials are now in progress (RF Schwan. It is rather difficult to assess this stage since the state of the beans . unpublished data). SCHWAN AND A. Reducing the amount of free carbohydrate will undoubtedly affect the growth and biochemistry of the bacteria but it is unclear what effect this will have with respect to quality and flavor. dried. Knives. A more precise investigation needs to be done on this in order to establish a clear endpoint to the fermentation when harvesting is optimal. the pectinase-producing variety of Saccharomyces cerevisiae (var chevalieri) was found. For example. WHEALS roasted and chocolate was produced by the usual means.1. brewing. starting with a high density will help the new population to establish itself but it will certainly be necessary to reduce the host microbiota. inoculation with a defined starter culture may be an important way in which reliable fermentations may be achieved relatively quickly. Defined cocktails should always be more reliable if only because of the lack of spoilage organisms and the fermentation time was less than the normal fermentation. F. It also suggests that the physiological analysis presented in section 3 is correct and that the consortium of microorganisms was correctly chosen with respect to their physiological roles. For example. In the other studies both Saccharomyces and Candida genera were found. With respect to the bacteria. These were encouraging results since the fermentation occurred normally and the product was more than acceptable. Manipulation of the Fermentation Depulping is an approach that is advantageous to the course of the fermentation because excess pulp can lead to an over-acid fermented bean but the precise design of the depulping machine is important. A potential problem with using starter cultures is that the resident microbial flora will compete with. Clearly. New designs may be needed and certainly the cost needs to be kept as low as possible. 3.8 (the optimum for aspartic endopeptidase). Attempts have been made to stimulate polyphenol oxidase activity that decreases during fermentation and drying.40. If the pH becomes too acid too soon (pH < 4. It is clear that the research done by major manufacturers on roasting and processing has enabled them to produce good quality products from inferior sources. Early results show that it can mimic the natural conditions of fermentation boxes and produce fermented beans for making good quality chocolate in five days (Freire.91 Both of these enzymes are very pH-dependent in their activity. However.4. has been reported to be a significant contributor to flavor.90 and it would be premature to say that it is fully understood. Inoculum. and UIT1). NJ) has patented a method to maintain high levels of putatively beneficial polyphenols. simultaneous.88 Alternatively Mars Inc. This has had some success in ameliorating the effect in some poorer quality Indonesian beans. more hydrophobic oligopeptides and less free amino acids are produced.23) genes that are expressed during seed development have been cloned from T. oxalic acid. In other words. all had similar potential for producing raw cocoa with high aroma potential. unpublished data). controllable. it is the acidity during the fermentation that is more crucial for flavor development. cacao93 and thus prospects for the identification of the flavor precursors are now good. The aspartic endopeptidase splits VCG at hydrophobic amino acid residues. one of the endogenous acids. which of the hydrophobic amino acid residues and hydrophilic oligopeptides are responsible for the cocoa aroma is not yet known. Experiments with alternative. and . (Hackettstown. and mechanized fermenters in an otherwise very traditional industry. When the pH during proteolysis approaches pH 3. related storage proteins or alternative peptidases both fail to produce appropriate flavor precursors.97 Initial results also show that it performed well in comparison with traditional fermentations although the process was stopped after four days.96 5.4. Use of a defined starter culture so that there is a well ordered succession and timely production of acids diffusing into the cotyledons. Fermenter Design Further work along these lines will produce a more precise understanding of the entire process and elucidate how the various microbiological factors determine the final outcome of the fermentation. by acidic incubation.39.44 In essence.15 Chocolate flavor chemistry is very complex and is determined by both the cocoa plant variety and the fermentation and roasting process89. Flavor Under-fermented beans have an astringent and bitter taste due partly to the presence of high levels of polyphenols. and dynamic analysis of all aspects in controlled conditions if a really good understanding of the process is to be achieved such that defined changes will have predictable and quantitative outcomes. Criollo. Combined with defined inocula there is the prospect of producing the best quality chocolate reliably and in less time. Downloaded by [Universidad Austral De Chile] at 16:10 31 January 2014 5. and turn rate can be controlled. 217 Although final acidity can be reduced by slow (sun) drying to allow the acids to volatilize94 and produce a less acid product. Roasting of these precursors in the presence of reducing sugars produced significant cocoa aroma.5) there will be both a final reduction in flavor precursors and an over-acid product. particularly by the Technical University of Braunschweig. On the other hand pH values close to 5. aeration.44 This is further emphasized by an analysis of VCG proteins and their proteolytic degradation products amongst five widely used cacao genotypes (Forastero.92 Two aspartic proteinase (EC 3. duration of the period of optimum pH and final pH are crucial for optimum flavor. attention to the primary aspects of good quality plant material and well controlled fermentations will lead to both improved final products and the need for less processing. temperature monitored and samples taken at intervals. SCA 12.95 Although acetic and lactic acids produced during fermentation are the key determinants. Although they can give rise to different quality chocolate. Research with such fermenters will accelerate the number of variables that can be studied and allow earlier use of this information by farmers. has resulted in the determination of the more important chocolate flavor precursors. timing of initial entry. Trinitario. Both the biology and the chemistry are complex and it will need a comprehensive. Thus. with respect to the organic acids that diffuse slowly into the cotyledons. 6. fermentation may still be the key to cocoa quality. on vicilin (7S)-class globulin (VCG) storage protein that produces the cocoa-specific precursors. aspartic endopeptidase and serine carboxy-(exo)peptidase. The products of this hydrolysis are substrates for the serine exopeptidase that removes the hydrophobic amino acid residues at the carboxyl terminus of the hydrophobic oligopeptides. However in a consumeroriented world where less processing and additives are desirable. To achieve some of these aims a sterilizable stainless steel vessel of novel design capable of turning a 50 kg load of beans has been constructed.8 (the optimum for serine exopeptidase). Schwan and Serodio.41. lead to an increase in hydrophilic oligopeptides and hydrophobic amino acids.MICROBIOLOGY OF COCOA FERMENTATION throughout the box is not uniform but delay in termination could lead to loss of quality. These are the first reports of making largescale. but work over the last thirty years. it is proposed to be the combination of two proteases. Another approach has been to modify a rotary drier enabling it to ferment up to 9 tons of beans. CONCLUSIONS We propose that reliable fermentations giving consistently high quality products can be achieved by a combination of three procedures: • • Control of the amount of free pulp at the start of the fermentation such that the final pH is not too acid. . amount of pulp. and Keen. 382:677–678... J. This has a different and more fruity flavor but can be processed in much the same way98 to produce “cupulate... J. [7] Barrile. A. 175:147–155. Eds. All aspects of the process need attention. M. [4] Lopes. Intern. 2002. . Flavanols and procyanidins of cocoa and chocolate inhibit growth and polyamine biosynthesis of human colonic cancer cells. [14] Kris-Etherton. C.. from raw materials to microorganisms to fermentation vessels to post-fermentation processing. F. 130: Supplement 8S. Romanczyk. and Piomelli. Geyer. 2002. breweries developed their own strains for use as starter cultures. and Keeney.. P. (eds). H. Mars incorporated.. M. Nguyen Ba1..M. Y. T...312. 375:542–543. [3] Erdman. L.J. N. Food Control. Anticlastogenic activity of cacao: Inhibitory effect of cacao liquor polyphenols against mitomycin C-induced DNA damage.D. Natsume. is very closely controlled.L. Kumon.. The authors thank CNPq. [8] Cordier.F. 2002. [13] Carn´esecchia. United States Patent 6.W. Brain cannabinoids in chocolate. Tew.. There is relatively little control over the raw materials and perhaps not even the best varieties of Theobroma cacao have been planted— quality may have taken second place to quantity in choice of tree. etc) can lead to understandable and reasonably predictable effects—but this is only a start. Schneidera. R.H.. Med.J. Microflora of cocoa beans before and after roasting at 150◦ C. 2000. Pharmacother. Science. 2001. S. R. D. The true history of chocolate. The clearest demonstration that the basic physiology and ecology of cocoa fermentation is understood is that biotechnological manipulation of the component parts (microorganisms.. M.. 9. Reed.A.. The fermentation process itself has been largely neglected. WHEALS Improved fermenter design to optimize the physical aspects of the process especially aeration Another approach to improving flavor would be to make chocolate from Theobroma grandiflorum (cupua¸cu). K. 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Cacoai Kloeckera apiculata Kloeckera apis Kluyveromyces fragilis Saccharomyces chevalieri Saccharomyces fragilis Schizosaccharomyces malidevorans Torulopsis candida Torulopsis castelli Torulopsis holmii Bacteria Gluconobacter oxydans subsp. suboxydans Lactobacillus acidophilus Lactobacillus casei pseudoplantarum Lactobacillus cellobiosus Lactobacillus kandleri Lactobacillus plantarum Current name Dekkera anomala Candida guilliermondii var. membranifaciens Issatchenkia orientalis Pichia norvegensis Pichia anomala Metschnikowia reukaufii Pichia farinosa Hanseniaspora uvarum Hanseniaspora guilliermondii Kluyveromyces marxianus Saccharomyces cerevisiae Kluyveromyces marxianus Schizosaccharomyces pombe Candida saitoana Candida castelli Saccharomyces exiguus Gluconobacter oxydans Thiobacillus acidophilus Lactobacillus paracasei subsp paracasei Lactobacillus fermentum Weissella kandleri Lactococcus plantarum 221 .