[advances in food research] advances in food research volume 8 volume 8 || fermentation, drying, and...

FERMENTATION. DRYING. AND STORAGE OF CACAO BEANS

BY P . A . ROELOFSEN

Laboratory of General and Technical Biology. Technical Uniuersity. Delft. Netherlands

I . Introduction . . . . . . . . . . . . . . . . . .

1 . Fermentation or Sweating . . . . . . . . . 2 . Drying . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

1 . Organisms Founcl . . . . . . . . . . . a . Fungi . . . . . . . . . . . . . b . Yeasts . . . . . . . . . . . . . c . Lactic Acid Bacteria . . . . . . . . . . d . Acetic Acid Bacteria . . . . . . . . . . e . Other Bacteria . . . . . . . . . . .

2 . Composition and Variation oE the Microflora during Fermentation . 3 . Changing Environmental Conditions in the Pnlp during Fermentation

a . Temperature . . . . . . . . . . . b . Aeration . . . . . . . . . . . . c . Chemical Factors . . . . . . . . . . .

Effect of External Conditions . . . . . . . . .

I1 . Essentials of Methods Used in Cacao Processing

111 . Histology of the Seed IV . External or Microbiological Ferinentation

4 . Explanation of the Seqnencc of Microorgnnisms and the

a . Normal Fermentation . . . . . . . b . Abnormal Fermentation and Effect of Changing the

External Conditions . . . . . . . c . Addition of Chemicals . . . . . . d . Use of Pure Cultures . . . . . . .

5 . Small-Scale Fermentations . . . . . . . V . Internal or Enzymatic Fermentation and Conscquences

1 . Death of the Cotyledons

2 . Enzymes in the Cotyledons

4 . Polyphenols in Fresh Cotyledons

. . . . . . . a . Criteria of Death in Cotyledons . . . . . b . When and Why the Cotyledons Die . . .

. . . . . . 3 . Changes in Nonpolyphenols in the Cotyledons during

Fermentation and Drying . . . . . . . . . . . .

5 . Changes in Polyphenols during Fermentation . . 6 Changes in Polyphcnols during Drying 7 . Special Oxidation Period and Postfermentation . .

b . Postfermentation . . . . . . . .

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a . Special Oxidation Period . . . . . .

225

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Page 226 227 227 228 228 230 231 231 232 234 234 235 235 238 238 238 239

240 241

242 244 245 246 248 249 249 250 253

259 264 269 270 276 276 277

226 P. A. ROELOFSEN

8. Flavor and Aroma . . . . . . . . . a. Suppositions on the Constitution of Flavor Precursors . b. Foniiation of Flavor Precursors during Cacao Processing

. . . 1. Moldiness . . . . . . . . . . . 2. Attack by Insects . . . . . . . . . 3. Control Measures . . . . . . . . .

VII. Nceded Research . . . . . . . . . . References . . . . . . . . . . .

VI. Storage of Commercial Cacao in TropicaI Climate

Puge . . 278 . . 279 . . 281 . . 286 . . 287 . . 288 . . 289 . . 289 . . 290

I . INTRODUCTION

The curing of fresh cacao seeds entails the processes of fermentation and drying. The manner in which this is performed determines the flavor and aroma of the cocoa' and chocolate obtained on roasting and processing the cured cacao. For this reason the curing of cacao is always described, although superficially, in books on cocoa and chocolate manufacture (e.g., Fincke, 1936; Chatt, 1953).

Scientific research on cacao curing began about half a century ago. This is evident from articles written by different authors, who were or had been working in Ceylon, Puerto Rico, German West Africa, Surinam, and St. Lucia respectively and which were compiled in a book edited by Hamel Smith (1913).

These scientists were aware that during fermentation, microbiological processes broke up the juicy tissue that covered the seeds and killed the kernels. They also knew that during drying enzymatic browning of tannins occurred, called internal fermentation.

Much earlier, experience had shown that the roasting of unfermented dry cacao did not result in the desired flavor but in a strong odor reminiscent of broad beans, Moreover, the kernels of such cacao did not possess a chocolate coIor, but were slaty-colored or, if they belonged to a variety with white cotyledons, grayish-white.

About 1927 results of microbiological research appeared in the form of three doctoral dissertations prepared under the direction of Henne- berg in Kiel and two papers by Ciferri from Puerto Rico. In 1935, Knapp, a research chemist of Cadbury Bros. Ltd., England, who had investi- gated the subject a t intervals in several countries producing cacao, published an admirable review of the status of our knowledge of cacao curing at that time. His essays, first published in the Bulletin of the

1 Unfortunately cocoa and cacao are often used to denote identical material. We will use cacao when referring to the beans, either fresh or the raw product as used by manufacturers or roasted, cocoa when referring to cocoa powder, the manufactured product for making the beverage, and chocolate when referring to the manufactured preparation, rich in fat and used for eating.

CURING OF CACAO BEANS 227

Imperial Institute, appeared in 1937 in a slightly elaborated form as a book, which ever since has been highly valued as a standard work of reference and information.

In 1935 cacao producers in Java requested that a thorough study of curing be made. This was carried out for two years by the present author and subsequently for two years by Giesberger. It was the first and is still the only research covering the whole field of cacao curing. Restric- tions on the communication of the results were withdrawn in 1946 but since the publication which then appeared ( Roelofsen and Giesberger, 1947) was in Dutch, the present author may be excused for paying considerable attention to this investigation, Perhaps the most characteristic feature of the literature on cacao curing i s that it is widely scattered in local journals and consequently of many of the authors are not aware of work performed elsewhere.

The synopsis given by Knapp (1937) has helped the situation as far as the literature up to 1936 is concerned, It is hoped that this review will have comparable value!

II . ESSENTJALS OF METHODS USED IN CACAO PROCESSING

1. FERMENTATION OR SWEATING The ripe fruits, called pods, are picked and broken or cut open after

which the seeds, called beans, are extracted by hand. The seeds are then transported in boxes or baskets to a central fermenting house containing a series of special wooden boxes with perforated bottoms to allow juice from the fermenting mass to run off. The first of this series of boxes is filled with beans to a depth of 20-90 cm. (commonly to 60 cm.). The mass of beans is then covered with jute bags, large leaves, or a wooden lid to retain the heat. After $4 to 154 days, the mass is transferred with wooden shovels into a second box and covered again. This process is repeated at fixed intervals, e.g., every day or every other day until the fermentation is discontinued. The duration of the process varies in different countries, seasons, and estates from 2 to 12 days. CriolIo cacao with large, round beans and white kernels, or cacao of Forastero-Criollo hybrids having a mixture of white and purple (violet) kernels, is usualIy fermented for 2-4 days. The bulk of the world's crop, consisting of Forastero-cacao with smaller, flatter beans having only purple kernels, is usually fermented for 5-12 days. Detailed descriptions of some procedures are given by van Ha11 (1932) and Knapp (1937). However, as Ball (1951) has recently stated for Trinidad conditions, these procedures differ even an adjacent estates and may not be changed for decades.

228 P. A. ROELOFSEN

As a result of the action of microorganisms the temperature of the seed mass rises to 3a35OC. (86-95OF.) after 1 day, 35-45OC. (95- 113OF.) after 2 days, and thereafter to 45-50°C. (113-122"F.) until a decline sets in after about a week. The temperature depends primarily on the degree of aeration but also on details such as the method of covering, frequency of turning, ripeness of the seeds, outdoor temperatures, etc.

On the small farms, which actually produce the greatest part of the world's crop of cacao, the quantities are often too small and the insula- tion too inadequate for proper fermentation. This results in the drying of living seeds and is deleterious to flavor development on roasting. Often immature, overripe, or even germinated beans are mixed, turning is neglected, contamination with earth occurs, beans are damaged while cutting the pods, and sometimes fermentation is even performed in holes in the earth.

In several countries it is customary to wash the fermented beans prior to drying, e.g., in Java and Ceylon, mainly because this improves the appearance of the raw cacao. However by washing, the seed coats become more fragile and a further inconvenience is the loss in weight of about 4.5%.

2. DRYING

Subsequent to fermentation the water content of the beans (about 60%) must be reduced to Iess than 7.5%. (Some estates and practically all small farms use sun-drying for 1-4 weeks, but on many estates hot air drying in kilns or rotary driers for 2 4 days is applied after 1 or 2 days of sun-drying.) The evaporation rate is limited by the diffusion rate in the kernels. This is determined mainly by the temperature of the material, The air temperature, however, should not exceed 6OOC. ( 140OF.) since this will impart a burnt odor to the cacao (see however, Vos, 1956).

Because the sun-drying of thin layers of beans is very rapid, the process is economical and, moreover, an attractive red-brown color is produced in the seed coats as a result of photochemical oxidation of tannins. Usually, the dry cacao, commonly called raw cacao, is graded before bagging and shipment.

Ill. HISTOLOGY OF THE SEED

The microscopic anatomy of the seed was described by Tschirch (1887) with respect to alcohol-treated material and raw cacao. Since some details are different in the living material, a brief description will be given here (see Fig. 1 ) .


The oval, more or less flattened seed has an embryo, usually called the kernel, which consists of two irregularly formed cotyledons and a radicle, the latter being called the germ by manufacturers. Apart from some small vascular bundles and the epidermis, there are two types of cells in the cotyledons. The first type is small and contains starch granules, one or more aleurone grains, and many small fat droplets

FIG. 1. Histology of the tissue of cotyledon, beeswing, seedcoat, and endocarp of the cacao seed.

(partly crystallized in alcohol-preserved material). The bulk of the tissue is formed by this type of cells. The second type of cell is found scattered among the first type, often in groups. These latter cells are larger and completely filled with flavonoid polyphenols giving a bright red color with vanillin-hydrochloric acid and demonstrating other catechol-tannin reactions. In purple (violet) cotyledons these cells also

230 P. A. ROELOFSEN

contain anthocyanine. More of the second type of cells are contained in the outer than in the central part of the kernel. According to Brown ( 1954), who isolated these cells from dried cotyledons, they also contain all of the theobromine and caffein and their total dry weight is 1&13% of that of the cotyledon tissue. The radicle contains some starch, tannin, and fat (different from cotyledon fat), but no pigments.

Between embryo and testa and also between the convolutions of the cotyledons in a filmy membrane, the so-called beeswing, consisting of flattened endosperm and, peripherally, a very thin layer of perisperm. It does contain some fat, starch, and protein, but no tannin.

The testa or seed coat sometimes called skin, shell, or husk, is com- posed of the two integuments grown together. The outer one contains some vascular bundles and longitudinal rows of big cells with very thick mucilaginous cell walls. Both integuments contain some scattered colorless cells with catechol-tannin and other cells having very little fat and starch. The outer epidermis of both integuments is con- spicuously thickened. In unripe seeds the dried seed coat is distinctly thinner and more fragile. Skin rigidity varies greatly with different clones. The seed coat is stretched by the growing embryo, and sometimes the thickened epidermis of the inner integument is torn locally; The whole seed coat may even be split open at the edge of the seed, resulting in brown spots and broken skins.

Connected with the outer integument is a thick white layer of endocarp, consisting mainly of big tubular cells with large intercellular spaces. In unripe seeds this layer is turgid, but in ripe ones it is col- Iapsed and juicy, the so-called pulp. It contains much sugar and citric acid, but no starch, fat, alkaloids, tannin, or pigments. During fermentation its cells die and are disconnected, forming a coating of a somewhat granular paste, loosely adhering to the seedcoat.

Polyphenol oxidase, the enzyme catalyzing the browning of the cacao kernels, is found in the tannin-free tissue of the cotyledons, in the endosperm, and in small quantities in the outer integument near the vascular bundles.

IV. EXTERNAL OR MICROBIOLOGICAL FERMENTATION

Scientists investigating cacao fermentation early realized that the microflora was responsible for the maceration of the pulp and for the killing of the pulp and the seed. As a result of pulp maceration, the drying rate is increased and if desired, the pulp may be washed off easily, The death of the pulp cells results in the dripping off of much juice, the so-called “sweatings.” Killing the kernels by fermentation starts the production of substances which may later develop flavor and

231 CURING OF CACAO BEANS

causes them to become brown during drying. Some of this flavor production occurs during drying and storage, but most of it during roasting. This flavor development is considered by some to be influenced by metabolic products of microorganisms, both by changing the conditions in the dead kernels (acetic acid formation) and, more directly, by producing odorous substances such as esters,

The changing smell of fermenting heaps and the examination of microscopic slides of fermenting pulp give evidence of radical changes in the composition of the microflora. However, until 1935 microbiological research consisted of little more than the isolation and the cIassification of some of the organisms found in fermenting masses or on raw cacao. A good review of these early works is given by Knapp (1937, p. 16) . This discussion will be confined mainly to two investigations, namely those of Roelofsen and Giesberger (1947) (carried out in Java during the 1935 and 1936 seasons mainly on the Siloewok Sawangan estate, on the north coast near Semarang) and of Rombouts (1952) performed in Trinidad (British West Indies) during the 1949-1950 seasons.

1. ORGANISMS FOUND The seeds in the healthy pod are sterile, but when extracted by hand

are inoculated with a variety of microorganisms. These contaminants are the main ones found on the pods although the microorganisms living in pulp adhering to baskets and boxes used for transporting the seeds to the fermentation house also occur commonly. Sometimes there is contamination with soil and hence soil microorganisms. In the ferment a t’ ion house numerous Drosophila flies also contribute to the inoculation with microorganisms. Nicholls ( 1913) proved their potential role, but their actual effect is uncertain. Numerous types of organisms will grow initially on the pulp juice, but these are soon overgrown by other organisms more adapted to the conditions during fermentation. Thus, organisms isolated at the very beginning may be disregarded.

a. Fungi (Exclusiue of Yeasts)

In several cacao producing countries species of Aspergillus, Mucor, Penicillium, and Rhizopus have been isolated from fermenting beans. However, visible mycelium is found only on outer parts of fermenting heaps which have been turned infrequently or not at all. Knapp (1937, p. 32) and Dade (1929) give pictures of such occurrences. On Java estates mycelium may be found onIy on the top Iayer of the cacao mass, provided it has not been turned during the last 24 hours and has a thickness of less than 30 cm. Evidently fungi are of no importance in the normal process, but in unturned heaps they may cause secondary mold

232 P. A. ROELOFSEN

infections in the raw cacao. In small-scale fermentations the skins are sometimes eaten through by fungi ( DeWitt, 1952a). Fungi attack cell walls much more rapidly than the other microorganisms found in fermenting cacao.

b. Yeasts

The odor of aIcohol and baker’s yeast leave no doubt that yeasts are very active in fermenting cacao, especially during the first l$$ days. Rombouts (1953) critically reviewed the description of the species of cacao yeasts previously isolated. To these may be added four yeasts from Java cacao, identified by the Central Bureau for Yeast Cultures at Delft (Roelofsen, 1953) and yeasts from Trinidad isolated and identified by Rombouts ( 1955).2 Many of these yeasts have also been found by Joly (1955) on the surface of different tropical fruits (not cacao) in Brazil, which confirms the view that cacao seeds are inoculated primarily by the fingers of the laborers who open the pods. Isolated in different countries and found in great numbers are: Sncchromyces cerevisiue var. ellipsoides and other Sacch. sp., Candih krusei, Kloeckera apiculata, Pichia fermentans, Hansenula nnomala, and Schizosacchuromyces pombe.

In the very beginning many species occur. After 1/!L to 1 day those requiring little oxygen dominate, e.g., Sacchuromyces sp. After two days of fermentation, when conditions become more aerobic and when alcohol and acetic acid are the main sources of carbon, aerophilic, pseudomycelium forming yeasts (e.g., Candida krusei) constitute the main part of the yeast flora. These are thermotolerant, living in the pulp at 45-50°C. (113-122OF.). Aerophilic yeasts prefer the outer parts of the fermenting heap and semianaerobic ones dominate in the center. In Trinidad, at all stages of fermentation, the cells of nonsporulating species outnumber those of spore forming ones.

Viable yeasts (probably as spores) also occur on raw cacao, but outside the fermentation boxes they grow only on partially dry or rewetted beans. In heaps of these, a white bloom of yeasts may be found within 12 hours. If heating has occurred, theobromine crystals may be found between the yeasts (Roelofsen and Giesberger, 1947). Seeds with torn seed coats may have yeasts between seed coat and kernel, but whole seeds remain sterile internally.

Of greater importance than the names of the yeasts is their role. 2 In order of decreasing frequency: Candida krusei, Tomlopsis rosei, Pichia

farinosa, Sacch. cereoisiae var. ellipsoides and other var., Kloeckera apiculata, Han- sen& anomala, Schizosacchammyces pombe, Pichia fermentans, Trichosporon pul- lulans, Pichia membrunaefaciens, Candida catendata.


Twenty-four strains of yeasts obtained from fermenting cacao in Java and West Africa and several species from the Central Bureau of Yeast Cultures a t Delft (not isolated from cacao) were prown by Roelofsen and Giesberger on fresh sterile cacao beans in flasks. These flasks were buried up to their tops in the bulk cacao crop of the same day and fermented along with it. The following phenomena were observed:

1. In all cases the pH of the pulp juice rose from 3.7 to 4.0 after two days. Apparently all could dissimilate citric acid. This was confirmed by aerobic growth in a synthetic medium with sodium citrate as the sole carbon source.

2. In all cases the cotyledons of the beans were killed after 30-35 hours, a few hours later than in the surrounding mass. The cotyledons were considered dead when tannin diffusion in them was apparent. Some substance produced by the yeast was the killing agent since ( a ) sterile beans died after 45 hours and ( b ) the outer cotyledon parts died earlier than the center.

3. Although at that time yeasts were thought to be incapable of macerating plant tissue, about half of the strains used appeared to macerate the pulp cells; so the beans could be washed easily like the bulk of the cacao. Some of these strains came f ron Java or West Africa cacao, but others were not from cacao at all. The time needed to kill the cotyledons was similar with macerating and nonmacerating yeasts.

An enzyme capable of macerating both fresh pulp and the collen- chyma in transverse sections of plant stems (method described by SIoep, 1928) was demonstrated to occur in macerated pulp produced with introduced yeasts as well as by natural fermentation. It was absent in sterile pulp and nonmacerated pulp supporting yeast growth. Pectin esterase was absent or a t least negligible in fresh as well as in macerated pulp. Juice of molded papaw fruits and the culture liquid of Clostridium pectinovorum also macerated cacao pulp.

Since the pectin depolymerizing activity of macerating yeasts seemed negligible and since these yeasts did not dissimilate pectin or produce pectin esterase, which as far as was known always accompanies pectinase ( polygalacturonase), Roelofsen ( 1936) first thought the enzyme was the hypothetical protopectinase commonly postulated at that time. Later ( Roelofsen, 1953), using paper chromatography, he observed enzymatic production of uronic acid from pectin by culture liquids of several yeasts from Java and West African cacao. In recent years a pectinase from Saccharomyces fragilis has been extensively studied by Luh and Phaff (1954). They found a considerable decrease in activity when 25% of. the glycosidic bonds in pectin were broken. Again no pectin esterase was found. According to Luh and Phaff (1951) pectin hydrolysis is a

234 P. A. ROELOFSEN

rare property among the yeasts, which is a t variance with the findings just mentioned.

After washing and drying, the cacao fermented with pure cultures of macerating yeast strains did not differ fro- 1 raw cacao except for the more reddish coIor of the shells, developed during sun-drying, and the absence of the usual smell of acetic acid. Some yeast strains produced a fruity esterlike odor when growing on cacao beans which was still detectable after drying the beans. However, the flavor on roasting was normal.

c. Lactic Acid Bacteria

Some investigators claim to have recognized lactic acid bacteria on microscopic observation of fermenting pulp, but the isolation of these bacteria and the realization of their abundance was not proven until 1935 in Java. Rombouts (1952) confirmed their presence in Trinidad but stated that, unlike those in Java, they could not be cultivatGd aerobically on yeast agar plates with 2% glucose and 2% calcium car- bonate. Probably the reason is that in Java an extract of fresh baker’s yeast was used instead of mannite as in Trinidad.

All strains isolated in Java were of the same type: heterofermentative rods of the genus Betnbacteriwm ( Orla-Jensen) . Betacoccus was seldom found. In Trinidad only rod types were found.

In Java, when sterile beans were inoculated with lactic acid bacteria and fermented in flasks along with the mass of material, the pH of the pulp juice rose, the cotyledons died 10-20 hours later than normal and maceration of the pulp never occurred. Here too, a killing agent was produced in the pulp. I t was probably acetic acid which had a final concentration of 2.1% in the juice. Bacterial growth was slow with fresh beans, the pH of 3.7 evidently was too low.

d . Acetic Acid Bacteria

Because of the strong smell of acetic acid it is commonly accepted that acetic acid bacteria are present in abundance. In Germany, Eck- mann (1928) was the first to isolate them both from raw cacao and fermenting juice sent to him in bottles by boat from the Kamerun. He described eight different types, four of which were known. However, it is doubtful whether all were of importance in fermenting cacao since Eckmann isolated them from enrichment cultures in flasks with wort alcohol inoculated with the cacao material.

In Java, 14 strains were isolated from fermenting beans (12-60 hours), All belonged to the species Acetobacter rnncens Beijerinck ( Ber- gey ) . Acetobacter melanogenum Beijerinck, which produced a brown


pigment, was less frequently isolated. Rombouts (1955) found the same two species in Trinidad. In Java, the well known cellulose membranes of Ac. xylinum were observed in drains under the boxes made for transport of “sweatings.”

On sterile seeds fermenting in flasks along with the estate cacao, growth of acetic acid bacteria is slow since the pH is too low, but as it rises growth increases. Acetic acid, however, is not produced. The kernels die at the same time as in noninoculated sterile beans and the pulp is not macerated. Acetobncter melnnogenzim removed the pulp in pieces together with the outer epidermis of the seed coat, probably not enzymatically but by means of some metabolic product acting on the mucilage cells.

Eckmann (1928) and Rombouts (1952) found 44O and 45OC. ( 11l0 and 1 1 3 O F . ) respectively as the temperature limits for growth in CUI- tures although many occur in the cacao mass at 5OoC. (122OF.). Rom- bouts postulated a constant infection of the center by condensed water from cooler surface layers; however, it should be pointed out that in cacao pulp the temperature limit might simply be higher than on plates.

e. Other Bacteria

Spore-forming aerobic bacilli have been found in Jamaica and West Africa (Knapp, 1937, p. 29) . In Java they rarely occurred and then only at the start of fermentation (probably from spores), but in Trinidad they were abundant after 256 or more days. None were identified. Anaerobic spore-forming bacteria have not been observed in fermenting cacao in Java or Trinidad.

Nonspore-forming bacteria of the genus Aerobacter were found both in Java and in Trinidad, but in appreciable numbers only after more than six days. This was simultaneous with the onset of putrefaction.

2. COMPOSITION AND VARIATION OF THE MICROFLORA DURING FERMENTATION

Studies on the actual composition of the microflora and its variation have been performed only in Java (Roelofsen and Giesberger, 1947) and Trinidad (Rombouts, 1952). The mean maximum number of organisms per bean after 1$$ to 2 days was estimated in both cases to be two million.

Figure 2 demonstrates the change in composition during 254 days of cacao fermentation in Java, the start being at the time the beans are put into the fermentation box, which is about 6 hours after opening the pods.

During the greater part of the harvesting seasons, yeasts dominate

236 P. A. ROELOFSEN

during the first day. In the beginning there are more species than later. Eventually they nearly disappear, as indicated by both plate counts and microscopic observation. They are succeeded by lactic acid and acetic acid bacteria, the former dominating until the end of the second day, and the latter thereafter until the fermentation ends after Z$$ days. By then most of the remaining yeasts are of the aerophilic type. This sequence is typical for all Java estates, but on some it is retarded due to abnormal procedures.

1 2 DAYS 28

2 lo9 PER BEAN

. 4.1

- 4.0 * 3.9

- 3.0 3 3.7

- 3.6 -

0.5 1 4.5 2 DAYS 2.5

FIG. 2. Variation in temperature, pH of the puIp, and composition of the microflora in fermenting cacao in Java (Roelofsen and Giesberger, 1947).

At the end of the harvesting season in certain years, the pulp is less juicy, resulting in more aeration, and hence higher temperatures. The roles of the yeasts and of the acetic acid bacteria are reduced under such circumstances.

In Fig. 3 the data of Rombouts have been plotted. Here also the start is about 6 hours after the pods are opened. There are some differ-


ences as compared with the Java fermentations. Acetic acid bacteria dominate one day earlier; the activities of lactic acid bacteria are sub- ordinated. There is a dramatic decline in the total number of organisms after 2 days.

In Trinidad the microflora has been followed up to the 8th day; the composition apparently is established after 3 days. From that time until the 8th day spore-forming bacilli dominate, after which they are mixed with non spore-forming types. However, Rombouts remarked that the former were mainly present as resting spores,

': ---OTHER TYPES 1.6 t- / ' L - S P O R E FORMING BACILLI 1.4 - 1.2 - 1.0 - 0 8 - 0.6 -

ACETIC A.B.

0 0.5 1.5 2 DAYS 35

FIG. 3. Variation in temperature, pH of the pulp, and composition of the microflora in fermenting cacao in Trinidad (Rornbouts, 1952).

The question arises as to how in Trinidad a temperature of 45-5OoC. (113-122OF.) can be maintained for 5 days with so few organisms, so many of which are originally present as spores. Are there exothermic enzymatic processes to bridge the gap between 4 5 O and 5OOC. (113- 122OF.) as supposed by Wadsworth and Howat (1954)? Wadsworth

238 P. A. ROELOFSEN

(1956) has observed that in a carefully insulated mass of sterile beans, 52OC. ( 1 2 5 O F . ) may be .reached although the beans die long before that. This might have been caused by oxidation of polyphenols and other enzymatic processes.

Both in Java and in Trinidad it was found that during the first two days great differences may exist between the upper and the central parts of the fermenting mass as a result of lower temperature and more aeration in the former.

3. CHANGING ENVIRONMENTAL CONDITIONS IN THE PULP DURING FERMENTATION

Knowledge of changing environmental conditions in the fermenting pulp is essential for a deeper insight into the causes of the changing composition of the microflora.

a. Temperature

Numerous observations of the course of the temperature rise in fermentation boxes have been published. The graphs in Figs. 2 and 3 may be taken as typical for normal estate fermentation in bulk, Max- imum temperatures of 45-5OoC. (113-122OF.) are reached in 2 to 21/, days followed by a decline after 5 days. On turning over the mass of beans, there is a temporary cooling, followed by a more rapid rise of temperature. A t the beginning of the harvesting season the mass warms up more slowly than it does later on.

b. Aeration

No analysis of the air between fermenting beans has been recorded. Roelofsen and Giesberger (1947) observed a rapid rise of the temperature in the tube of an inverted large funnel placed on the mass in a fermenting box having a temperature of 48OC. (118OF.). This did not occur when the perforations in the bottom of the box were closed. Apparently, air ascends through the mass.

Aeration increases during fermentation since 7-14 kg. juice (5-12 kg. water) per 100 kg. fresh cacao drips off in 2% days (most of it during the first day). About 5 kg. dry matter disappears as a result of metabolism. Since in addition to metabolic water, about 2 kg. water evaporates (Roelofsen and Giesberger, 1947), at least 120 cubic meters of air must have passed through 100 kg. cacao in 2% days.

After long periods of drought and with overripe beans, there is less pulp and consequently more aeration and a more rapid rise in temperature, Furthermore, turning the mass during the first 3 days increases aeration and temperature since the beans are piled more loosely and hitherto unexposed parts of the pulp are aerated.


During the first 24 hours aeration in the center of the mass is negligible since the temperature is low. The pulp is voluminous and has not yet coIlapsed. Moreover, air is kept out by the carbon dioxide produced during alcoholic fermentation. In this period, pulp colored with dilute methylene blue decolorizes. As the pulp collapses and dries out progressively, aeration is facilitated. After l$$ to 2 days, methylene blue is no more reduced in the outer parts of the pulp; however, the inner parts of the pulp will certainly be free of oxygen because of the large number of respiring organisms present at that time. This is confirmed by the fact that many lactic acid bacteria are present in the pulp. Since browning of the cotyledons does not occur, it is apparent that anaerobic conditions continue under the skins for several days.

c. Chemical Factors

The pH, sugar content, ethanol, and acetic acid of the juice are important factors in growth of the microflora and death of the beans. The data obtained by Roelofsen and Giesberger (1947) are given in Table I. Determinations of pH, total acidity, and acetic acid, obtained by earlier investigators, are similar.

TABLE I Changes of Cenditions in Fermenting pulp^

Hours fermented

0 19 23 39 46 80

Fermentable sugar b 10-13 11 4 0.3 0.2 0.7 Ethanol b 0 1.8 3.6 2.7 2.0 2.0 Volatile acid as acetic acidb 0.04 8.2 0.2 1.2 2.4 2.4 PH 3.6 3.8 3.9 4.1 4.3 4.5 pH (Rombouts, 1952) d 3.7 3.8 4.0 4.1 4.2 4.5 Titratable acid e 4.8 4.6 2.5 2.7 3.6

a From Roelofsen and Giesberger, 1947. b In per cent of water in pulp and skins. c Colorimetric. d Glass electrode. e Arbitrary units.

Of the 10 to 13% sugars in fresh juice, about 2/3 are monuses and 1/3 sucrose. Forsyth (1949), using paper chromatography, found only glucose, fructose, and sucrose. As is evident from Table I, the sugars have practically disappeared after l+$ days. The decrease is faster than the figures seem to indicate since the content is expressed in terms of the decreasing water content of pulp and skins. Later there is a small increase in sugars which is ascribed to the diffusion from dead kernels into the skin.

240 P. A. ROELOFSEN

When expressed on dry weight, polysaccharides in the pulp increase during fermentation. According to Saposhnikova (1953), this is due to enzymatic synthesis without help of living cells. However, even if there were an increase in absolute quantity, this might very well be cell wall material, glycogen, etc., of yeasts and bacteria.

The maximum quantity of ethanol (after one day) occurs when the number of yeasts present is highest. The unexpectedly high content of ethanol after 256 days is ascribed to the presence of ethanol in the kernels since whole beans were used for the determination instead of pulp and skins.

The early increase in acetic acid is attributable to lactic acid bacteria. The maximum is reached after 2 days, then it decreases in accordance with the abiIity of acetic acid bacteria to oxidize the acid (Knapp, 1937; Wilbaux, 1937). Forsyth (1953) found 2.5% acetic acid in the pulp after about 2 days and 1.6% later.

The pH gradually rises from 3.6-3.7 to 4.5 within 2j/, days and to 6.5 after 7 days (Rombouts, 1952). At the start pulp juice contains about 1% of citric acid (Knapp, 1937), which, apart from small quantities of amino acids, seems to be the only free organic acid present (Forsyth, 1949). Being metabolized by both yeasts and lactic acid bacteria, it disappears and is replaced by the less dissociated lactic and acetic acids. This explains the constant increase in pH despite a minimum of titratable acid after 156 days. The maximum titratable acidity is reached after 256-3 days and then is followed by a progressive decrease ( Knapp, 1937; Knaus, 1934).

After 30-36 hours following the death of the kernels, polyphenols, alkaloids, etc., diffuse from the kernel into the seed coat and pulp, where the polyphenols are oxidized, thereby causing a progressive browning. When the seed coat is torn, juice oozes out, producing a conspicuous brown spot. It is not known whether or not polyphenols or other substances from the kernel have any influence on the microflora. MacLean (1953) found that molds invading cacao pods and beans utilize theobromine but it is not known whether acetic acid bacteria and yeasts also do so.

4. EXPLANATION OF THE SEQUENCE OF MICROORGANISMS AND THE

EFFECT OF EXTERNAL CONDITIONS

Experiments on changes in the fermentation procedures have been carried out in many of the producing countries. The purposes of these experiments were to obtain a greater yield or a more valuable product with more flavor, less acetic acid, more plump beans, more uniform appearance, etc. The acetic acid of cacao often has been considered

CURING OF CACAO BEANS 24 1

objectionable or even detrimental to flavor production during roasting. For this reason and perhaps to obtain more esterlike substances which are supposed to improve the flavor, a prolongation of the period of yeast domination has often been attempted. Because of the lack of information on the composition of the microfloro and the ecological factors which determined it, most trials met with little success, even those conducted for relatively simple purposes such as elimination of acetic acid bacteria (Knapp, 1937). More recent trials have been more successful in changing the microflora in special cases such as when the beans were unripe. These experiments, however, have not produced a better product than that obtained by the planter by following his traditional method.

a. Normal Fermentation

Roelofsen and Giesberger (1947) gave an explanation for the sequence in the composition of the microflora ordinarily found in Java. In fresh pulp, the rather low pH of 3.6, high sugar content, and the low oxygen supply favor yeasts, which outnumber all other organisms during the first day. The oxygen supply in the center of the piles is sufficient for true semianaerobic yeasts, but aerophilic species prefer the surface layers, On the outermost beans true molds grow until the mass is turned.

As a result of citric acid consumption, the pH rises gradually, becom- ing progressively more favorable for the development of lactic acid bacteria. The environment is suitably anaerobic for these organisms since oxygen consumption by the yeasts is so intensive that even methylene blue is reduced. In the meantime temperature increases and when it exceeds 3OOC. (86OF.), it probably becomes less favorable for yeasts and more favorable for lactic acid bacteria. This, at least, has been the experience in the fermentation of wine, Moreover, the heterofermentative lactic acid bacteria produce acetic acid, which under anaerobic conditions is unfavorable for yeasts. Thus after 24 hours, when the pH is nearly 4.0 and the temperature is between 32 and 36OC. (89 and 96OF.), lactic acid bacteria quickly outgrow the yeasts.

However, the pH rises further and when it passes 4.0 it becomes favorable for acetic acid bacteria which are able to grow on ethanol. This substrate is unsuitable for lactic acid bacteria which cease growing due to the lack of sugar. As aeration becomes more intensive, the acetic acid bacteria multiply quickly after 30 hours and outnumber the lactic acid bacteria after 2 days. With the development of acetic acid bacteria, the accompanying yeast population changes its character. There are more aerophilic, pseudomycelium-forming types, most of which can likewise use ethanol as a substrate.

242 P. A. ROELOFSEN

The pH of the pulp continues to rise because both acetic and lactic acid are respired, except for that which diffuses into the dead kernel. At pH values above 5.0 Aerobncter appears. These organisms produce amines and ammonia from amino acids and when the pH is at neutrality, the cacao has a putrefactive odor which attracts carrion flies. The color of the skins blackens as a result of heavy polyphenol oxidation and chinon condensation.

Naturally there are variations of this general picture. In Trinidad, Rombouts (1952) found much less lactic acid bacteria than normally occurs in Java. He also observed a rapid reduction in the total number of organisms after 2 days, probably as a result of the high temperature. Subsequently, aerobic spore-forming bacilli increased in numbers.

Even in the same country, variations occur in different seasons and with different methods of fermentation. For instance, in the later harvesting season of 1935 in Java, conditions were found to be less favor- abIe for yeasts, and hence because of the low production of alcohol, there were also fewer acetic acid bacteria. This resulted in the abnormal predominance of lactic acid bacteria shown in Fig. 2.

b. Abnormal Fermentation and Effect of Changing the External Conditions

More extreme conditions regularly occur when unripe beans are fermented in bulk. This is normally done in Java at the end of the harvest when all pods are picked in order to prevent a moth (Acrocer- cops crumerelln Sn.) from infesting the pods. Unripe beans have much pulp which is stiff and contains less sugar acid (pH 3.8-3.93). Initially, aeration is very good and the respiration of the yeasts results in a very rapid increase in temperature. In Java a temperature of 4145OC. ( 106-113°F.) was noted after only 15 hours (Roelofsen and Giesberger, 1947, p. 110). In Trinidad Knapp (1926, 1937) observed a temperature of 4OOC. (104OF.).

When the mass is turned after 15 hours, the pulp collapses and becomes gummy. The juice is ropy and will not run off; consequently, aeration is greatly reduced. The temperature remains at 3!5-40°C. (95-104OF.) and even drops following the second turning after 36 hours. As a result of the anaerobic conditions and a pH value ns high as 4.0 after 15 hours, yeasts are quickly outnumbered by lactic acid bacteria. This situation remains constant for several days. Acetic acid bacteria are practically absent and since the period of yeast growth is SO

short, the pulp is not macerated. According to Roelofsen and Giesberger (1947) a more normal

sequence of microorganisms and better cacao are obtained by ferment-


ing unripe cacao on bamboo matting in 5-cm. layers for 2 days. The beans are then killed by holding them in boxes in the usual manner for 2 days. A still better procedure is to expose unripe pods for a week to the sun’s heat, prior to fermentation. Many beans will ripen during such a treatment.

Differences in aeration also explain the slow increase in temperature and the slow change of the microflora of fermenting underripe beans as compared with these changes with ripe and overripe beans; overripe beans have less pulp and produce less sweating. When the pulp is too dry, however, artificial wetting may give a better fermentation. (Rom- bouts, 1952).

Of course, in superficial zones of fermenting masses, acetic acid bacteria appear earlier and in greater numbers than in the center, where more lactic acid bacteria occur. The same differences are found between thin layers of fermenting cacao and thick ones (20 and 60 cm.) With very thin layers ( 5 cm.) on bamboo matting, it is possible to preserve a yeast-dominated microflora. This may be done prior to fermentation with normal layer thickness as is desirable for unripe cacao. It may also be done for I f $ days subsequent to a normal fermentation of ripe cacao in order to kill the beans. In the latter case, yeasts will dominate the acetic acid bacteria for several days until putrefactive bacteria appear. Apparently low temperature in the thin layers favors the yeasts in their competition with acetic acid bacteria. Somewhat thicker layers may be used when air is forced through the mass (Roelofsen and Giesberger, 1947).

By turning the mass of normal cacao during the first 2 days, when access of air is a limiting factor, fermentation will be accelerated and the temperature will rise. When turned and unturned masses of cacao are fermented for the same periods of time the former will appear as if it has been fermented some hours longer.

Another indication that aeration is a limiting factor for fermentation during the first 2 days is the variable rate in different types of boxes. When the bottom and sides are tight, fermentation is slow. On the other hand when the perforated bottom of the box is covered with a coarse bamboo matting, the rate of fermentation is increased. Furthermore com- pact coverings retard fermentation. When no covering is used, fermentation is also retarded because of cooling by evaporation and heat transmission. The most effective covering is an insulatins space of air that permits aeration. This may be obtained by use of a thick layer of frayed and curled old banana leaves covered by dry gunny bags, or gunny covered bamboo roofing located 10 cm. above the cacao, or a perforated wooden lid.

244 P. A. ROELOFSEN

Further examples of the effect of heat transmission on the rate of fermentation are to be found in the observations of Roelofsen and Gies- berger (1947) on experimental and commercial fermentations on different estates in Java. On estates located at a higher altitude (400 meters as against 20 meters above sea level), fermentation was retarded. This could be prevented by placing the fermentation boxes in a closed rather than open shed. Rain lowers air temperature in the tropics but increases the humidity so it takes up less water upon passing through the fermenting cacao. This explains the retarding effect of rains in one country and the accelerating effect in another. Examples are given by Rombouts (1952), who also points out that universally valid interpretations of the influence of weather are not possible. Evidently, fermentation is accelerated somewhat if the space under the box is low and not ventilated since air passing through the cacao under these conditions will be humidified and preheated. If this space is dry and open the rate of fermentation will be retarded (i.e., 4 hours in the first 60 hours).

Spraying warm cacao with water (i.e., 20 liters per 100 liters cacao) after 2 days of fermentation markedly retards the process, Fermentation is almost completely stopped by submerging the cacao in water. This is a procedure often used in Java to keep the cacao during the night until sun-drying is possible without the disadvantage of prolonged fermentation,

c. Addition of Chemicals

As long as scientists have been interested in cacao fermentation, they have tried to influence the microbiological process by adding chemicals or by inoculating the beans with certain microorganisms.

Addition of sulfur dioxide, as a means of helping the yeasts to com- pete with bacteria, has been tried in different countries by Steinmann (1927), Busse et al. (1929), Knaus (1934), Wilbaux (1937), and Roelof- sen and Giesberger (1947). About 5 liters of a 0.5% solution of sodium hydrosulfite added to 100 liters of cacao after 1 day of fermentation will retard the process, but not change the microflora. The prevention or even reduction of the growth of acid bacteria was not accomplished in any experiments involving the use of sulfur dioxide.

The addition of acid was equally effective in retarding bacteria but more beneficial to the growth of yeasts. Zeller (see Busse et al., 1929) and Wilbaux (1937) used lactic and citric acid respectively, but the quantities used were too small. According to Roelofsen and Giesberger (1947) 5-10 liters of 0 5 1 % sulfuric acid per 100 liters of cacao, mixed with the beans after 1 day is more effective. This decreases pulp pH by 0.4-0.8 units and markedly favors the growth of yeasts. By repeating


the procedure the period of yeast growth may be prolonged considerably. This is especially true if after 136 days the procedure is combined with fermentation in thin layers or with forced air circulation. The dried raw cacao is practically free of acetic acid, and has an esterlike odor caused by yeasts, but chocolate prepared from it is not considered sig- nificantly different from other cacao fermented by the usual procedure in the same period of time. Recently Dittmar (1954) also claimed to have lengthened the period of yeast growth by adding hydrosulfate as judged by the decrease in volatile acid content of the fermented kernels. However, his experimental results are not decisive since he used 5-kg. experimental samples in bags, buried in the untreated bulk cacao.

The effect of acid confirms the important role of pH as described earlier. The difference in microflora of fermenting cacao and coffee beans is mainly a matter of pH of the pulp, which at the beginning is 3.6 and 5.5 respectively. By adjusting the pH to 6.0 and 3 3 respectively, a typical coffee fermentation may be induced in cacao and a typical cacao fermentation in coffee (Roelofsen and Giesberger, 1947).

Knapp (1937) discussed the effect of adding sugars. Roelofsen and Giesberger (1947) added 0.6-2 kg. sucrose per 100 kg. beans after 15 hours of fermentation. This increased the concentration of sugar in the pulp juice by 2 7 % . As was observed earlier there was little increase in temperature, confirming the view that aeration is the limiting factor during the first day. Yeast growth was favored, more alcohol was produced and hence acetic acid bacteria were also favored. MacLean and Wickens ( 1951) added 600 ml. of 5% glucose to 40 lb. lots (about 0.5% in pulp juice) which was fermented in baskets. They observed no effect when the glucose was added at the start and a drop in temperature when it was added after 2 days. The amount is small and the drop probably results from the addition of water.

In Java the addition of sugar, together with ammonium sulfate and potassium phosphate was tried by Roelofsen and Giesberger (1947) but apart from a very small acceleration, no effect of the salts was apparent.

d. Use of Pure Cultures

The addition of pure cultures to fresh cacao has been often tried to prevent growth of acetic acid bacteria or at least to increase the growth period of yeast. Busse et al. (1929) failed to achieve success by the addition of the lactic acid bacterium, Thermobacterium delbriicki. Chierichetti (1939) advocated the use of Termobacterium ( Z ~ Q - m o w ) mobilis, the alcohol-forming bacterium occurring naturally in palm wine, but this did not meet response.

Preyer (1901), followed by several others (Schulte im Hofe, 1908;

246 P. A. ROELOFSEN

Nicholls, 1913; Knapp, 1924; de Haan, 1928; Busse et al., 1929; Ficker and von Lilienfeld-Toal, 1930; Wilbaux, 1937; and RoeIofsen and Gies- berger, 1947) tried the addition of pure or mixed cultures of yeasts. Several believed that the difference in flavor of Venezuela and Africa cacao might be due in part to the kind of yeast prevailing during sweating. Most investigators did not realize that the species used should macerate the pulp.

In Java a yeast factory prepared pressed yeast froin a locally isolated cacao yeast,3 from Zygosaccharomyces mrx ianus (Hansen), and from Snccharomyces frugram (Beijerinck) , All three macerate the pulp and the latter has a strikingly high optimum temperature. At the start of the fermentation 10 kg. yeast suspended in 30 liters of water were mixed with lo00 kg. cacao, thus providing 0.4 million yeast cells per bean as against 0.2 million of other organisms already present. The temperature rose more quickly; the period of yeast domination was lengthened by half a day and the pulp was macerated better than usual. However, after 2 days the composition of the microflora was again as usual, and the dried product as judged by several Dutch experts did not appreciably differ from normal cacao except that the shell was more reddish in color. The two yeasts mentioned last had been chosen also for their capacity to produce an ester odor in cultures, but this did not occur in fermenting cacao.

5. SMALL-SCALE FERMENTATION More than 60% of the world’s cacao is produced by farmers with

small holdings. Even though they do not hesitate to mix unripe, ripe, and overripe beans, the quantity of a day’s harvest is often too small to be fermented properly.

Even with care, a small-scale fermentation cannot be maintained easily, with the result that slaty beans are produced. When treating 1000 liters in wooden boxes, the same fermentation rate is obtained as with larger quantities. With 100 liters the temperature will be only 1-2OC. ( 1 . 8 - 3 . 6 O F . ) lower. With much smaller quantities the use of specially insulated baskets, a box in a box, or a box with a glass lid for trapping the sun’s heat have been advised. The best solution, however, would be for the small farmers to pool their crops and practice coopera- tive fermentation.

Since the breeder must examine the product from different trees, fermentation on very small scale, such as the contents of one, or at most a few pods, is a necessary consequence of cacao selection. In recent years especially, the selection of swollen-shoot resistant varieties in

9 Identified later as Saccharomyces cerevisiae ( Hansen), but apparently cliffer- ent from normal baker’s yeast which does not macerate cacao pulp.


Africa has been very intensive. The assessment of the quality of the dried fermented cacao of these varieties is necessary before their use on farms and estates may be advised.

The oldest method for such very small-scale fermentation involves the use of muslin bags buried in the fermenting heap. In Java, Roelofsen and Giesberger (1947) improved the method by using very wide open- mesh bags, specially knitted from cotton yarn with meshes 1 x 1 cm. The beans in the bag were inoculated by rubbing with cacao beans from the day’s harvest, spread out flat in the center of the fermenting heap and replaced when the mass was turned. It was found that the microflora on the beans within the bag was always the same as in the bulk materials. During washing and drying the beans were kept in the bag. There was no detectable difference in outer and inner appearance when compared with the bulk beans provided drying was done at the same rate as for the bulk material. This method was used for selection purposes by Ostendorf and Roelofsen ( 1938).

There is one objection to this method. If fermenting pulp produces some flavor precursor or substance influencing flavor development, these could vary with the varieties, and hence the bulk could influence the contents of the bag, However, since the chemical composition of the pulp of varieties differs very little and since even the addition of considerable amounts of sugar, salts, organic and inorganic acids, sulfur dioxide, yeasts, etc., has not thus far produced a different cacao, the objection, in the opinion of the author, is academic, This also applies to the theoretical possibility that a flavor precursor produced within one kind of beans might diffuse into other beans.

MacLean and Wickens (1953) used the same method but spread the beans on top of the fermenting mass, separated by a layer of leaves to prevent contamination but still allowing transmission of heat. Under these conditions the microflora must be greatly different from that which would be found in bulk fermentation since the sample is so heavily aerated. Nevertheless, the results were considered satisfactory.

For selection and fermentation research, beans have been fermented in flasks or very small boxes and kept in a thermostat under constant or progressively increasing temperature conditions ( Hoynak et al., 1941; Roelofsen and Giesberger, 1947). In order to prevent a premature domination of acetic acid bacteria, the latter authors had to reduce the aeration by compressing the bean. In the experiments by Hoynak et al. an abnormal microflora, more like that occurring in coffee fermentation, was found. It reduced the pH, which was originally as high as 4.2. Prob- ably the beans had been killed in the pods since these were kept in a refrigerator during transport from Costa Rica to Pennsylvania.

Special setups for laboratory fermentation were described by Mac-

248 P. A. ROELOFSEN

Lean (1950) and DeWitt ( 1952a). Both temperature and aeration were regulated, but evidence of a normal microflora was not given. Wads- worth and Howat ( 1954), using the setup of DeWitt and inoculating the beans with a yeast and an acetic acid bacterium, observed a fall of pH from 4.0 to 2.5, which seems to indicate abnormal conditions. Although in this case the beans certainly were alive, the original pH of the pulp was unaccountably high.

V. INTERNAL OR ENZYMATIC FERMENTATION AND CONSEQUENCES

As previously indicated, death of the kernels during fermentation is essential for the biochemical processes which together are called internal or enzymatic fermentation. However, as already Loew (1907) stated, most enzymes should remain active. When destroyed by heating at 75°C. (167OF.), browning and flavor development do not occur. Internal fermentation starts following the death of the kernels in the fermentation heaps, and proceeds during drying and, to a limited extent, during storage of the dried product.

The consequences of processes induced by internal fermentation are listed in Table 11. The most conspicuous difference, except for the flavor

TABLE I1 Differences between Fermented and Unfermented Dry Cacao

Dry cacao Unfermented Fermented (nonsweated) (sweated)

Shell condition

Color on inside shell Radicle condition Consistence of cotyledons Cotyledon section orig-

inally white Cotyledon section orig-

inally purple Taste Aroma (unroasted) Aroma (roasted)

Soft and close fitting

Clean and pale Sections, white Leathery or cheesylike Dirty white or grayish

Slaty, blueish grey

Bitter and astringent Faintly earthy Resemble broad bean

Crisp, either loose or close

With dark brown deposits Sections, dark brown Crisp Cinnamon brown or cream-

colored or patches of both Dark brown or violet or

both Less so the more browned Acid and faint fragrance Chocolate odor

fitting

on roasting, is the slaty color visible on cutting beans which originally were purple and were dried without having been sweated. When living beans are dried the anthocyanin remains in the tannin cells which, on the yellowish background of the other cells of the cotyledon tissue, produces the slaty color. In dead beans this substance has diffused throughout the kernel and this, when dried, becomes violet or brown. The dirty white or grayish color of unsweated dry beans which orig-


inally had white cotyledons is less conspicuous though easily distinguished from the cream color of sweated and dried unoxidized white beans.

In commerce, beans with completely violet or creL.n-colored sections are likewise termed unfermented, and those with violet or cream-colored parts in addition to brown parts are termed partly fermented or under- fermented ( Fig. 7 ) . This terminology causes much confusion. They should be called completely unoxidized and partly unoxidized beans respectively.

In the following sections chemical changes occurring in the kernels during fermentation and drying will be discussed, but not the literature on the chemistry and analysis of cocoa and cocoa products. For these subjects the reader is referred to Fincke (1936) and Chatt (1953).

1. DEATH OF THE COTYLEDONS Since internal fermentation starts when the cotyledons are killed,

it is important to know when this happens and why.

a. Criteria of Death in Cotyledons

At first the loss of capacity of germination was the only test for detecting the death of cacao seeds, but since the growth tip of the embryo is the most easily damaged part, failure to germinate does not imply the death of the cells of the cotyledon tissue. The modern tetra- zolium test as applied by Wadsworth and Howat (1954) on cacao seeds likewise is of limited significance since in living beans only the plumule and the radicle color clearly. Realizing the limits of the germination test, Busse et al. (1929) thought that the cotyledons were only partially killed during fermentation. Browning was considered a criterion for death; on the other hand the occurrence of purple and white patches on sections of commercial cacao was considered proof that these parts were alive when the bean was dried.

Knapp (1924) was the first who noted the diffusion of the purple pigment out of the isolated cells into the surrounding tissue and who rightly considered this diffusion an indication of death. Hardy (1925) observed the diffusion of a colored substance out of whole purple cotyledons kept in acetic acid at p H 3.8 or lower. However, he wrongly thought it was caused by increased permeability of living ( ! ) proto- plasm below its isoelectric point. Hence he assumed that all acids would show the same effect, Unfortunately he did not try other acids, for then he would have seen that it was not a matter of pH. He would probably have concluded that acetic acid is a poison and that the increase of permeability was the result of death.

250 P. A. ROELOFSEN

Knapp’s diffusion criterion was improved and moreover made ap- plicable to beans with white cotyledons by Roelofsen and Giesberger (1947) who applied on the sections a reagent (10% sodium bichromate in dilute acetic acid) that colored and precipitated the tannins. In both white and purple living cotyledons, brown specks consisting of groups of tannin cells are visible with a hand lens, whereas dead tissue is homogeneously dark brown. Sections of dried nonsweated cotyledons although dead will become speckled, but not when previously moistened since the tannins will have diffused. When sections of purple living cotyledons are embedded in paraffin on a slide and heated gently, diffusion of the color may be followed microscopically. Sections of beans killed at O°C. (32OF.) or lower will likewise become speckled with the reagent since diffusion is slow at lower temperatures.

Other visible criteria are: the more vitreous appearance of dead cotyledon tissue (which apparently is optically more homogeneous), and after several hours, the exudation of a milky white or purpIe juice into the interstices of the cotyledons and between the cotyledons and the shell, Apparently the shell acts like a semipermeable membrane since the fermenting bean absorbs water from the pulp and eventually becomes so turgid that if the shell is pricked with a pin, juice spouts out. This juice turns brown rapidly on exposure to air. In dried beans, the juice dries to dark brown patches on the inner side of the skins, which therefore is characteristic for well sweated cacao. Probably the particles in the milky juice consist of complexes of tannins and purin bases (Knapp, 1937, p. 68).

b. W h e n and Why the Cotyledons Die

Roelofsen and Giesberger (1947) found by using the tannin test that the cotyledons of normal ripe cacao fermenting in bulk die in 30-36 hours except in the top and the bottom layers. The temperature in the middle area is usually 3436OC. (93-97OF.). After 36 hours the cacao is usually turned and within a few hours the remaining living beans also are killed. On two estates in Java, using thin layers, airtight boxes, and no covers, fermentation was retarded, and death in the central layers occurred after 50 to 60 hours. Purple and white beans died simultaneously.

According to Knapp (1937), diffusion of color in GoId Coast and Trinidad fermentations usually coincides with a temperature of 44-47OC. (111-116OF.) which is reached after 48-60 hours, About the same rate of rise in temperature is found in Java. Therefore the difference in time of death probably is not real but rather the result of difference in judgment.


Opinions regarding the cause of death of the beans have been at variance for many years. For instance, Busse et al. (1929) took it for granted that alcohol was the cause. They criticized Schulte im Hofe (190s) who thought the alcohol was only a precursor of acetic acid, which according to him was the real cause of death. Actually most of the early investigators considered temperature to be the primary killing agent. Using germination capacity as a criterion for death, it was found that 2 hours at 45OC. (113"F.), 6 hours at 44OC. (111°F.) or 9 hours at 43OC. ( 1 0 9 O F . ) will suffice to kill (see Knapp 1937, p. 61). However, when color diffusion was used as a criterion, Knapp found that 24 hours at 45OC. (113OF.) were needed. H e also noted that when the pigment diffuses during fermentation at 45OC. (113OF.), it is more reddish violet. Hence, he concluded that while the observed temperature during fermentation is almost sufficient to account for the death of the cotyledons, there is Iittle doubt that acid and perhaps also alcohol are contributory causes,

However, since the observations in Java showed that during normal fermentation cotyledons are already dead at 35OC. (95OF.), this conclusion was rejected by Roelofsen and Giesberger (1947). The fact that with fermenting beans the outer parts of the cotyledons invariably died earlier than the central part (as shown in Fig. 4A), was taken

FIG. 4. Sections of fermenting beans after applying tannin reagent. The outer parts of the kernels have already been killed. A. Singlc bean, B. adhering pair of beans.

as evidence that the killing agent is some substance occurring in the pulp. In beans killed by heat or by freezing, all parts died simultaneously, Hence the peripheral layers were not more sensitive than the ccntral part, Moreover, the topography of dead tissue in adhering pairs of beans, as shown in Fig. 4B, excluded this possibility.

Experiments with sterile beans in flasks buried in bulk cacao of the day's harvest (fermenting along with it) led to the conclusion that

252 P. A. ROELOFSEN

acetic acid is the primary cause and temperature and alcohol are contributory ones. These experiments will be discussed in more detail.

The cotyledons of sterile beans held in an atmosphere of nitrogen, hydrogen, or carbon dioxide in cotton or rubber stoppered, sterile flasks fermenting along with the estate cacao died after about 45 hours. How- ever, if such flasks were removed when the cacao was turned after 36 hours and then buried in the next day’s harvest; repeated again the third day, the cotyledons were still alive after 84 hours. This undeniably proves that neither lack of oxygen, accumulation of carbon dioxide, nor the temperature reached within 36 hours in normal sweatboxes, are primary killing agents,

However, if the sterile beans in cotton-stoppered flasks were inoculated and then fermented along with the estate cacao, the cotyledons died earlier (see Table 111). The topography of the dead tissue indicated

TABLE I11 Influence of Type of Fermentation on Time Required for Death of the Cotyledon

Number Inoculation Time required for

death (hr. )

1 Yeast and acetic acid bacterium 23 2 Yeast and lactic acid bacterium 26 3 With pulp from estate cacao 30 4 Yeast (macerating or nonmacerating) 35 5 Lactic acid bacterium 40 6 Acetic acid bacterium 45 7 Sterile beans 45

the presence of a poisonous substance in the pulp except in the case of acetic acid bacteria where heat evidently was the cause of death. Further experiments with sterile beans in solutions of different acids and alcohol in varying concentrations (also buried in the sweatboxes) disclosed that neither temperature nor lactic or citric acid, ethanol, hydrogen ion or acetate ions are primary causes of death when present at the concentrations occurring during a normal fermentation.

Evidently molecular acetic acid is the main cause of death. Under sweatbox conditions, 1% of acetic acid is sufficient to kill. This amount is actually found in fermenting pulp at the moment the beans die. Temperature is a contributory cause since 3% acetic acid is required to kill the cotyledons in beans at room temperature within the same period. No difference in sensitivity was observed between white and purple beans of the hybrid cacao in Java. However, there might be some difference between true Forastero and true Criollo beans since the former have thicker skins which are less permeable to acid (Knapp, 1924).


In sweatboxes in Java, acetic acid responsible for the death of most beans is produced to a large extent by lactic acid bacteria since a t the time of death the number of acetic acid bacteria present is too small to account for the acetic acid. In Trinidad, however, it obviously orig- inates from acetic acid bacteria (Rombouts, 1952).

At first sight some results obtained with inoculated sterile beans, as mentioned above, seem to contradict the conclusion that acetic acid is the killing agent. The slowness of death in No. 3 (see Table 111) as compared with No. 1 and 2 is explained by the fact that the latter were more heavily inoculated. Numbers 5 and 6 lag because the pH 3.6 is unfavorable for these bacteria and moreover acetic acid bacteria do not produce acetic acid if alcohol is absent. The rapid death in flasks with yeast (No. 4) as compared with sterile beans (No. 7 ) indicates that alcohol may also kill cotyledons. In such beans the purple pigment is more bluish than in beans killed by acetic acid. However, it does not prove that under sweatbox conditions alcohol is a cause of death for with the pure cultures in the flasks the alcohol concentration is higher. Apparently maceration of the pulp does not make much difference insofar as the death of the beans is concerned.

For practical purposes the important conclusion is that the shortest sweating time possible (without risk of causing slaty beans) is about 9 hours following the turning of the mass after death of the center beans. On most Java estates this amounts to a total of 45 hours. If the zero hour is at 6 P.M. on the day of harvest, the fermentation should not be terminated before 3 P.M. on the third day. However, since sun- drying is desirable, the cacao is either sweated until the next morning (60 hours) or steeped in water to stop the process. If the central part of the cotyledons in some beans is still alive when drying begins, these will not become slaty, for the center will die by the time the drying process starts. The effects of such a short fermentation period on flavor development and browning will be discussed later.

2. ENZYMES IN THE COTYLEDONS

Chemical changes in the beans that require enzymes are browning, proteolysis, and very probably production of flavor precursors, and possibly many more. Thus far, however, mainly two scientists (Brill, 1915, Ciferri, 1931 ) have tried to obtain information concerning the enzymes that might be important, and they used only qualitative tests. Several of these are now considered either obsolete, too crude, or of no value. With Ciferri’s work, moreover, there is doubt with respect to the manner in which the blank tests were set up and whether or not an antiseptic was used. Both authors obtained the so-called fermented material from

254 P. A. ROELOFSEN

beans sweated in flasks at room temperature (20-30OC.) (68-86OF.) for 4 days, but evidently it is even doubtful whether or not death had occurred. Although they are far from satisfying, the results of these investigators are tabulated in Table IV. The present author has used the current enzyme nomenclature and has made some additional or critical remarks. In the last column is indicated if other literature permits any conclusion about changes of the enzyme substrate during fermentation or drying.

Evidently there are some contradictory results, but in view of our present knowledge concerning changes occurring in the substrates, the presence in fresh material of invertase (probably p-fructosidase ) , p- glucosidase, P-galactosidase, a-amylase, proteinase, asparaginase, phenoloxidase, peroxidase, and catalase may be accepted. Furthermore it is evident that some enzymes are inactivated by drying and more of thein by drying subsequent to fermentation. Enzyme inactivation by tannins is well known, so modern test methods prescribe their previous extraction. It may very well be that the inactivation of certain enzymes is of great importance for production of flavor precursors by others (see later).

Naturally there must be many other enzymes since the cacao embryo is not in a latent stage. Special measures must be taken to keep seeds viable for short periods of time, even in the pods (Evans, 1950). They germinate within 3 days at 3037OC. (86-99OF.) if the pods were opened, slower if the pods are cut off but left unopened. In the latter, Wilbaux ( 1937) found an increase of phenoloxydase activity. Probably many enzymes are activated during the first day of fermentation. Sterile beans produce much carbon dioxide both in air and under anaerobic conditions ( Wadsworth and Howat, 1954).

Aside from the work of Brill (1915) and Ciferri (1931), there have been few publications on cacao enzymes. Von Lilienfeld-Toal (1938) found catalase with a pH optimum of 7.0 in fresh and freshly dried cotyledons, as well as in fermenting ones, but none after 5 days of fermentation. Birch and Humphries (1939) could not find lipase in fresh, dried, or fermented beans. Since they did not eliminate the possibility of inactivation by tannins and since lipase is likely to occur in fat-containing seeds, definite conclusions should await further experimentation. Vilstrup et al. (1950) found a milk clotting enzyme (proteinase) in commercial cacao. It was very heat stabile since there was some activity in roasted beans. Like most plant proteinases, it was activated by cyanide and inactivated by monoiodic acetic acid. DeWitt (1952b) was the first to use fat- and polyphenol-free powder of fresh cotyledons prepared by extraction with cold acetone and ether. He


claimed to have found peroxidase, catalase, ascorbic acid oxidase, and enzymes catalyzing the aspartic-malic- fumaric- acid equilibrium. Aspar- aginase has been demonstrated in living embryos.

Von Lilienfeld-Toal ( 1938) described the disappearance of the color in purple beans during fermentation, 4 days being sufficient for complete disappearance. Forsyth ( 1952b, 1953, 1957 ) has demonstrated the presence of enzymatic destruction of the violet cyanin pigments, a galactoside and an arabinoside, in anaerobic conditions in fresh, fermenting, and dried unfermented cotyledons. Hydrolysis by P-galactosidase is the first stage. It hydrolyzes both cyanins with equal velocity. The cyanidin then is converted by an unknown process into colorless leucocyanidin which subsequently is converted into complex leucocyanidin. A similar decolorization of fruit anthocyanins by fungal glycosidases has been described recently by Huang (1955).'

The most studied enzyme of cacao is phenoloxidase. The physiolog- ical role is as a terminal oxidase in respiration (James, 1953). As early as 1907, Loew demonstrated this in the cotyledons by use of the guaiacum test. He also stated that the blue color obtained disappeared in an aqueous suspension of ground tissue as a result of reduction. Although some fungi secrete a phenoloxidase (Bavendamm, 1928), the microorganisms in fermenting cacao do not, or if they do, their enzyme does not affect the cotyledons, since heated beans completely fail to brown when fermented and dried in the normal manner. The guaiacum test showed the enzyme to be present in all cells of the cotyledon tissue except in the tannin cells (Forsyth, 1955). Phenoloxidase also occurs in the endosperm and traces of it in the testa, especially in the vascular bundles ( Roelofsen and Giesberger, 1947).

Phenoloxidase apparently has a metal as a prosthetic group since it is blocked by cyanide (Roelofsen and Giesberger, 1947; Forsyth, 195213). Moores et al. (1952) purified the enzyme and found only phos-

4 Recently, Forsyth and Quesnel (1957) showed that the cacao glycosidase is optimal at pH 4 and 45°C. (113"F.), occurs only in the tannin free cells, starts its activity as soon as the cotyledons have died and the pigments have migrated to the site of the enzyme, and then converts the two cyanins into cyanidin within the next 2 days, provided the temperature exceeds 40°C. (or F.). It is only active under anaerobic conditions since it is blocked by oxidation products of e.g. epicatechin. It is inhibited by the complex phenolic fraction even in the unoxidized state. There- fore little activity is folmcl in the brown and violet parts of raw cacao btlt consiclcr- able activity in slaty beans.

The liberated cyanidin spontaneously forms a colorless pseudo-base which, contrary to earlier findings, does not undergo further alteration under anaerobic conditions, but is readily oxidized to a brown substance. Unbleached purple beans may also turn brown, but with n residual purple cast.

!2 TABLE IV Results of Tests on Enzymes in Cotyledons a

Unfermented Fermented

Enzyme material material

Fresh Fresh Dry Not dried Dried Known changes

in substrate

(Brill) (Ciferri) (Ciferri) (Brill) (Ciferri)

Invertase b P-Fructosidase ( inulinase )

a-Glucosidase ( maltase )

a-Glucosidase ( trehalase)

a-Galactosidase ( melibiase )

a-Galactosidase (raffinase) c

p-Glucosidase (emulsin) d

p-Galactosidase ( lactase) *

a- Amylase Proteinase e

Asparaginase ( aminoacidase ) f Phosphatase ( glycerophosphatase) Phytase g

Lipase h

Phenoloxidase i Peroxidase i

+ +

+ + + +

+ + +

- t -

- -

Disuppears Little or no production of reducing

sugars Little or no production of reducing






sugars Little or no change Proteolysis

No change Oxidation

Catalase k + + + Methylene blue reduction 1 + Enzymatic sulfide production m + +

- + - - -

n c 2: 0 0 q n P n 9 0 W M *

a From Brill ( 1915) and Ciferri (1931 ). Footnotes inserted by present author. b Probably p-fructosidase, perhaps a-glucosidase. c Might have been p-fructosidase. d In fresh cotyledons already found by Sack (1913).

f Method apparently unsuitable; however, found by DeWitt ( 1952b). g Not proved that phosphate formed originated from substrates given.

i In fresh beans first demonstrated by Loew (1907).

?!

e f = contradictory results; however, found by Vilstrup et al. (1950) in commercial cacao.

h Not found by Birch and Humphries (1939) in fresh, dried, or fermented beans.

i In fresh beans also by DeWitt ( 1952b ) . kBut for dry fermented, confirmed by von Lilienfeld-Toal (1938) and DeWitt (1952b).

mProves reduced condition but origin from sulfur given is doubtful; might be caused by cysteine from proteolysis. n Found by Forsyth (1957) in fresh beans.

5 Nonenzymatic since also in boiled controls; Ciferri used higher concentration.

258 P. A. ROELOFSEN

phate and copper as inorganic constituents, thus definitely proving thkit copper is the active element. In line with this, DeWitt (195%) found that cacao trees lacking copper contain less of the enzyme in their leaves. When cotyledons from different trees are killed at 50OC. ( 132°F.), the rate of browning is found to be very different, but the cause of this variation is not necessarily due to copper deficiency (Roelofsen and Giesberger, 1947).

For obvious reasons, some interest has been focused on the influence of pH and of acetic acid. Loew (1907) observed that beans im- mersctl in 1-4% acetic acid with 3-5% ethanol at 42OC. (108OF.) for 2 0 3 0 hours (prcsuinably being killed) still browned on sectioning. This did not occur, however, when 4% acetic acid was applied on sections. Most authors agree that the optimal pH is about 7; at 5.0 activity is much lower. This is a t variance with DeWitt (1952b), however, who found the optimal pH to be 5.0. At pH 4.0 activity is still present, but at 3.0 the enzyme is inactive ( Roelofsen and Giesberger, 1947). Forsyth (1952b), who determined the activity on the separate polyphenols of cacao, found that when cotyledons were ground, all polyphenols are oxidized so rapidly and extensively that they become insoluble within an hour. Below pH 5.5, activity rapidly decreases but is still presei?t a t 4.0. Epicatechin and complex tannins are less readily oxidized than the cyanin pigments and leucocyanidin. In fermenting beans, the initial pH of about 6.3 falls but seldom below 5. This explains why naked cotyledons, killed by heat, brown more rapidly than fermented ones, and why fermented beans, steeped in water containing calcium car- bonate, brown more completely than when steeped in plain water ( Roelofsen and Giesberger, 1947).

The heat stability of the enzyme is considerable as is usual with phenoloxidases. For instance, in cotyledons the enzyme withstands long periods at 60OC. (140°F.), one hour at 70OC. (158OF.), but less than 5 minutes at 75OC. (167OF.) (Knapp, 1937). In extracts, however, it is more labile ( DeWitt, 1952a). Low moisture content of course enhances the heat stability. In a dry powder of unfermented beans Moores et d. (1952) found a loss of only 67% after 20 minutes at 90°C. (194OF.), but total destruction occurred when 20% of water had been previously added.

During fermentation, 5OOC. (122OF.) is seldom exceeded. In some cacao driers, however, inlet temperatures over 100°C. (212°F. ) occur. Evidently, this will destroy the enzyme in beans which are still moist internally. Such cacao does not brown as much as normal raw cacao when postfermented (this process is discussed in Section V, 7 ) . Roiled and then dried beans do not brown at all during postfermentation. These facts suggest that phenoloxidase does occur in normal raw cacao al-


tl~ougl~ Knapp (1937) c d d not demonstrate it and altliorigh Moores ct d. (1952) hesitated to ascribe the very low activity found in raw cacao ( 6 units, as against 300 in fresh tissue) to enzyme action.

With regard to the influence of water on the activity of cacao phenoloxidase, Knapp states that activity is rapid when 20% of water is present and very slow with only 10% However, it is doubtful whether or not the slow progressive browning of tannin occurring in raw cacao during storage is enzymatic.

The main substrates of cacao phenoloxidasc are undoubtedly the polyphenols, which will be discussed later in more detail. In addition, phenolic amino acids (such as tyrosine), which are supposed to be set free by proteolysis during fermentation, will be oxidized. As is known, enzymatic oxidation of free tyrosine occurs widely in nature. In the opinion of Sizer (19S3) even tyrosine groups in proteins may be oxidized, but this is doubted by others.

3. CHANGES IN NONPOLYPHENOLS IN THE COTYLEDONS DURING FERMENTATION AND DRYING

Knapp reviewed most of the information on this subject. It consists, to a large extent, of a comparative analysis of unfermented and fermented dry cotyledons made by Harrison (1896-97) and by Churchman (see Knapp, 1937, p. 8) . More recent work includes only that of MacDonalcl (1937) and Humphries (1939, 1944b) on fat and theobromine and of Birch (1941) on nitrogenous compounds. Roelofsen and Giesberger ( 1947) studied the external conditions affecting devclop- ment of flavor precursors but not their chemical constitution.

Drawing conclusions from analytical differences, in specific compounds, between fermented and unfermented cotyledons is difficult because of the simultaneous occurrence of exchange of compounds between pulp, skin, and kernel, loss of weight as a result of metabolism, increase of weight by oxidation, precipitation by tannins, etc. The only constant reference available for expressing the amounts of substances present is on the basis of the number of beans, e.g., 100. If analyses are expressed in per cent of dry weight, they first must be converted in weight per 100 beans, using data pertaining to the change in dry mattcr per 100 beans, before conclusions can be drawn with respect to quantitative changes (Humphries, 1944b ). Possibly a certain weight of fat or of raw fiber may also prove to be suitable as constant reference.

As already mentioned, the pH in the cotyledons decreases during fermentation. According to von Lilienfeld-Toal ( 1938), it decreases from 6.25 to 4.5 in 7-8 days then increases to 5.5 after 13 days. This, however, is seldom observed in practice, According to Forsyth ( 1953),

260 P. A. ROELOFSEN

it decreases to only 5.4 in 6 days. As a result of volatilization of acetic acid, the pH in moistened raw cacao is higher (5 .c6 .2) . Older data were reviewed by Knapp (1937).

The main acid causing these changes is presumably acetic acid. In Java, dry cacao fermented for 2% days contained 0.&1.6%, against 0.1% if fermented for 154 days (Roelofsen and Giesberger, 1947). The highest amount in Java cacao noted on arrival in Holland was 0.7% (Ultee, 1936). The highest acetic acid content found in Trinidad was 0.&0.9%; however, this was reduced to an average of 0.2% upon arrival in Europe (Knapp, 1937). In Brazil, Dittmar (1954) found up to 1.3% of acetic acid (dry weight basis) in wet beans fermented for 51,$ days. Two-thirds of this disappeared on drying. The volatilization of acetic acid from raw cacao is not only apparent from the smell in warehouses, but also from the formation of a “bloom” of calcium acetate crystals on floors under the stacks. Mosimann (1944) found in Europe 0.1-0.44% free acetic acid in commercial roasted cacaos and 0.2-0.78 when tartaric acid had been added to aid the distillation. Apparently 2040% of the acetic acid is in the salt form. This was overlooked by most of the previous authors, who determined free acetic acid only. On heating with mineral acids much of the bound acetic acid is freed.

The only other non-nitrogenous organic acids found in (roasted) cacao were oxalic (0.3-0.5%) and citric (0 .44475%) (Mosimann, 1944). The absence of lactic acid is striking. It is not known which acids occur in fresh beans. No data are available on ethanol in fermenting or dried cacao kernels.

Soluble substances from the dead kernels diffuse into the shell and pulp during fermentation and the early stages of drying. These are mainly polyphenols, theobromine, and salts. The shell which is nearly free of purin bases may contain 2% of theobromine when dried after 6 days of fermentation. This exceeds the percentage occurring in the kernel and equals it if expressed on a fat-free basis (Knapp, 1937). The content in the fat-free kernel declines about 0.7%. The loss is masked by a decrease in dry matter, but the absolute loss (per 100 beans) is as much as 40% of the amount originally present in the kernel (Humphries, 194413). Accumulations of theobromine crystals sometimes may be found on the outer and inner surfaces of the skin and on the cotyledons, The latter phenomenon is known as “white spots.” Knapp (1937) supposed that such beans have been killed in the pod by very dry weather; however, white spots also occur in wet climate and on estates where the occurrence of dead beans in the fresh crop is out of question. Such accumulations of theobromine crystals are produced by heating of piles of partly dry beans and obviously is related to the greatly increased


solubility of theobromine in hot water ( Roelofsen and Giesberger, 1947). The possibility of theobromine being used as a source of nitrogen by the microorganisms of the fermenting pulp cannot be excluded since fungi are known to do this ( MacLean, 1953).

There are indications that theobromine, which originally is localized in the tannin cells (Brown, 1954), may form complexes with certain polyphenols but not epicatechin (Knapp, 1937). The complex of caffein and epicatechin also is very loose (Forsyth, 1 9 5 2 ~ ) . In the milky juice around dead cotyledons, the particles consist of theobromine mixed with tannin. On cooling a concentrated filtered infusion of cocoa powder, a precipitate is formed as in the “creaming down” of tea, which is ascribed to “caffein-oxytheotannate.” The thcobromine-tannin complex, if present, is supposed to be dissociated by acetic acid.

The amount of diffusion of polyphenols out of the kernel during fermentation may be estimated at one-third of its original content (k lo%), epicatechin being the most mobile fraction. No data are available on loss of inorganic constituents.

The loss of dry matter is caused not only by diffusion into the shell, the pulp and sweatings, but also by metabolism. According to Humph- ries (1944b, 1952) and Kuppers (1951), the loss from the cotyledons is practically constant during fermentation and amounts to 11% in one week, or 1.6% per day. Although these data cannot be used in the calcu- lation of the loss of weight in beans during normal processing, it is clear that there is a considerable loss, and the producer expects a premium for prolonged fermentation in order to balance the loss in weight.

During drying, acetic acid and alcohol are lost, polyphenols dehydro- genate, and probably decarboxylation and deamination also occur. In fact, the dry weight decreases a little, resulting in a slight increase on dry weight basis of some components such as theobromine.

Evidently, the water content of the cotyledons increases considerably during fermentation. In Trinidad it amounts to 3.6% in 3 days and 4.9% in 6 days of fermentation (Birch, 1941), in Columbia 3.0% in 4 days of fermentation (Platone, 1951), and in Java as much as 5% after 60 hours. A minor part of the increase will be due to metabolism, but the main causes are diffusion of dry matter into skin and pulp and uptake of water from skin and pulp, the latter being demonstrated by the pressure in the juice under the skin. Consequently the increase in water content mainly takes place following the death of the beans.

The composition of the cacao butter is not changed during fermentation and drying (Knapp, 1937; Dittmar and Raimann, 1956). Since fat droplets evidently do not diffuse readily, the fat content on dry weight basis naturally rises during fermentation. Knapp (1937) mentions an

262 P. A. ROELOFSEN

increase of 1.5-2%, but, of course, this depends on the duration of fermentation ( MacDonald, 1937; Humphries, 1939, 1944b ) , Humphries calculated an increase of 5% after 1 week, but per 100 beans there was an increase of only from 50 to 51 grains after 3 days, and after S days a decrease to 48.5. This would suggest a slight synthesis followed by either diffusion of fat or some kind of dissimilation without previous lipolysis. However, since this is improbable in fermenting dead beans, the question rises as to whether the slight changes were real or caused by such processes as incomplete extraction, contamination with varying amounts of other lipids, or fixation of the fermenting beans by boiling.

With respect to carbohydrates the following data are available. Disappearance of sucrose was postulated by Harrison ( 189697) , who found 1.4% in fresh Forastero and none in fermented kernels. Likewise Knapp (1937) found up to 1% in unfermented Arriba and none in fermented cacaos. In fresh Calabacillo, Harrison (1896) found traces of sucrose and in fresh Amelonado, MacLean (1953) found 2.4%. However, by use of paper chromatography it has been shown that besides sucrose, other saccharides occur in raw cacao that will hydrolyze in the determination of sucrose and hence have been determined as such, Both Thaler (1954, 1957) and Cerbulis (1954, 1955) found sucrose as the main sugar, but also raffinose and stachyose in raw as well as roasted cacao. Arriba containcd more sucrose than usual. In addition to these, Cerbulis found melibiose, probably originating from raffinosc, a inan- ninotriose which was possibly produced from stachyose, ’ several nn- known oligosaccharides, iiiositol, probably originating from phytin and glycerol. Since he used roasted cacao it is not certain that these substances occur in raw cacao. In view of these facts, the previously observed “disappearance of sucrose” during fermentation shonld he interpreted as “partial disappearance of sucrose and oligosaccharides.” Presumably they are lost partly in the shell and partly by hydrolysis and dissimilation. According to Diemair et d. (1958) there is 1.9% sucrose in raw Arriba cacao, but little in Accra and Bahia cacaos.

Similar amounts of reducing sugars have been found. According to Knapp (1937) it varies between 0.1 and 2%, but what happens during fermentation is uncertain. Thaler ( 1957) found 0.25-2.2% in raw cacao, Dicmair et nl. (1958) 0.8-1.24, According to Thaler (1954), CerbuIis (1954) and Dieinair et aZ. (1958), glucose and fructose are the main components; traces of galactose, a pentose and a methylpentose were found in addition. The latter authors found abont 0.5% glucose and 0.4-0.8% fructose in raw Accra, Arriba, and Rahia cacaos.

According to Knapp (1937) starch is not changed during fermentation. Neirinckx and Jennen (1952) found a substantial decrease, hut the fact that no maltose has been found casts doubts on this claim.

CURING OF CACAO BEANS 26x3

Knapp (1937) found a high increase in pectin, mucilage, and gums. However, Dopplcr (1936) has shown that these materials, when extracted from cacao, usually are contaminated with considerable amounts of starch. Probably the increase found by Knapp is the result of a higher degree of contamin a t’ ion.

The insoluble protein-nitrogen content of fresh cotyledons of Trin- idad Criollo hybrids is about 2% and about 2.78 in Forastero (Hardy and Rodrigues, 1953). These authors found O.1-0.2% more protein nitrogen in purple than in white cotyledons of the same mixture of cacao seed- lings. In different Forasteros a higher protein content is correlated with lower grade. Less protein was found by Birch (1941) in the same cacaos. Total nitrogen seems to be higher in raw Criollo hybrid cacao than in Forastero (Becker and Stelling, 1952), but this might be the result of diffusion of theobromine to the skins during the more prolonged fermentation of the latter.

The need of a longer period of fermentation for Forastero has been correlated with its higher protein content (Hardy and Hodrigues, 1953),

Substantial changes occur during fermentation. Proteolysis, postulated by Harrison in 1896, was confirmed by more recent analysis ( Knapp, 1937; Birch, 1941; Neirinckx and Jennen, 1952). Birch found that 94% of the protein loss occurred during the 24 hours following death of the cotyledons. Soluble amino and amide-nitrogen increased althouqh these compounds were lost from the cotyledons by diffusion. The rate of loss, however, was exceeded by the rate of production, owinq to rapid protein breakdown. Unaware of these findings, Becker and Stelling ( 1953) rediscovered the increase of soluble nitrogen during fermentation. They also found four amino acids (aspartic and glutamic acid, alanine and isoleucine) in extracts of fermented raw cacao but none in unfermented beans (that none are present seems doubtful). Also Maly (195s) confirmed the proteolysis by finding nine free amino acids in fermented cotyledons and again in unfermented ones. The amino acids found were aspartic and glutamic acid, alanine, cysteine, leucine, serine, threonine, methionine, and arginine. In the present author’s laboratory the same except methionine were recently found in raw cacao and in addition phenylalanine, probably also tyrosine. There was no cliffcrence between Java, Accra, and Venezuela cacao. Cysteine is of interest because of Ciferri’s test for production of sulfide from sulfur.

Although not occurring in the cotyledons, the increase of vitamin D content of the shells on sun-drying of fermented beans deserves mention here. Knapp and Coward (1935) showed that up to 28 Inter- national Units per gram is produced by ultraviolet irradiation of erqos- terol originating from the yeasts in the pulp. Evidently, this is greater the longer the beans are sun-dried (Scheunert, 1937). Kon and Henry

264 P. A. ROELOFSEN

(1935) showed that on feeding cacao shells to milking cows, the vitamin D content of the milk increased. This, however, does not justify its use as cattle feed since there are other cheap sources of vitamin D. Further- more, more than 0.025 grams of purin bases per kilogram body weight per day produces adverse symptoms. The nutritive value is doubtful ( Bartlett, 1945).

4. POLYPHENOLS IN FRESH COTYLEDONS

Knapp (1937) and Doppler (1936) have reviewed the early literature on this subject. At that time it was known that the cacao polyphenols were of the catechin type because they gave a red color with vanillin-hydrochloric acid reagent, and could be precipitated with formalin and hydrochloric acid. Moreover, ( - ) epicatechin had been crystallized, identified, and shown to be readily oxidized in the presence of phenoloxidase. In addition, a colorless water-soluble amorphous tannin had been obtained. This could be oxidized readily to the so-called cacao-brown. This substance was insoluble in water but soluble in alkaline solution and was considered to be a mixture of condensation products of oxidized catechin derivatives.

As early as 1893 the pigment of purple beans had been extracted and called cacao-red. On heating the alcoholic extract with acid, more cacao-red was produced and later on it was found that the same tannin that could be oxidized to cacao-brown was convertible into cacao-red by heating with acid. Epicatechin, on the other hand, colored very slowly under these conditions. This cacao-red, as well as the natural pigment, was readily and irreversibly oxidized to brown products.

The natural purple pigment was identified as cyanidin-3-monoglyco- side by Lawrence et al. (1938). Knapp and Hearne (1939) showed that the “cacao-red” obtained from white beans by treatment with acid, also had an anthocyanidin-like, in fact a cyanidin-like character, and they concluded that the colorless tannin precursor contained leucoanthocyanidin.

Leucoanthocyanidins were first detected by Rosenheim ( 1920) in leaves and fruits of the grape vine, Later G. M. Robinson and R. Robin- son (1933) found that they were widely distributed in nature. According to Bate-Smith (1954) they are very probably even responsible for reactions in plant tissues commonly attributed to tannin, They often occur free but sometimes as glycosides. Structures I and I1 in Fig. S have been proposed by Robinson for the pyran ring. By dehydration in the case of structure I and by oxidation and subsequent dehydration in the case of structure 11, they are convertible into anthocyanidins ( I V ) , Pig- man et al. (1953) favored structure I1 for a leucoanthocyanidin from Picea bark. Tayeau and Masquelier (1948) decided upon structure I for a chromogen in peanut testa. In recent years the structure of the

OH

H o w OH

HO HOH

OH

HO H OH

OH

W O H

H OH leucoan thocyanidins

..@ HO H,

(- ) epicatechin

c1

cyanidin-3-glucoside (oxonium chloride)

so, H glucose

colorless pseudobase

HCI

NaAe - OH

violet color base

Na&Q 1

blue Na-salt

FIG. 5. Structures of ( - )epicatechin and cyanidin, proposed structures of leucoanthocyanidins and transformations of anthocyanins.

266 P. A. ROELOFSEN

pyran ring as in I1 was demonstrated for two chromogens from trees (King and Bottomley, 1954; Swain, 1954). According to Hillis (1955), it depends on certain conditions as to whether catechin, anthocyanidin, anthoxanthidin, or tannin is produced on heating the leucoanthocyanidin with acid.

In recent years success has been achieved in separating natural polyphenol mixtures, by use of paper chromatography, from tobacco and tea (Roberts and Wood, 1953) and catechol tannins (White et al., 1952). In contrast with the current view that the major part of these mixtures consists of high molecular complexes of condensed catechin deriv a t‘ ivcs, it was shown that most of it consists of relatively small molecules of the Clj flavan type, viz., catechins, catechin esters, leucoanthocyanidins, an- thoxanthidins, and their glycosides.

FIG. 6. Two-dimensional paper chromatogram of polyphenols of purple cxxo cotyledons (from Forsyth, 1955); for symbols see Table V.

Applying this method as well as countercurrent separation to extracts of dried purple cotyledons and of separated tannin cells at least nine polyphenol components were separated and determined quantitatively (Forsyth, 1952a, b, 1954, 1955, 1957) (see Fig. 6 and Table V ) .

There are four catechins, of which 92% is ( - )epicatechin, making up 35% of the total polyphenol. The other catechins were provisionally identified. The caff ein-catechin complex, described in early literature, splits up when chromatographed ( Forsyth, 1 9 5 2 ~ ) .


TABLE V Polyphenols of Cacao Cotyledon a

Symbol Substance

C, c, c 3

c, L1 L2,3 A 1 A2 C.T.

( - ) Epicatechin ( + ) Catechin ( ? ) ( + ) Gallocatechin ? ( - ) Epigallocatechin ? Lencocyanidin Leucocyanidins 3-a-L- Arabinosidyl-cyanidin 3-P-D-GaIactosidyl-cyani~iI~ Complex “tannin”

Percent of

Dry weight Total polyphenols

33-42 2.75 35

0.25 3 t 1 2 5 3 9 1.6 21 0.8 10

3: 0.3

2 26 ( 24-40 )

Total polyphcnols 7.8 100 ( 7 . 1 4 1 )

From Forsyth ( 1955).

The leucocyanidins were at first considered to be partly glycosidic (Forsyth, 1952a), but this is not the case. All produce cyanidin on heating with butanol-hydrochloric acid and give the color reactions of catechins.

There now appear to be two cyunins, a galactoside and an ar a b’ ino- side ( Forsyth, 1957). Anthocyanidins in nature always occur as glycosides. They may be in different stages: as red oxonium salts ( IV) , violet color bases (V) , blue phenolic alkali salts (VI ) , and colorless pseudobases (VII ) (Blank, 1947). Forsyth and Rombouts (1952) indicated the occurrence in fresh cotyledons of color bases, pseudobases and probably colorless complexes with other polyphenols.

The nonmobile tannin fraction is nondialyzable, contains a trace of anthocyanin, is glycosidic, gives a strong leucocyanidin reaction, but a negligible titration with permanganate. Presumably the 0-dihydroxy grouping is not available for reaction.

It is striking that all polyphenols seem to be based on epicatechin. This also applies to tea polyphenols except that here gnllate esters of catechins occur. Bate-Smith (1954) has stated that, up to the present, no other hydroflnvans than catechin and gallocatechin have been found in nature.

The amounts of the components, mentioned in Table V apply to Forastero cacao. Two white Criollo samples gave lower figures (totaling 6.0 and 4.6%) although the same components were found, except for cyanins. The leucocyanidins were present in relatively larger amounts.

268 P. A. ROELOFSEN

The same compounds were found in several other varieties including the species pentagcna and Zeiocarpa ( Cheesman, 1944). With varying pigment content, the relative proportion of the two cyanins remained the same.

Many methods of determining totaI polyphenols in cacao have been used by different authors. Their results differ considerably due either to incomplete extraction or to inadequate methods of determination. Complete extraction of unoxidized cacao has been obtained with hot water at constant pH ( MacDonald, 1937; Roelofsen and Giesberger, 1947) and acetic and dilute hydrochloric acids (Hallas, 1939, 1949; Forsyth, 1955). Oxidized tannins dissolve more readily in more alk a 1’ me liquids. The use of organic solvents gives incomplete extraction.

Only part of the polyphenols are precipitated with hide powder (Wilbaux, 1937), gelatin (with tea: Shaw and Jones, 1935), and cinchonine ( MacDonald, 1937; Duthie, 1938; Humphries, 1944a). For example, with cinchonine all except catechin are precipitated ( Forsyth, 1953). More complete determination is possible by oxidation with permanganate (Humphries, 1944a; Forsyth, 1955) or hypoiodite (Roe- lofsen and Giesberger, 1947; Shaw and Jones, 1935), but the conversion factor depends on extent of previous oxidation. Precipitation with formaldehyde-hydrochloric acid is the best method ( Roelofsen and Giesberger, 1947; Duttie, 1938; Humphries, 1944a; Hallas, 1949; Forsyth, 1955). However, dispersed fat may coprecipitate and should be extracted. Fur- thermore the factor for conversion of precipitate to polyphenol may vary. Forsyth (1955) obtained a factor of 0.725 for epicatechin but Deys (1938) 0.95. For tea “tannin” Deys obtained 0.97, Shaw and Jones (1935) 0.98.

In Forastero cacao, Forsyth (1954) found 7.1-9.1% total polyphenol (in Criollo 4.6--8.0%). This seems low since Duthie (1938), although extracting incompletely, found 7 .243% and Hallas (see Chatt, 1953) found about 9% although precipitating with cinchonine. Wilbaux ( 1937), using the hide powder method, found 8-12%.

In Criollo-Forastero hybrids, Roelofsen and Giesberger ( 1947) found 8-13% formaldehyde precipitate which in their opinion need not be corrected for condensed formaldehyde. With these hybrids they found about 1.05-1.36 times as much polyphenol in purple beans as in white beans although from the same pod. The outer parts of the fresh kernels contained about 1.3 times as much as the central part (1.5 times as much if expressed on fresh weight basis since the outer part contains 7-12% less water). Hence flat beans contained relativeIy more than plump ones.


5. CHANGES IN POLYPHENOLS DURING FERMENTATION In determining the changes in polyphenols during fermentation, the

peeled kernels must be fixed quickly (e.g., in boiling water) prior to mincing and extraction to prevent oxidation. Thus after 60 hours of fermentation, Raelofsen and Giesberger (1947) found a decrease of 2540% in polyphenol content. This must have occurred subsequent to the death of the cotyledons after a period of 30 hours. From the determinations of Forsyth (1952b, 1955), who fixed the cotyledons in dilute hydrochloric acid, losses in total polyphenol can be calculated as 24% after 60 hours, rising to 58% after 8 days. Diffusion out of the cotyledons accounts for the losses observed. Without precautionary fixation, Mac- Donald ( 1937) found an increase during fermentation presumably since the oxidase is more active in fresh than in fermenting beans. The increase may also be the result of determining the cinchonine precipitate rather than total polyphenol.

FIG. 7. in Trinidad,

fermentation

Detailed information about the changes in the individual components has been provided by Forsyth (1952b, 1953, 1957). Typical results are shown in Fig. 7. As soon as the cotyledons die (i.e., after one day), all components start to diffuse out of the cotyledons, especially the catechins. In addition, the cyanins are enzymatically hydrolyzed into sugars and cyanidin, and this is converted into leu~ocyanidin.~ Hence, the leucocyanidin fraction increases, but it soon drops as a result of diffusion and conversion into a more complex tannin fraction, thereby

5 See however, footnote 4.

270 P. A. ROELOFSEN

retaining its leucocyanidin character. According to von Lilienfeld-Toal (1938) 4 days for fermentation suAice for the complete disappearance of cyanins. In laboratory experiments under optimal conditions ( 44OC. ( lll°F.), pH 6.0), Forsyth (1952b, 1953, 1957) found total disappearance within 3 days. He stated that some conversion also occurs in boiled beans, especially at temperatures exceeding 45OC. ( 1 1 3 O F . ) . This is nonenzymatic conversion of cyanidin into the colorless pseudobase which is reversible on strongly acidifying. The enzymatically induced conversion, however, is irreversible.

Except when fermentation is prolonged unduly, no oxidation occurs in the cotyledons since the microorganisms in the pulp and the juice under the shell act as barriers against entrance of air. The changes described are anaerobic.

6. CHANGES IN POLYPHENOLS DURING DRYING

During drying, air enters through the shell, the juice under the shell of fermented beans turns brown and forms a brown deposit on the inside of the shell. Gradually, the cotyledons also turn brown internally.

Little is known of exactly what happens to the polyphenols during drying and oxidation. If living cotyledons are dried in the sun or at low temperatures in a well ventilated oven, the polyphenols remain in the tannin cells. They are not oxidized and do not change. All components are readily extracted with dilute acid and the cyanins are soluble in absolute ethanol. However, if as a result of death, the diffusion has occurred prior to drying, the components are less readily soluble. According to Forsyth and Rombouts (1952), the colored part of the cyanidin (free color base) is adsorbed, probably on polysaccharides and is not extractable with absolute ethanol. This procedure, however, does dissolve the colorless cyanidin pseudobase fraction. With aqueous alcohol both are soluble. In fermented beans no such incomplete adsorption occurs because of the acetic acid present. These facts explain the puzzling observations of Steinmann (1931) on a supposed photochemical production of pigment within the cotyledons during sun-drying.

Humphries (1944a, b ) and Forsyth (1952b) studied the oxidation process with minced aerated fresh cotyledons, but evidently this is not wholly comparable with oxidation in fermented beans. Chemical conditions are very different, and the polyphenols have not only decreased but also have changed in relative proportion, in degree of polymeriza- tion, and (with respect to cyanins) in structure. In minced cotyledons the soluble fraction drops within an hour to about 28% of the original


amount according to Humphries (1944a, b ) and to zero according to Forsyth (1952b), who showed that catechins become insoluble less rapidly than all other components. When the minced cotyledons were covered with water, a rather puzzling temporary increase in soluble tannins during the first 15 minutes was found by Humphries.

Hathway and Seakins (1955) showed that between pH 4 and 8, pure catechin (without enzyme) is oxidized with formation of o-quinone and then condenses, involving C-C bond formation. Without oxygen, only epimerization occurs. It is not known whether this reaction also happens in the cacao bean or how the other polyphenols are oxidized and made insoluble. One can only say that since the cacao-brown produced in purple beans is always darker than in white beans and since the total polyphenol content shows little difference, the oxidation product of cyanidin is probably a much darker one.

FIG. 8. Transverse sections of raw cacao beans. A. Unoxidized flat, wrinkled bean. B. A partly oxidized pair of plump beans which have stuck together. Note location of browning and of the air space in B (from Roelofsen and Giesberger, 1947).

Presumably the general trend of oxidation will be the same as that found during oxidation of other flavonoid polyphenols; formation of quinones, hydroxyquinones, and somehow a condensation to high-molecular brown products with high astringency and, hence, low solubility ( Joslyn and Ponting, 1951; Roberts, 1949; Roux, 1955).

Only epicatechin and complex “tannin” can be extracted from sun- dried, browned cotyledons. In the case of Fig. 7 this was about 1 and 2% respectively. The amount varies with the extent of browning since

272 P. A. ROELOFSEN

the oxidized and condensed mixture called cacao-brown is soluble only in alkali. Cyanins are found if unoxidized parts are present in originally purple cotyledons.

Commercially, such beans are called “partly fermented and if practically no browning has occurred, “unfermented.” The location of the unoxidized parts in the “nibs” as seen in transverse section is illus- trated in Fig. 8. Since such incomplete oxidation usually reduces the market value, the circumstances enhancing and restraining oxidation have received attention from various investigators, especiaIIy Roelofsen and Giesberger (1947). According to these authors, since sections of fermented beans brown rapidly, absence of browning in dry beans must be due to lack of oxygen during the period prior to inactivation of oxidase due to the lack of water. The slow diffusion of oxygen into drying wet kernels is explainable on the basis that it is hampered by a steady flow of water in the opposite direction through the shell and through the cotyledon tissue. If the cotyledon tissue were rigid (like wood), all evaporated free water originally located in vacuoles, intercellular spaces and pores, would be immediately replaced by air. How- ever, as indicated by data in Table VI, the cotyledon tissue shrinks

TABLE vr Water Content and Shrinkage of Central and Outer Parts of Kernel of

Beans Fermented for 2 Days a

Cotyledon Water Air tissue

( 100 ml. ) Water evaporated Shrinkage penetrated

( ml. ) ( ml. 1 ( ml. ) x

Central part 48.7 52.3 49.7 3.6 Outer parts 37.1 39.8 36.0 3.6

a After Roelofsen and Giesberger ( 1947).

considerably. Under these circumstances, air-filled spaces will appear only in the final stages of the drying process. However, by then, oxidase apparently is inactive because of lack of water. Evidently, it is not the penetration of air as such, but rather the diffusion of dissolved oxygen that is the limiting factor in the. browning process. The extent of browning, therefore, is a function of the extent to which dissolved oxygen diffuses into the tissue before the oxidase is inactivated by drying.

The shell is the main barrier against access of oxygen during drying, for beans with punctured shells or with an injection needle inserted under the skin seldom remain unoxidized.

It is clear that oxidation usually has been found to be more complete with slow than with rapid drying. This is indicated also by the


location of unoxidized parts of the “nibs” as seen in transverse sections of so-called twin beans (Fig. 8b). Such an asymmetric oxidation may be produced artificially by a one-sided application of sealing wax on superficially dried beans. This proves that with twin beans the phenomenon is not caused by a difference in pH or in the moment of death (see Fig. 4). Kernels of beans fermented with pure yeast cultures have a higher pH and hence a more active oxidase. Still these may likewise show unoxidized parts, again implying that pH and oxidase activity are not limiting the oxidation.

Less rapid drying does not always imply a more complete oxidation since very rapid drying tends to produce more plump (less wrinkled) beans, and since plump beans tend to be better oxidized. This will be discussed more extensively later on.

Invariably the experiments performed have confirmed what has been known as a result of practical experience, i.e., browning is more complete the longer the beans have been fermented. There are at least two reasons for this. The cotyledon tissue seems to be more permeable since the naked cotyledons, if kept in moist air, brown more completely and rapidly if the beans have been fermented for a longer period even though their pH is a little lower.

A second and probably more important reason is that the shells are more permeable to air when fermented for a longer period of time. There are several indications of this. In the first place it can be demonstrated by pressing air into fermented beans while being steeped in water. The production of pectinase by yeasts, by spore-forming bacilli, and by bacteria of the Aerobacter type, readily explain the increased permeability. Furthermore shells of some Java cacao clones are very thin and in these the kernels were usually well oxidized. This was also observed in beans washed so intensively that the outer layers of the testa had been removed. The improvement of oxidation and “plumping up,” by steeping beans in water prior to drying, was also ascribed to an increase in shell permeability as a result of loss of soluble matter. Two hours of steeping sufficed to obtain the maximal effect on subsequent oxidation to be acquired by this procedure (Roelofsen and Gies- berger, 1947).

Another factor determining the access of air is the shape and volume of the bean, From data such as those in Table VI, it appears that the outer parts of the kernel shrink less than the center. The outer zones of the closely fitting drying nibs are likely to constitute a kind of “shell” that more or less resists unproportional decrease of its surface. Therefore, a central cavity tends to be produced. From there, oxygen evidently diffuses more easily into the nibs of the kernel than from the

274 P. A. ROELOFSEN

outside (see Fig. 8); probably not only because the inner tissue is softer, but also because the flow of water and water vapor is outward.

A central cavity does not always occur, however. I t is more often found in plump beans than in flat ones. This is readily understood. In the first place, the amount of central tissue is relatively greater in plump beans. Second, as a result of the more circular shape of the “shell,” as a result of closely fitting outer parts of the nibs caused by drying, the plump beans collapse less readily. Flat beans, on the other hand, often collapse during drying as shown in Fig. 8A. In the latter, all browning must proceed from the outer surface inward. With rapid drying browning may be restricted to a thin outer zone, These are the so-called unfermented beans of commerce which have closely fitting nibs. They are lacking in brittleness and ease of “nibbing,” which adversely affects the ease of grinding in the factory.

The “caving-in” is caused by a suction force developed in the cmter of the kernel as a result of differential shrinkage (see Table VI) . In flat beans, only a small suction force is developed because there is obviously less resistance to collapsing. A central cavity is rarely found since this is only developed when the suction force is high enough for air to be sucked in through the shell. This concept is supported by the fact that cavities nearly always develop in flat beans if an injection needle has been stuck into the bean in such a manner that its central canal is not blocked. It also explains why very rapid drying tends to increase plumpness ( Roelofsen and Giesberger, 1947; Wilbaux, 1937). The moisture gradient tends to be greater, and the “shell” more rigid.

Beans without a central cavity are not only flat or even “caved in,” but often their shells stick to the kernels and are wrinkled. This adher- ence is evidently another result of absence of air since it never occurs with beans having punctured shells. Because the shell shrinks less than the kernel, adhering shells will be wrinkled, whereas smooth shells are loose.

In Java the first wrinkled beans could be observed during drying when the original water content of 60% of the total weight had dropped to 40%. Then more wrinkled beans appeared progressively until the end of the drying process. In plump beans, browning also started a t an average water content of 40%. When dry, plump beans contained about 0.5% less water than wrinkled beans, but of course would be equalized during storage.

The correlations between plumpness, loose shells, and well browned kernels on the one hand and between flatness, wrinkled shells, and incomplete oxidation on the other have been noted by many investigators, most recently in West Africa by MacLean and Wickens ( 1951 ) .


Wohlf‘lrth (1924) seems to have been the first who proposed the use of plumpncss and volume-weight of cacao beans as indexes of commercial value. Others improved on his nicthod of determining the volume. Fincke (1924) and Sam (1929) used sand as a pycnometric medium. Recently Dittmar ( 1954) proposed determination of the ratio of plump to wrinkled beans by flotation of the former in a 5% solution of salt.

No correlation has been observed between purple or white color of cacao from Java Forastero-Criollo hybrids and the tendency to produce wrinkled unoxidized beans. This is apparent since there is also no correlation between color and flatness. However, with mixtures of varieties in which color is correlated with shape and volume, a correlation evidently exists between color and extent of browning.

As stated earlier, the amount of extractable polyphenols in d ry cacao decreases with the extent of browning and correlates with flatness. Sam- ples of wrinkled, mostly unoxidized kernels of Java cacao that was fermented for 60 hours gave 6% formaldehyde precipitate, whereas in plump ones about 4% was found. Since the original content of about 10% may be supposed to have been reduced to 7% by diffusion during fermentation, oxidation in wrinkled beans is responsible for a decrease of only about 1%. Obviously the tannin content is just as good (or as bad) ;in index of quality as plumpness, and accordingly several authors have advised its use ( Kaden, 1939; von Lilienfeld-Toal, 1938). The latter also proposed the use of the extractable cyanin pigment as an index. Unaware of this, Kaden ( 195%) rediscovered this possibility. However, Hallas (see Chatt, 1953) found small differences in soluble cyanin content between purple and brown fermented, dried beans, viz., in different series: 0.06 and 0.01, 1.00 and 0.77, 0.38 and 0.02. This does not encourage the use of this procedure, which moreover is very elaborate. It seems to be still unknown to what extent this decrease is due to oxidation to brown substances, or conversion into colorless leueoanthocyanidin or into colorless pseudobase.

As a result of this disappearance of cyanin, said to be completed after 4 days of fermentation (von Lilienfeld-Toal, 1938), one would expect a frequent occurrence of “white” parts in incompletely oxidized, raw cacao prepared from purple beans fermented for at least that period. The greater part of the world’s cacao crop is fermented longer.

However, unoxidized parts in oriqinally purple beans are without exception purple. Are beans which discolored during fermentation always completely browned, so that the absence of purple pigment never becomes apparent, or is the bleaching partly reversed during drying?

The production of a reddish brown color in the shells on sun-drying

276 P. A. ROELOFSEN

is a well known phenomenon. If dried in the dark, the shells become light brown in color. Steinniann (1931) observed that the precursor was colorless and that acetic acid shifted the color to a more reddish one. Roelofsen and Giesberger (1947) stated that polyphenols and oxygen are necessary (but no enzyme) and that the photochemical reaction also occurs in air-dry shells. In addition they observed that beans fermented with yeasts became more reddish, as if they had been treated with acid, and that the pigment was not cyanin but more like cacao- brown since it did not dissolve in water or alcohol. In sections of the sun-dried shells, the color appeared to be located in the outermost layers, which suggests that the active rays do not penetrate far. Stein- mann’s claim that ultraviolet rays are inactive requires confirmation.

Recently Hillis (1954) found a correlation between the tendency of catecholtannins to redden and their leucoanthocyanidin content. Oxygen was needed in most cases; with more oxygen the color turns a more intense brown. Photocatalysis of this reddening is well known among tannin producers and leather manufacturers. Pigman et al. (1953) observed surface reddening of a nearly pure leucoanthocyanidin in diffuse sunlight. The structure of the colored compound is unknown.

According to Obata and Sakamura (1953), photooxidation of tyrosine produces the same substances as dark oxidation. However, with “catecholtannins” the color becomes more reddish in the light.

Obviously, sunlight not only catalyzes the oxidation of a colorless compound to one of a reddish brown color, but also of “cacao-red” (now known to be cyanidin) to “cacao-brown” (Doppler, 1936).

7. SPECIAL OXIDATION PERIOD AND POSTFERMENTATION

a. Special Oxidution Period

Tea fermentation is not a microbiological fermentation but merely a special oxidation process that occurs subsequent to the killing of the cells of tea leaves by rolling. Schulte im Hofe (1908) tried such an oxidation period with partly dried cacao beans. H e claimed to obtain more complete browning and more flavor by keeping the partially dried and still warm beans in a covered box during the nights of the sun- drying period. Alternatively the beans may be kept in the box con- tinuously for 23 days at 455OOC. (113-122OF.), but in that case the water content should not exceed 15% because of the development of fungi and yeasts.

The laborious packing in boxes made the method impractical, but in many countries the beans are piled or banked up during the night. Sometimes the piles are covered and a temperature of more than 4 O O C .


(104OF.) may be reached. Knapp (1924) suggested that this heat is produced by oxidation. Vyle (1949) mentioned two alternatives while describing the processing of good Venezuelan Criollo cacao, as practiced on the plantation of a well known British chocolate factory. One method is sun-drying for a week, but during the mornings only, In the inter- vening periods the cacao is piled and covered with sacks or put into boxes. The other method requires a special heating and drying appa- ratus. Here the beans are first heated for 6 hours at 60-66OC. (140- 151OF.) in an atmosphere saturated with moisture. Then follows a slow drying to a moisture content of 1520% at 50-6OoC. (122140OF.) for 3 days, and finally a rapid drying. A similar procedure used in Samoa was described by Eden (1953). He kept partially dried beans in a heated revolving drum for about 12 hours. Essentially, these are the methods advocated by Schulte im Hofe in 1908.

b. Postfermentation

Eden (1953) also applied his oxidation method to incompletely oxidized dry cacao which had been remoistened. This too was a modern version of a very old method. About 1927, several chocolate manufacturers in Germany discussed in the journal Gordkzn the pros and cons of the postfermentation processes which were then in use (Rieck, 1927). Fincke (1952) discusses several of these methods, the first of which had been advocated as early as 1818. This so-called Nachfermentierung or Behelfsfermentation was applied to insufficiently oxidized cacao and consisted of steeping in water, draining, curing with moderate heat, and drying.

A process, similar to the “Behelfsfennentation” was advocated in Java (Roelofsen and Giesberger, 1947) to be applied to wrinkled beans, obtained separately by the customary grading used on estates, It did not involve heating, but merely steeping overnight and then redrying. To check the darkening of skins the use of 0.5 to 1% sodium bisulfite in, the steepwater was advised. It greatly improved browning and also flavor on roasting (see page 285). Of course the content of soluble tannin and of acetic acid decreased, Even “nonsweated” dried beans become normal in appearance. Most important was the “plumping” of 70-90% of these beans all of which originally were wrinkled and flat. Disadvantages were the loss of about 2.5% of weight, the inevitable breakage of some shells, and additional labor costs. It would be necessary to obtain a premium for plump grades.

Manufacturers have shown a recent increased interest in postfermentation. Kempf and Murer (1951) were assigned a patent for a process similar to the Behelfsfermentation, the only difference apparently

278 P. A. ROELOFSEN

being that it was to be used on intentionally nonsweated dry cacao. This is washed free of pulp, then kept at 55-66°C. (131-140°F.) for several hours (with or without prior addition of alkali) and then dried. The main purpose was to get nearly sterile cacao for use in ice cream. The flavor, however, is poor (see page 282). It appears that there are spores in normal raw cacao that survive roasting. Presumably raw cacao that is fermented for a short time and dried in filtered air will contain very few spores and will do as well as unsweated cacao but will produce a better flavor.

In Germany, Kaden (1952a) obtained a patent on a similar postfermentation to improve slaty and incompletely browned beans with application of pressure and vacuo and of dilute alcohol and acetic acid. This process has been christened Okadierung. Taubert (1955) and Baiicker (1954) indicated that in order to obtain revolutionary results, the beans must be steeped in dilute solutions of organic acids instead of in water. Taubert applied for a patent on the use of a temperature of 90°-1050C. for curing the wet beans (see Fincke, 1952).

8. FLAVOR AND AROMA

Obviously, chocolate flavor and odor developed on roasting is by far the most important characteristic of cacao beans. However, we do not know anything about the chemistry of this process. We know only the several conditions to be realized in order to obtain good flavor.

Numerous trials by interested planters and by scientists have been made to find a substitute for the “sweating” process. They killed thc beans by methods which left oxidase unimpaired, i.e., freezing. heating at a moderate temperature, and steeping in dilute organic acids (see Knapp, 1937). Heating at 50-6OoC. ( 122-140°F. ), as suggested by Fickendey (1913) and strongly advocated by Stevens (1925) was tried several times with both fresh beans and beans that had been fermented for one day to loosen the pulp. While external and internal appearance of these experimental products could be as good as or even better than those of normal cacao, the flavor on roasting was invariably 1 ac k’ ing or insufficient. This also was found with beans killed by freezing, steeping in acetic acid, or by drying.

We must assume that during fermentation and drying, a substance “A” or a complex of substances is converted into a substance or R complex “B,” which on roasting produces chocolate flavor. Killing is necessary but not sufficient for production of “€3”; the killed bmns must be kept or brought under special conditions which are realized durinq fermentation. In addition, these conditions insure the elimination of a substance “C,” perhaps identical with “A,” which on roasting produces


a disagreeable beany smell. Enzymes are commonly supposed to be necessary for these processes, but this has never been proved. Perhaps both the action of some enzymes and the inactivation of others are required,

a. Suppositions on the Constitution of Flavor Precursors

One might suppose that the chemical constitution of the essential oil obtainable from roasted cacao should disclose something about the constitution of its precursor. Sack (1913) distilled 1 ml. essential oil with chocolate odor from 20 kg. unroasted commercial cacao but did not study it. Practically all we know of the distillate of roasted cacao, we owe to Bainbridge and Davies (1912), who obtained 24 ml. oil from 2000 kg. of beans. They identified 50% as a-linalol, over 10% as alkyl esters of lower fatty acids, and also found some free acids. Schmalfuss et ul. (Schmalfuss and Barthmeyer, 1932; Schmalfuss and Rethorn, 1935) determined diacetyl and acetylmethylcarbinol in commercial cocoa powder and finding that it was twice as much as in good butter, postulated its participation in chocolate flavor. This was considered doubtful by Fincke (1936) since addition of diacetyl to cocoa had little effect.

Fincke also doubted the presence of linalol. If present, it might have originated from sterols. The occurrence of esters made many suggest that the ester-producing yeasts in the pulp were of certain significance but, as will be indicated later, this was not confirmed.

Another line of approach is the isolation and identification of the flavor precursors from the raw cacao bean, If these were known, flavor might be changed and increased by adding them, which of course would be of paramount significance for chocolate manufacturers (but not for producers of high quality raw cacao!). Knapp (1937) stated that cacao butter from unroasted cacao did not produce flavor on roasting, whereas the residue of an alcoholic cacao extract did, Hence, starch, proteins, cell-wall material and fats may be rejected as primary flavor precursors although they might exert minor effects, They might also be necessary for absorbing and preserving the flavor.

The polyphenols have been supposed to be precursors of aroma in tea, coffee, and cacao. Shaw (1934) announced that tea tannin, boiled with acid, produced an odor like that of methylsalicylate, and the boiling of tea tannin with caffein in his opinion produced a tea-like aroma. Oxidation of tea tannin was supposed to produce aromatic cleavage products, With coffee, chlorogenic acid is thought to participate in aroma production ( Roelofsen, 1939). Taubert ( 1955) claims to have obtained a cocoa-like odor when keeping gambir catechin in dilute acid at 90°C. (194OF.) for 14 hours. As stated earlier, cacao, when post-

280 P. A. ROELOFSEN

fermented and dried after soaking in acid, is said to develop special flavor precursors. The hypothesis that browning of cacao is directly connected with precursor development was rejected as soon as it was observed that beans killed by heating, freezing, or mincing were per- fectly brown but without any flavor. Moreover, the reverse situation of good flavor without browning also occurs. This will be discussed later.

Forsyth and Rombouts (1951) confirmed Knapp’s statement concerning flavor production of an alcoholic extract of cacao. This did not occur, however, when the purin had been removed. The flavor was restored when theobromine (not caffein) was added again prior to roasting. Certain fractions, containing complex leucocyanidins, when roasted with theobromine, were also stated to develop an aroma sug- gestive of chocolate. However, these authors no longer hold this view ( Forsyth and Rombouts, 1956).

This is in line with experiments performed in the present author’s laboratory by van der Veken in 1952. Residues of ethanol and methanol extracts of raw cacao when roasted in glass-stoppered bottles developed flavor more or less reminiscent of cocoa, especially when the pH had been adjusted to 5.8. Previous removal of theobromine did not affect the flavor on roasting. As a matter of fact, it is known that theobromine changes very little on roasting of cacao beans (Chatt, 1953). An alkaloid in cacao which does diminish during roasting is trigonellin, from which niacin is produced as in the roasting of coffee (Adamo, 1955).

Since in the present author’s experience it was known that sucrose in coffee does affect its aroma, sugars were removed by van der Veken from the residues of methanol extracts of raw cacao by treatment with baker’s yeast. Two-thirds of the total of the sugars disappeared, but the aroma on roasting the residue was unaffected. Apparently free sugars are not important. Glycosides were absent, According to Diemair et d. (1958) about one-third of the reducing sugars disappear on roasting, whereas sucrose is unaffected. A Maillard-type of reaction is anticipated.

When polyphenols, organic acids, etc., had been removed from the alcoholic cacao extracts by precipitation with lead acetate, no flavor developed on roasting; however, addition of the precipitate after removal of the lead failed to restore flavor on roasting. Apparently the aroma precursors had been destroyed and no conclusion could be drawn as to their constitution.

Not only are the flavor precursors A and B unknown, but also substance(s) C, which affects aroma adversely on roasting. Hardy and Rodrigues (1953) advanced the hypothesis that C might be a protein. They found less in fresh Criollo than in Forastero beans, and the more present the lower the grade of Forastero. In the opinion of the present


author this hypothesis is by no means unlikely. Protein certainly is not a flavor precursor since it does not dissolve in alcohol. Moreover, the unpleasant odor it develops on pyrolysis is well known. There is extensive proteolysis during fermentation. It is possible that the anaerobic condition in the fermenting bean considered essential for ff avor precursor production by Forsyth and Rombouts (1951), is needed for the promotion of proteolysis, because plant proteinases are commonly activated by reducing conditions. Furthermore oxidized polyphenols become more astringent and by reacting with the proteins (tanning) will inhibit enzymatic hydrolysis more than unoxidized polyphenols will.

Tobacco is another product which porduces aroma on pyrolysis, and one of the main changes in curing and fermentirig of tobacco is known to be proteolysis (Frankenburg, 1946, 1950). With certain types of tobacco a correlation has been found between low protein content and high commercial value (Vickery and Meiss, 1953) just as in cacao. Bokuchava and Popov (1954) claim that tea tannin reacts with amino acids to produce tea-aroma constituents, which in view of Heimann’s (1955) findings of the oxidative deamination of amino acids by quinones is by no means unlikely. Furthermore, amino acids are known to produce aroma when subjected to Maillard type re? c t’ ions.

The amino acids formed in tobacco, tea, and cacao, and the products of their deamination and decarboxylation might be flavor precursors, as they are known to be in malting and the baking of bread. In fact they are alcohol-soluble, However, preliminary experiments in the present author’s laboratory indicated no change in the pattern of free amino acids during roasting and only a small decrease, but Diemair et al. (1958) observed a considerable decrease.

b. Formation of Flavor Precursors during Cacao Processing

Determination of the conditions enhancing development of flavor precursors is of the utmost importance. A primary requirement is a method to assess flavor. Brokers often assess ffavor by smelling the nibs after rubbing them between both hands. This is definitely inadequate. Flavor should be assessed after roasting the beans by a standard method and tasting a cocoa liquor or preferably a piece of chocolate. Even this procedure is not foolproof. There is always a lack of flavor in handling small samples as compared to that of full-scale factory preparation. Furthermore, an experienced taste panel is required for rating the experimental samples. Statistical analysis of results and frequent repeti- tion of the assessment are needed if experience is insufficient. Even then, only marked differences can be distinguished (MacLean and Wickens, 1953; Wadsworth, 1953; DeWitt, 1954).

282 P. A. ROELOFSEN

Roelofsen and Giesberger ( 1947) could draw important conclusions although using another method. Not having acquired satisfactory results by chemicaI analysis of steam distiIIates, they resorted to olfactometric measurement of the aroma strength of the air in a series of bottles containing a progressively diluted aqueous extract of powdered, roasted kernels. The air from the bottles was sniffed by means of a gIass tube with nose fitting ends. The first sample tested was odorless water and then progressively from the less diluted samples to the more concentrated ones until a faint sweetish odor indicated to the tester that the threshold value of the sample had been reached. This usually happened at a dilution equivalent to 0.006-0.01558 powdered cacao suspension. For reliable results, samples had to be tested repeatedly and without dis- tinctive designations on the bottles. The beans of samples to be compared were roasted as a mixture after having been marked on the shell with ink to make possible their subsequent identification. The method has the advantage of being simple, rapid, and relatively accurate, provided the experimenter has good sensory perception. It is well known that in general by determining the threshold values, much smaller differences between samples can be detected than by assessing differences in strength of odor.

However, the value of this assessment is limited: first, odor is assessed, not flavor; second, not its quality, but only the strength of its dominating component is assessed. Still, this determination of “aroma number” apparently is correlated with commercial evaluation of flavor, as judged from the well known differences in flavor as a result of the variables: long and short fermented cacao, stored and fresh cacao, inadequate and adequate roasting, the high priced San Felipe cacao, and the common Accra. The method seems unsuitable for assessment of quality of flavor in cacao varieties but proved to be of value for determining the conditions of processing which enhance flavor in one and the same cacao. Such conditions, partly well known, partly as revealed by use of the olfactometric method in Java (Roelofsen and Giesberger, 1947) or in other experiments, now will be summarized:

1. Beans killed by mincing, freezing for 1 hour a t -4°C. (25”F), or submersion in hot water for 30 min. at 55OC. (131OF.) prior to drying, brown very well but produce no aroma. Neither do heated beans fermented in a wide-meshed bag along with the bulk, or beans fermented for 15 hours (still living), then heated, and then fermented further. Heating at 55OC. (131OF.) is less detrimental if applied to beans that have already been killed in the sweatbox and progressively less the later the heating is applied (e.g., after 235 days of fermentation). However, heating at 90°C. (194OF.) after 255 days adversely affects the aroma strength. This, however, is not the case, after 4% days of fermentation.


Apparently the precursor development occurs only in cotyledons killed in a special way, i.e., by the combined action of acetic acid, alcohol, and moderate heat as occurs during fermentation. When the beans are first killed at 55OC. (131OF.) and then exposed to these special conditions in the sweatbox, no precursor is developed. However, according to Forsyth and Rombouts (1951) and Wadsworth and Howat (1954) flavor precursor is produced by beans that have been killed slowly at 48°C. (118OF.) and 50°C. (122°F.) respectively and kept at that temperature for several days, Another method for obtaining precursor development without fermentation is the postfermentation of beans that have been dried when alive. However, a full flavor has never been produced in this way.

It is clear that the precursor-developing reactions resist moderate heating (provided the special conditions in the cotyledons have already been established), but they do not resist heating at 90°C. (194OF.). This is completely in line with the application of heat during the special oxidation periods, as described earlier. For example, in Venezuela (see Vyle, 1949), Criollo cacao is heated at 50-66OC. ( 122-151°F.) for several days subsequent to sweating for 40-60 hours in order to obtain the desired Venezuelan flavor. The precursors themselves resist 90OC. (194OF.) in wet beans. In dry ones, they resist even higher temperatures since they are not lost during roasting but rather develop flavor at 130OC. (266OF.).

2. In Java, beans killed by steeping in 1% acetic acid at 25OC. (77OF. ), then dried normally, did develop aroma. This was also observed by Knapp (1937) although the aroma was considered of low intensity. However, acid (let alone acetic acid) is not a conditio sine qua T I Q ~

since sterile beans fermented with pure yeast cultures in flasks buried in the sweatbox develop a normal amount and a normal quality of aroma. The unchanged color of the cyanin in such beans (normally fermented ones become more reddish) suggests that little acid, if any, penetrated. As a matter of fact, beans submerged in water of pH 7.0 and “fermented” in the sweatbox produced more aroma than if submerged at pH 4.0. However, Forsyth and Rombouts (1951) obtained the opposite result, so further experiments are needed.

3. Aromatic substances produced by yeast did not take part in flavor development on roasting, even if the growth of yeast had been enhanced and lengthened to such an extent that the dried cacao obtained a flower- like odor (see p. 245). Chocolate made from such cacao was not considered to be different in any way from the normal product. It is now generally assumed that the microflora has no influence on aroma ( DeWitt and Cope, 1951).

4. At one time it was thought that the purple beans in a mixture of

284 P. A. ROELOFSEN

hybrid cacao required a longer fermentation than the white ones. How- ever, as stated earlier, there is neither a difference in the time of death, the ratio of plump beans, nor the extent of browning. Furthermore, no difference could be detected in strength and quality of aroma of purple and white (incompletely oxidized) beans selected either from raw Java cacao or from a single pod from a hybrid tree. Hence, the anaerobic disappearance of pigment in purple beans during fermentation cannot in itself be of significance for aroma.

This, of course, does not mean that different varieties do not vary in flavor and in their time requiremmts for fermentation. Both in Java and elsewhere it was found that there may be great differences in strength and quality of aroma and flavor between clones of the same variety (Roelofsen and Giesberger, 1947; MacLean and Wickens, 1951; Wadsworth, 1955; DeWitt, 1954), as well as between beans of the same pod (Wadsworth, 1955). A mixture of clones seems to produce a better flavor than the constituent clones taken separately (DeWitt, 1954; Cope and Jolly, 1955; Fennah, 1955). This is in line with findings of Roelofsen (1939) with coffee. Moreover, it is well known that manufacturers of tea, coffee, cocoa, tobacco, etc., blend their product in order to acquire the best aroma,

5. As is demonstrated by kernels killed by heat, freezing, etc., browning may occur without any development of flavor precursor; however, the lower price received for wrinkled, incompletely oxidized beans, as compared with plump beans, has established the opinion that oxygen in some way or another enhances precursor development. In Java no difference in strength or quality of aroma was found between such grades selected from the raw product. This has been confinned in studies with West African cacao using a taste panel (MacLean and Wickens, 1951; Wadsworth, 1955).

Apparently oxygen is needed neither for precursor development nor for the elimination of the substance that produces the beany smell. Forsyth and Rombouts (1951) even postulated the importance of the absence of oxygen; in fact, it is absent during fermentation and the first stage of drying. On the contrary, Wadsworth (1953, 1955) found that air must have free access (or at least that carbon dioxide must be removed), during both fermentation (aseptic) and drying. This point certainly requires further attention.

6. In Java, maximal aroma strength was obtained by lengthening the fermentation up to 4lh days, which is a good 3 days beyond the time at which the cotyledons are killed. Furthermore, Wadsworth (1955) found that this is the optimal period for keeping the dead beans prior to drying. This is fully in accordance with the world-wide experience, i.e., that more flavor is obtained when fermentation is prolonged.


In Java, with 2 days of fermentation, the same level could be reached by drying more slowly, i.e., by sun-drying for a week instead of artificially drying in 254 days. However, a fermentation for only 156 days (although all beans were killed) could not be compensated for by slow drying. Apparently precursors are also developed during drying, provided the moisture content does not drop below 15-20%, and provided the fermentation is not shorter than about 20 hours after the time the beans were killed. Whether this minimum period of fermentation is needed to establish the optimal chemical conditions in the cotyledons, or whether it is needed because of the higher temperature attained, is still an open question. In Wadsworth's experiments, 5OOC. (122OF.) was the optimal temperature.

7. No difference in strength of aroma was found between ripe and unripe beans in Java if both had been fermented in wide-mesh bags in the sweatbox. However, quality was found to be best, in accordance with common experience, with ripe beans. If fermented in bulk, overripe beans develop more aroma than ripe ones. This is ascribed to their earlier death as a result of more aeration and quicker fermentation of the mass. Hence, the difference will be detectable only if the fermentation is too short and the drying too fast to get full precursx development.

According to Wadsworth and Howat (1954) and Wadsworth (1955) a "germinaticn" period of 3 days at 35OC. (95OF.) is needed for full flavor development in laboratory experiments, However, the following facts are a t variance with this. If picked ripe pods are left unopened for several days, the beans begin to germinate. Such beans are commonly said to lack flavor. In normal sweatbox fermentation, the beans live for only 30 days, which is about 36 hours after opening the pods. On some estates and on most small holdings, this period of incipient germination lasts longer. Thus far, however, there are no indications that this in- tended or unintended delay in the death of the beans produces better flavor. Further experimentation is essential before Wadsworth's conclusions can be accepted.

8. Washing or steeping fermented beans prior to drying has often been considered to reduce flavor on roasting. In Java it was found that washing may indeed diminish the aroma strength, but only because it hastens drying and hence is not apparent when drying is slow or the fermentation time long, Steeping fermented beans for 12 hours did not appreciably affect aroma strength as compared with unsteeped fermented cacao. Apparently precursor development proceeded uncurtailed, which was surprising since the temperature was about 20°C. (36OF. ) lower. According to Forsyth and Rombouts (1951), steeping entails a loss of flavor.

9. Steeping dry beans followed by redrying (i.e., postfermentation)

286 P. A. ROELOFSEN

increased aroma strength. in both slaty and normal Java cacao. Some precursor must have been dissolved in the steepwater since its residue developed flavor on roasting; however, this was probably mostly extracted from the shells. As mentioned earlier, precursor development during postfermentation is a generally accepted phenomenon, but it is uncertain as to whether it happens in beans that were fermented long enough or dried slowly enough to produce the full amount of precursors.

10. Storage of raw Java cacao at 76% relative humidity for several months markedly increased strength and quality of aroma; however, this was not the case when stored at 43%. The advantage of storing cacao is well known to manufacturers.

‘41. STORAGE OF COMMERCIAL CACAO IN TROPICAL CLIMATE

In tropical climates, raw cacao is much more liable to deterioration by fungi and insects than in moderate climates. The occurrence of moldy beans,

- much more than of grubby ones, is a common defect - much more than of grubby ones, is a common defect

100- % EQUILIBR.REL.HUH.

40.

-BEAMS (XYLCN! nrmOD) -KERNELs ( M L N E METHOD) M S H C L U (XVLENC METHOD)

44

a0

“O4 s 6 T e s lo 4 1 qn Q 44 41 i 4 (T 18 40

of cacao

FIG. 9. Sorption isotherms at about 26°C. (79°F.) of beans, kernels, and shells of cacao (from Scott, 1929 and Roelofsen and Giesberger, 1947).

which is dried either too slowly or insufficiently, or which is stored too long in a humid, hot climate without special measures. The main factor determining the liability of cacao to these defects is the equilibrium relative humidity (e.r.h.) of the cacao beans, which on prolonged storage is equal to the mean r.h. of the atmosphere.

Usually the moisture content rather than e.r.h. is determined. This may be converted into e.r.h. for comparison with other materials. Most other materials contain less fat and hence more water than cacao at the same e.r.h. The e.r.h./moisture content isotherm at about 26OC. (79OF.) for Accra cacao beans was determined by Scott (1929) and for kernels and shells of Java cacao by Roelofsen and Giesberger (1947) (see Fig. 9) . From the latter data, the graph has been constructed for beans having a shell fraction of 11%. The graphs for Accra and Java beans


do not coincide because in the latter case the xylene distillation method for moisture content was used with powdered material. This results in about 1.3% more water than the oven methods commonly used for whole beans [heating for 3 hours at 105OC. (221°F.)]. Scott did not mention which procedure he used, but presumably it was an oven method.

1. MOLDINESS

Several investigators determined the critical e.r.h. or the critical water content for mold growth in and on cacao. Dade (1929) could not detect internal molding of commercial Gold Coast beans with 7.9% water (81% e.r.h.) after 11 weeks, nor with 8.4% (84%) after 4 weeks, but beans with 9.1% moisture content (86% e.r.h.) molded after 2 weeks. Ciferri (1931) observed growth of mold on raw Puerto Rico beans kept at 79% r.h. after 16 weeks. The relative humidity, however, may have gone higher for short periods since initially the e.r.h. of the beans was more than 90%. Roelofsen and Giesberger (1947) placed washed raw Java cacao with an initial moisture content of 4 5 % as well as ground nibs of the same beans in flasks at a constant r.h. of 76% (controlled by moist salt). No mold growth occurred on or in beans after 5 months at about 27OC. ( 8 1 O F . ) but ground kernels supported visible growth after 4 months. At 85%, undamaged beans became molded externally after a few weeks whereas the ground nibs were heavily molded.

An e.r.h. of 828, corresponding to 8.0% water (9.491; with xylene method) was considered a safe limit by Dade, but since damaged beans are liable to decay on prolonged storage even a t 76% r.h., a safer limit for long storage seems to be 72% r.h. The latter corresponds to 7.2% moisture by the oven method. Even a small amount of growth must be prevented, since the moisture content increases as a result of metabolism. Aspergillus glaucus has been observed to thrive even below 70% e.r.h. on more favorable substrates. This, however, does not happen with cacao.

It is well known that beans with broken shells or shells perforated by the radicle are much more liable to moldiness. Plump beans have more broken shells and hence become moldy more often than wrinkled beans (Renaud, 1954). Intact beans are practically always invaded at a point in the testa opposite the radicle although no structural difference from other parts of the testa are apparent (Dade, 1929). If this point is covered by sealing wax, a much smaller number of beans decay even at a high humidity (Laycock, 1930). The testa opposite the radicle is further weakened during incipient germination ( Renaud, 1954).

Shells of nonfermented dry beans are practically never invaded by mold (Laycock, 1928; Dade, 1929; Knapp, 1937). Presumably the testa tissue is somewhat macerated by pectinase produced by yeasts during fermentation.

Renaud (1954) reviewed the literature on the fungi and actino-

288 P. A. ROELOFSEN

mycetes isolated from moldy cacao beans and identified. As with other plant products, the most common molds are species of Aspergillus (i.e., glnucus, fumigatus, flavus, tnmarii ) which are the most xerophilic fungi known. Species of PenicilZium and Mucor buntingii also are commonly encountered. This species of Mucor and A. furnigntus are reguIarly found in the outer zones of unturned fermenting piles. These and other species of fungi invade the beans even during the drying process if drying is too slow.

The optimal temperature for most of these fungi is 30°C. (86°F.). Penicillium seems to prefer somewhat lower and M . buntingii higher temperatures. In moderate climates, the less favorable temperature requires a higher humidity for growth, so even without taking special measures, mold growth usually stops when the cacao arrives there. Actinomycetes, bacteria, and yeasts require a higher humidity than fungi. They do not normally grow in stored cacao. Yeasts and molds may produce a slight external bloom on the shells during the nights of the sun-drying period, but it is quite harmless. White spots on the cotyledons are sometimes taken for molds, but usually these are due to theobromine. However, when the shells are damaged, yeast colonies may cause similar spots.

Moldy cacao has a musty odor which does not disappear on roasting. Moreover, it lacks flavor. Cacao butter and probably theobromine are decomposed ( MacLean, 1953). Postfermentation is said to lessen this damage.

2. ATTACK BY INSECTS

One of the more recent investigations on insects in commercial cacao was made by Nicol (1941) in England. Moths are most common. The only moth found in samples from Africa was Ephestia cautella Wlk., whereas in samples from America, E . elutella Hb. was more common. In English warehouses, both insects may occur, with E . elutella dominating. The eggs are deposited on the shells, but larvae live in the kernel and form extensive mycelium-like webs. They never penetrate unbroken cacao shells.

Two beetles and their larvae, Araecerus fnsciculatus De G. and Lmioderma serricorne F., are fairly common. These species do not grow in moderate climates. Other insects or insect parasites are seldom found. The larvae of the beetles may attack unbroken beans although they prefer broken ones.

The critical e.r.h. for attack of cacao nibs was determined by Roelof- sen (1959) for Arnecerus and by Roelofsen and Giesberger (1947) for E . cautella. Both these insects barely survived at 63% r.h. and about 26OC. (79OF.). At 76% r.h., multiplication was still slow but at 85% it was very good, The lower humidity requirement of insects as compared


with fungi is well known. In grains a certain species thrives at even 40% e.r.h. This explains the occurrence of moths in cacao stored in moderate climate. As with fungi, insects may attack the beans during the drying period, but more often invasion occurs in the stacked bags.

3. CONTROL MEASURES

In warehouses on the Gold Coast, a mean relative humidity of 80% has been observed (Steemson, 1929) and 6045% in Java, depending on season and altitude ( Roelofsen and Giesberger, 1947). Since the cacao tree requires a humid climate, similar conditions will prevail in other producing countries. Although cleanliness in buildings in which cacao is stacked is important, it is evident that everywhere in the tropics stored, cacao will be infested by insects in time. Moldiness will also appear in most, but not all, countries. However, even in a humid tropical climate, a hot and dry warehouse may pervent moldiness.

Of course, the primary condition is reasonably fast drying to an e.r.h. of 70% or less. For storage, the buildings should be as hot as possible (i.e., low, with iron roofing, and not shaded ). The floors should rest on piles. If the floor is in contact with the soil, the bags should be put on platforms since water vapor ascends even through concrete floors and will affect the bags in the bottom layer. Only asphalted floors are moisture-proof.

In order to prevent moldiness, the cacao simply may.be redried as soon as the e.r.h. has risen to 75 or 80%. However, redrying entails breakage of shells. In some cases it might be feasible to warm the air in the buildings a little during the night until the mean r.h. is below 70%.

More rigorous measures must evidently be taken to prevent insect infestation. In Java, during the Second World War, storage in calico “chambers” erected in the warehouse all around the stacks proved to be effective for prolonged storage. The calico should nowhere touch the stacks. Infested cacao must be freed previously of all insect stages, which object may be attained either by heating up to 55OC. (131OF.) or by fumigating, e.g., with methyl bromide, which does not affect flavor ( Spoon and Sesseler, 1955).

VII. NEEDED RESEARCH

Research will naturally very much depend on whether the investi- gator’s interest is purely scientific or whether it is aimed at a practical problem. In the first case, nearly every point reviewed offers oppor- tunities for further research, but very few scientists will be in a position to perform such work on the spot. In the second case, the present author is of the opinion that further research might be mainly focused on: ( I ) The best conditions for natural flavor precursor development, ( 2 ) The chemical nature of the precursors.

290 P. A. ROELOFSEN

As to the first point, the results of Roelofsen and Giesberger (1947), obtained with the olfactometric method should be checked by including the assessments of a taste panel. As has been discussed above, there are indications that good flavor may be developed in cacao without microbiological fermentation, viz., by killing the beans with acetic acid or with moderate heat, followed by a period of storing the dead beans before they are dried. This should be checked and amplified with trials of other methods to kill the beans, e.g., freezing and mincing, and of the best conditions for keeping the dead beans in order to obtain the maximum precursor developments. There are several still obscure points, such as the supposed necessity of anaerobic conditions in the cotyledons, of incipient germination, and of a lowered pH in the cotyledons. Many questions arise, e.g., are precursors developed in artificially killed beans kept under anaerobic conditions, or aerobically with inhibited oxidase, or when subsequently fermented in the sweatbox? What happens with flavor if proteolysis is enhanced in minced cotyledons by adding proteinases or reducing substances such as cysteine, or when it is checked by mild oxidation with flour improvers?

It is not impossible that research along these lines might lead to a modified method of processing cacao, but the author believes that the chances are poor since the methods developed through an experience accumulated after half a century of trial and error will be difficult to improve.

Elucidation of the chemical nature of the flavor precursors presumably is of more interest for chocolate manufacturers than for cacao producers, for if these should be detected, the former will perhaps be less interested in good natural cacao flavor since they might add artificial flavor to inferior cacao by steeping in synthetic precursor solutions. This problem might perhaps be tackled by proceeding along the lines indicated earlier, viz., by fractionating extracts of cacao. However, testing the flavor production of roasted extracts is a problem of its own. Possibly the extract should be roasted after absorption by exhaustively extracted dry cacao nibs. In addition, substances thought to be precursors might be added to cacao lacking in flavor, e.g., cacao protein hydrolyzates, saccharides, and polyphenol fractions.

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