Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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for cobalt speciation analysis. The cyanocobalamin

form of cobalt has been measured in blood serum,

seminal fluid, and milk serum. Vitamin B

analogs

have also been determined in animal carcass tissue.

These speciation techniques should see intense devel-

opment in future.

0028 Neutron activation analysis can be used to

determine the amount of cobalt in biological and

environmental samples; typical detection levels are

0.03 mgg

1

. This technique usually requires a post-

irradiation period of around 3 weeks before g-ray

emission from the

Co isotope can be measured.

Radiation flux levels are usually around 10

–10

neutrons cm

2

1

. Concentration techniques using

dithiocarbamate complexation are usually applied

to improve detection levels.

See also: Chromatography: High-performance Liquid

Chromatography; Cobalamins: Properties and

Determination; Physiology; Mass Spectrometry:

Principles and Instrumentation; Applications

Further Reading

Barceloux DG (1999) Cobalt. Journal of Toxicology –

Clinical Toxicology 37: 201–216.

Brown S and Savory J (1984) Chemical Toxicology and

Clinical Chemistry of Metals. New York: Academic

Press.

Schneider Z and Stroinski A (1987) Comprehensive B

Berlin: Walter de Gruyter.

Smith IC and Carson BL (1981) Trace Metals in the

Environment. Cobalt, vol. 6, pp. 1202. Ann Arbor, MI:

Ann Arbor Science.

Smith KA (1990) Soil Analysis, Modern Instrumental

Techniques, 2nd edn. New York: Marcel Decker.

Smith RM (1987) Cobalt. In: Mertz W (ed.) Trace Elements

in Human and Animal Nutrition, 5th edn. London:

Academic Press.

Underwood EJ and Suttle NF (1999) The Mineral Nutrition

of Livestock, 3rd edn. Wallingford, UK: CAB Inter-

national.

Ysart G, Miller P, Crews H et al. (1999) Dietary Exposure

estimates of 30 elements from the UK Total Diet Study.

Food Additives and Contaminants 16: 391–403.

Cockles See Shellfish: Characteristics of Crustacea; Commercially Important Crustacea; Characteristics of

Molluscs; Commercially Important Molluscs; Contamination and Spoilage of Molluscs and Crustaceans;

Aquaculture of Commercially Important Molluscs and Crustaceans

COCOA

Contents

Chemistry of Processing

Production, Products, and Use

Chemistry of Processing

B Biehl, Technische Universita

t Braunschweig,

Germany

G Ziegleder, Fraunhofer-Institut fu

r Verfahrenstechnik

und Verpackung, Freising, Germany

Introduction

0001 The most valuable constituents in cocoa are cocoa

butter and flavors. Both are specific for seeds of

Theobroma cacao L. Whilst the quality of cocoa

butter is not changed during processing, the particu-

lar flavor of cocoa depends on profound chemical

reactions which take place during processing and

manufacturing. The unprocessed cocoa seed would

not give the characteristic flavor when roasted unless

it was first fermented and dried. The sequence of raw

cocoa processing in the tropics and then manufactur-

ing is essential since reactions during fermentation

depend on water and enzymes of the living seed,

whilst manufacturing comprises nonenzymatic reac-

tions in a dry lipid phase. Raw cocoa beans are the

link between processing (normally in the tropics)

and manufacturing (normally in the consuming

1436 COCOA/Chemistry of Processing

countries). A large number of essential constituents of

cocoa flavor are produced during roasting of raw

cocoa from flavor precursors formed during tropical

processing. Reactions during fermentation and drying

resulting in raw cocoa beans and subsequent reac-

tions taking place during manufacturing are de-

scribed in this first article.

Chemistry of Raw Cocoa Processing

0002 Both the seeds and the pulp are essential components

in raw cocoa processing. However, the reactions

which are essential for raw cocoa quality take place

within the seeds alone and not in the pulp. Enzymes

of the seeds are directly involved but microorganisms

only indirectly, in that bacteria and fungi are respon-

sible for the degradation of the pulp, and their

metabolites, especially acetic acid, which are instru-

mental in the subsequent changes in the seeds. Organ-

isms participating in the direct attack on the nibs

during the late stage of fermentation and overfermen-

tation are also important.

0003 Processing depends on the chemical composition

and physiological state of the beans, their enzymes,

and their pre- and postmortem subcellular structures.

They greatly affect the final product quality.

Biology of the Seed

0004 Two commercially exploited types of the species T.

cacao, Criollo and Forastero, are distinguished on the

basis of fruit morphology and geographic origin. Cri-

ollo gives fine-flavor cocoas, although in low yields,

while the more vigorous, high-yielding Forastero

subtypes and hybrids predominate in plantations

worldwide. Cocoa pods contain about 35–45 seeds

consisting of the embryo and the shell, which is

covered when ripe by a mucilaginous pulp, the endo-

carp (Figure 1). The main parts of the embryo are two

folded cotyledons connected by a small embryonic

axis. A rudimentary, skin-like endosperm covers the

surface of the embryo. After fermentation the shell,

covered with residues of pulp, comprises about 12–

16% of the seed dry weight, depending on bean size

and pulp degradation during fermentation. The dry

weight of a seed varies considerably, but is ap-

proximately 1.0–1.2 g. Only the cotyledons are

used for cocoa and chocolate manufacturing and in

these the leaf mesophyll is the dominating tissue,

consisting of two types of cells which differ in com-

position.

0005 About 80% of the cells store lipid and protein, the

major volume of which is occupied by a large number

of individual lipid bodies of constant size (about

2 mm) surrounding one or more branched protein

storage vacuoles and amyloplasts. The remaining

20% of the cells are polyphenol storage cells. Their

lumen is almost entirely occupied by one central

vacuole containing all the stored polyphenols and

purines.

0006Subcellular structures in the lipid and protein stor-

age cells control pre- and postmortem reactions

during fermentation, which are not the same as in

cell free systems. On the subcellular level in vivo,

the situation can roughly be described as a dispersed

lipid phase (lipid bodies) in a continuous water phase

(cytoplasm; Figure 2). During fermentation, the cells

are killed by heat and acetic acid, but postmortem

structures are maintained. In the cell lumen, with low

concentrations of acetic acid, the lipid bodies fuse in

situ, and they form, more or less, a continuous lipid

phase which separates cytoplasmic inclusions as a

hydrophilic dispersed phase. In contrast, in the pres-

ence of a high concentration of acetic acid, fusion of

the lipid bodies is more extensive, causing complete

fig0001Figure 1 Unfermented cocoa seeds showing the pulp, the testa

and the cotyledons. Reproduced from Cocoa: Chemistry of Pro-

cessing. Encyclopaedia of Food Science, Food Technology and Nutri-

tion, Macrae R, Robinson RK and Sadler MJ (eds), 1993,

Academic Press.

COCOA/Chemistry of Processing 1437

segregation of the lipid in the cell center (Figure 2). In

the first case, intracellular diffusion of water-soluble

compounds is reduced by lipid barriers. In the latter

case, diffusion is not restricted. Proteolysis, browning

reactions, acid diffusion, and flavor precursor forma-

tion mutually depend on these structures.

Composition of Unfermented Cocoa

Seeds

0007 The prominent secondary compounds are flavonoids

and purines (theobromine and caffeine). The storage

reserves include predominantly cocoa butter but also

protein and starch. All these compounds are stored in

the cotyledons. The composition of ripe seeds is

shown in Table 1. Differences of cocoa butter, pro-

teins, and pigments are found between Forastero and

Criollo types.

Cocoa Butter

0008 Fat, the main storage component, on average com-

prises 53–58% of the cotyledon dry weight. It con-

tains about 95% triacylglycerols, 2% diacylglycerols,

<1% monoacylglycerols, 1% polar lipids, and 1%

free fatty acids (as percentages of lipids). Triacylgly-

cerols consist of about 37% oleic (O), 32% stearic

(S), 27% palmitic (P) and 2–5% linoleic (L) acids (as

percentages of total fatty acids). Other saturated and

monounsaturated fatty acids do not contribute more

than about 2% of the fatty acids. These values are

typical of fermented beans but do vary between

samples, and between genetic and geographic origins.

This is also true of the complex fatty acid composition

in triacylglycerols. In cocoa butter, the disaturated

triacylglycerols, mainly POS, SOS, and POP predom-

inate by far over monosaturated triacylglycerols,

tbl0001Table 1 Analyses of unfermented West African cocoa

Constituent Dried beans (%)

Cotyledons 89.60

Shell 9.63

Embryonic axis 0.77

Fat 53.05

Water 3.65

Ash (total) 2.63

Nitrogen

Total nitrogen 2.28

Protein nitrogen 1.50

Ammonia nitrogen 0.028

Amide nitrogen 0.188

Theobromine 1.71

Caffeine 0.085

Carbohydrates

Fructose (0.09)

Glucose 0.30 (0.07)

Sucrose 0 (2.48)

Starch 6.10

Pectins 2.25

Fiber 2.09

Cellulose 1.92

Pentosans 1.27

Mucilage and gums 0.38

Tannins (total phenolics) 7.54 (13.5)

Acids

Acetic (free) 0.014

Oxalic 0.29

After Rohan (1963) with permission of the Food and Agriculture

Organization of the United Nations, except data in parentheses for sugars

in Bahia from Berbert (1979). Total phenolics from Swain (1954) as cited by

Rohan (1963) assuming 55% fat; see Further Reading.

Acetic acid

0.035mol l

−1

Acetic acid

0.200mol l

−1

Premortem

Postmortem Postmortem

fig0002 Figure 2 Schematic representation of electron microscopic aspects of postmortem changes in mesophyll cells caused by different

concentrations of acetic acid during fermentation-like seed incubation. Spherical lipid globules in the living cell fuse in situ (left) or

form a central bulk of lipid (right). Hydrophilic compartments are indicated by dots or dashes. Reproduced from Cocoa: Chemistry of

Processing. Encyclopaedia of Food Science, Food Technology and Nutrition, Macrae R, Robinson RK and Sadler MJ (eds), 1993, Academic

Press.

1438 COCOA/Chemistry of Processing

mainly POO and SOO. The melting temperature of

cocoa butter at 31–34



C is an essential characteristic.

Differences of fat hardness are reflected in the iodine

value of cocoa butter and the ratio of disaturated to

monosaturated triacylglycerols (Table 2B, C).South

American cocoas are the softest compared with cocoa

from Asia and Africa. Storage fat accumulation

during seed development undergoes characteristic

changesinfattyacidcomposition(Table 2A).Cli-

mate, demonstrably the ambient temperature and

stress due to heat or drought, affects biosynthesis

and thus the final composition of triacylglycerols,

and the melting and crystallization characteristics of

cocoa butter. Whilst the amount of fat per seed

depends on both, genetics and physiology of pod

development, effects due to genetic differences or the

process of fermentation on cocoa butter quality are

not unequivocal. (See Fatty Acids: Properties; Trigly-

cerides: Structures and Properties.)

Polyphenols

0009 Flavonols are the major components. The order

of quantities in Forastero types is (approximate

weight percentages of total polyphenols): proantho-

cyanidins (58–65%), catechins (29–38%), then

anthocyanins (1.7–4.0%). Specific compounds

detected include: anthocyanins (3-a-l-arabinosidyl

cyanidin and 3- b-d-galactosidyl cyanidin); catechins

(()-epicatechin, (þ)-catechin, (þ)-gallocatechin,

()-epigallocatechin). Additionally, several (up to

eight) proanthocyanidins (di- and oligomeric) and

proanthocyanidinglycosides (predominantly based

on ()epicatechin) have been isolated and identified.

Three flavonols have been found as minor constitu-

ents: quercitin, quercitin-3-glucoside, and quercitin-

3-galactoside. Up to 17 phenolic acids and esters have

also been reported. The total amount of seven of

them comprise not more than 23 p.p.m. of the seed

dry weight (phloroglucinol, protocatechuic acid,

vanillic acid, o-hydroxyphenylacetic acid, p-couma-

ric acid, caffeic acid, ferulic acid). (See Tannins and

Polyphenols.)

0010Anthocyanins and their aglyca are lacking in Cri-

ollo-type seeds but provide the characteristic color of

Forastero seeds. They are also taken as an indicator of

the ‘degree of fermentation.’ Colorless proanthocya-

nidins and catechins are strongly astringent and are

effective tannins. Their enzymatic oxidation and

polymerization cause browning reactions after cell

wounding and reduction of astringent taste. Cocoa

polyphenols are strong antioxidants which are

increasingly considered as antioxidative protectors

in food. (See Colorants (Colourants): Properties and

Determination of Natural Pigments.)

Alkaloids

0011The composition of methylxanthines in cocoa seeds is

genetically variable but characteristic of cocoa. Theo-

bromine (1–2%, dry weight) and caffeine (0–2%, dry

weight), together with traces of theophylline and 7-

methylxanthine, are found. They are not metabolized

during fermentation. Theobromine plays a role in the

bitter taste of cocoa. Several phenolic amines and

alkaloids derived from tyrosine and tryptophan

have been detected at very low levels (3–40 mgg

1

)

in both unroasted and roasted cocoas. (See Alkaloids:

Toxicology.)

Sugars and Acids

0012Besides a total of around 12% (dry weight) of poly-

saccharides (Table 1), the free sugar content in cocoa

seeds is not more than 2–4% (dry weight). Sucrose is

the major component (about 90% of total sugars),

followed by fructose plus glucose (about 6%). In

addition, galactose, raffinose, melobiose, sorbose,

mannotriose, xylose, arabinose, mannitol, and inosi-

tol have been detected by several investigators in

tbl0002 Table 2 Data reflecting differences of cocoa butter hardness

A Ratio of saturated

to unsaturated

fatty acids in triacylglycerols in

developing cocoa seeds

Days after pollination Massratio

105 0.98

130 1.06

170 1.63

B Ratio of disaturated

to monosaturated

triacylglycerols in cocoa

butter from different origins

Provenance Massratio

South America 9.43

Africa 12.32

Asia 19.45

C Iodine values incocoabutterfromdifferent origins

Provenance Iodine value

Ghana

36.5 + 0.78

Ivory Coast

35.5 + 1.15

Brazil

37.5 + 1.31

Ecuador

36.7 + 0.04

Malaysia

34.7 + 0.34

A Data derived from Griffiths G, Harwood JL (1991) The regulation of

triacylglycerol biosynthesis in cocoa (Theobroma cacao)L.Planta 184: 279–

284; B and C

derived from Chaiseri S, Dimick PS (1989) Lipid and

hardness characteristics of cocoa butter from different geographic

regions. Journal of the American Oil Chemists Society 66: 1771–1776; C

from

Cocoa BeanTest 1961/62 (1962) Hamburg: Gordian. In A cocoa butter is from

unfermented seeds; in B and C it is from fermented seeds.

Palmitic acid

(P) plus stearic acid (S);

oleic acid (O) plus linoleic acid (L); Arachidic acid

(A).

POP, POS, SOS, PLP, PLS, SLS.

POO, SOO, SOA.

COCOA/Chemistry of Processing 1439

minor quantities. (See Carbohydrates: Classification

and Properties.)

0013 Volatile acids are not found in fresh cocoa beans,

but nonvolatile acids are found in low concentra-

tions. Phosphoric, malic plus tartaric acids were

found to make up 0.32% (dry weight), oxalic acid

0.35%, and citric acid 0.73%. These acids in unfer-

mented seeds are not metabolized during processing

nor do they contribute to cocoa quality.

Proteins and Amino Acids

0014 Correct analyses of proteins strictly depend on suit-

able protection against tanning, especially against

protein–quinone reactions. The protein content in

the cotyledons of ripe seeds varies between 10 and

16% (dry weight). Criollo cocoa seeds have been

reported to contain less protein than Forastero

seeds. The proteins of Forastero seeds reveal about

16 peptide bands in sodium dodecyl sulfate–polyacryl

amide gel electrophoresis (SDS-PAGE): four of them

predominate in amounts. They are subunits of vacu-

olar storage proteins. One of these polypeptides is

an albumin of 21 kDa, characterized as a Kunitz

protease inhibitor (52% (w/w) of total protein).

Three – the 47-kDa, the 31-kDa, and the 16-kDa

polypeptides – make up about 43% (w/w) of total

protein. They are subunits of the vicilin-type (7S)

globulin, a glycoprotein, each of them consisting

of multiple pI-forms. The 31-kDa and the 16-kDa

subunits are posttranslational processing products of

the 47-kDa subunit. The native (7S) globulin is

a trimer (139-kDa) composed of 47-kDa and (31-

kDa7 þ16-kDa) subunits. It is almost unsoluble at

pH < 5.0. The amino acid sequences of the major

polypeptides (47, 31 and 21 kDa) and their precur-

sors have been derived from the nucleotide sequences

of the corresponding cDNAs and genomic clones

from immature cocoa beans. The sequence of cocoa

(7S) globulin is unique but similar to that of cotton

seeds and is different from those of other plants.

Cocoa seeds do not contain legumin-like (11S)

globulin.

0015 The fresh cotyledons from ripe seeds contain about

5mgg

1

(dry weight) of free amino acids. Acidic

amino acids dominate. (See Amino Acids: Properties

and Occurrence.)

Enzymes

0016 Various enzymes have been identified in cotyledons of

fresh, ungerminated cocoa seeds which in part are

active during postmortem fermentation or drying,

including glycosidases, invertase, proteases, poly-

phenoloxidase, and peroxidase. Several proteases

have been established:

0017An aspartic endoprotease: its activity optimum is

at pH 3.5. It consists of two polypeptides, which

are obviously derived from a 42-kDa polypeptide

by self-digestion. It splits native (7S) globulin, and

its specific activity is 20–50-fold higher in recalci-

trant cocoa seeds compared with that in orthodox

seeds of several higher plants.

0018A cysteine endoprotease: its specific activity is

considerably lower than that of the aspartic endo-

protease. In contrast to an additional cysteine

endoprotease which is induced during germin-

ation, it does not split the native (7S) globulin.

0019A seryl exopeptidase: this carboxypeptidase

reveals optimum pH around pH 5.8 and substrate

specificity, preferentially liberating hydrophobic

amino acids.

0020A leucine-p-nitroanilide cleaving seryl exopepti-

dase displaying maximal activity at pH 6.8. (See

Enzymes: Functions and Characteristics.)

0021Glycosidases are responsible for hydrolysis of fla-

vonoid glycosides. Their highest activity in cocoa

beans was found between pH 3.5 and 4.5. Polyphe-

noloxidase (o-diphenol: O

oxidoreductase) in unfer-

mented seeds displays a high activity which is strongly

reduced after fermentation. Under a wide range of pH

optima reported by several authors, pH 6.0–6.4 is the

most probable range of highest activity. In wounded

plant tissues it effectively oxidizes polyphenols, and

the resulting quinones react spontaneously to give

brown polymerization products.

Composition of Pulp

0022The mucilaginous, sweet pulp firmly adhering to each

of the individual seeds is formed during pod develop-

ment from an endocarp meristem. Pulp from ripe

seeds contains high but variable amounts of sugars

(Table 3) which impair seed germination. Citric acid

is responsible for the low pH value (pH 3.5–3.8). The

pulp variably binds a lot of water due to pectins.

Additionally hemicelluloses, cellulose, and proteins

are constituents which are insoluble in water and

which resist hydrolysis in strong mineral acids.

0023Besides pectins in the pulp, the seed testa also con-

tains mucilage: both swell considerably after removal

of acid.

Changes During Fermentation

0024Changes in the seeds (cf. Figure 3 in Cocoa: Produc-

tion, Products and Uses) start in the organized sub-

cellular structure of the living seed. They proceed to

postmortem reactions which are caused by heat and

diffusion of acetic acid from the fermenting pulp

1440 COCOA/Chemistry of Processing

(Figure 2). These changes are controlled by the effect-

ive concentration of acetic acid and the nib pH value,

which drops to pH 5.0–4.0.

0025 Once the plasma membranes and tonoplasts are

destroyed, proteins, polyphenols, and other com-

pounds are released from their compartments and

come into contact with enzymes. The pH value estab-

lished in the cells controls the postmortem activity of

acidic hydrolases (proteases, glycosidases, invertase)

under anaerobic conditions.

0026 As a result of cell destruction, variable amounts

of soluble constituents, flavonoids, amino acids,

purines, and sugars are lost from the seeds due to

exudation. A loss of 10–40% of soluble substances

modifies the original composition of cocoa seeds.

Reported changes in the content of fat are in accord-

ance with this loss of soluble substances.

0027 Sucrose disappears to a large extent from the seeds

during fermentation, due to invertase activity. How-

ever, the amount of fructose plus glucose in the fer-

mented beans is not more than 50% of the original

amount of sucrose. Since their proportions are vari-

able, they obviously undergo further reactions.

0028 The flavonoid glycosides are hydrolyzed by acidic

glycosidases. The violet aglycon cyanidin subse-

quently undergoes bleaching by nonenzymatic trans-

formation to a colorless pseudobase. Leucocyanidins

and proanthocyanidins are transferred nonenzymati-

cally to more complex forms. Although no reactions

of the catechins have been established, the loss of 50–

75% suggests nonenzymatic oligomerization. These

strongly tanning compounds are released from poly-

phenol storage cells but they do not prevent proteoly-

sis in the absence of oxygen.

0029 Proteolysis in the beans is crucial for the formation

of cocoa-specific aroma precursors. It starts after

uptake of acetic acid. The extent and type of prote-

olysis, and thus the resulting aroma potential in fer-

mented beans, strongly depend on the pH value

established in the beans during this stage of proteoly-

sis. The significant details have been found by com-

paring changes during fermentation with aseptic

incubations of seeds, acetone dry powder, and of

purified cocoa seed proteins and proteases (Figure 3).

tbl0003 Table 3 Analysis of cocoa pulp mucilage

Fraction Unit Quantity

Sugars

Hexoses mg seed

1

104 (51)

Others (including sucrose) mg seed

1

61 (33)

Polysaccharides

Pectins mg seed

1

12–13

Water-insoluble

Polysaccharides (hemicelluloses and cellulose) mg seed

1

23–31

Nitrogen

In the pectin fraction %w/w 0.35

In the fraction of water-insoluble polysaccharides %w/w 2.28

Pulp fresh weight mg seed

1

900–1200

(800–1000)

Pulp dry weight mg seed

1

200–240

(120–180)

Unpublished data from Dziggel M and Biehl B (1986).

Pulp of seeds from Amelonado-type and in some cases Malaysian hybrids from

ripe pods and in some cases from

overripe pods were analyzed giving

one example of strongly variable quantities, depending on pod physiology and ripeness. Compare Table 1 in Chapter Cocoa – Production, Products and

Uses and see Biehl B, Meyer B, Crone G, and Pollmenn L (1989) Journal of the Science of Food and Agriculture 48: 189–208.

pH 4.0−5.5

pH 5.0−6.0

pH 4.0−4.5

Aspartic

endoprotease

carboxy-(exo)

peptidase

Nonenzymatic

Maillard reactions

during drying

and roasting

Cocoa aroma

−

Cocoa seed

storage

(7S) globulin

Hydrophobic

oligopeptides

Hydrophilic oligopeptides

Hydrophobic amino acids

fig0003Figure 3 Proteolytic formation of cocoa-specific flavor precur-

sors during fermentation and their transformation during drying

and roasting.

COCOA/Chemistry of Processing 1441

0030 All cocoa seed proteins, especially albumin and

(7S) globulin, are degraded by cocoa aspartic endo-

protease after strong acidification (pH 4.0). How-

ever, at higher pH values (pH 5.0–5.5), the (7S)

globulin is the only protein attacked by the aspartic

endoprotease. Cocoa-specific aroma precursors com-

prise characteristic patterns of hydrophilic oligopep-

tides and free hydrophobic amino acids resulting

from suitable degradation exclusively of cocoa (7S)

globulin. Neither cocoa albumin nor (7S) globulins

from other plants (so far as these have been investi-

gated) give the characteristics degradation pattern

and cocoa-specific aroma precursors depends on

the substrate specificity and the specific cooperation

of both the aspartic endoprotease and the carboxyex-

opeptidase. In the pH range 4.0–5.5, the aspartic

endoprotease cleaves (7S) globulin at the site of

hydrophobic amino acids in its chain, giving a con-

siderable number of hydrophobic oligopeptides. At

pH 5.0, carboxyexopeptidase acts near to its pH

optimum and specifically removes hydrophobic

amino acids from the hydrophobic oligopeptides. In

this way they are further converted to hydrophilic

oligopeptides and free hydrophobic amino acids,

especially leucine, alanine, phenylalanine, and tyro-

sine. After incubation or fermentation, their propor-

tions are significantly higher than in the proteins

(Table 4).

0031 Consequently, during strongly acidic fermentation

(around pH 4.0), almost no hydrophilic oligopeptides

and few free amino acids are generated and cocoa

aroma potential in fermented beans is low in contrast

to moderate nib acidification during fermentation.

At pH 5.0 both enzymes are sufficiently active.

0032During fermentation, about 2 days after seed

death, the rate of proteolysis decreases significantly.

This decrease is not due to tanning since it is also

found in the absence of polyphenols during acetone-

dry powder autolysis.

0033The liberated amino acids and peptides in the beans

do not seem to be metabolized during fermentation

unless the seed takes up oxygen during the final stages

of fermentation. They may now be exposed to direct

aerobic microbial attack, especially during subse-

quent prolonged (over)fermentation.

0034Changes in the pulp are considered below in

Microbiology of Pulp Fermentation.

Changes During Drying

0035Reactions during drying are an oxidative continu-

ation of fermentation. They are important for quality.

Only about 10–20% of catechins and proanthocya-

nidins present in unfermented beans are found in raw

cocoa after fermentation and drying. Polyphenols are

oxidized by molecular oxygen: the resulting quinones

undergo polymerization to give brown products. Oxi-

dation is brought about by residual activities of the

polyphenoloxidase. However, as it is optimum at pH

6, in acidic beans (pH 4.0–5.0) browning is impaired

during drying. At pH > 7.0 polyphenols are oxidized

nonenzymatically. Thus, overfermented cocoa easily

turns brown, giving an incorrect appearance in the

cut test of perfectly fermented cocoa beans. Since

tbl0004 Table 4 Amino acids in cocoa seeds

Free amino acidsin cotyledons of cocoa seeds Protein-boundamino acidsin unfermented seeds

Amino acid fraction

In cotyledons

after standard

fermentation

In cotyledons

afteraseptic

seed incubation

In vitro after

enzymatic digest

of purified cocoa

(7S) globulin

Total cotyledonprotein

Enriched (7S) globulin

Acidic

15.9 20.5 21.8 30.4 26.0

Hydrophobic

58.3 58.6 56.7 34.1 41.3

Portion of Leu, Ala, and Phe

under hydrophobic

40.9 41.3 36.0 18.8 23.6

Others

25.8 21.0 21.5 35.5 32.7

From 5 days heap fermentation in Ghana (Amelonado).

Unfermented ripe beans from Malaysia (hybrids) underwent aseptic incubation at pH 4.5 in acetic acid containing media for 20 h at 40



C and 40 h at 50



under nitrogen.

Purified cocoa (7S) globulin was incubated in dilute acetic acid at pH 5.2 at 50



C for 16 h in the presence of partially purified cocoa aspartic

endoprotease. Resulting peptide mixtures were incubated at pH 5.8 at 45



C for 16 h in the presence of partially purified cocoa carboxypeptidase.

Acetone dry powder from unfermented Malaysian seeds were hydrolyzed in hydrochloric acid.

Proteins in pH 8.9 buffer extract of acetone dry powder from unfermented Malaysian seeds were separated on a Sephadex G150 column. The fraction

containing the (7S) globulin was hydrolyzed in hydrochloric acid.

Values are mol% of total estimated free amino acids or protein-bound amino acids respectively.

Data from Kirchhoff PM, Biehl B and Crone G (1989) Peculiarity of the accumulation of free amino acids during cocoa fermentation. Food Chemistry 31:

295–311; and Voigt J, Heinrichs H, Voigt G and Biehl B (1994) Cocoa-specific aroma precursors are generated by proteolytic digestion of the vicilin-like

globulin of cocoa seeds. Food Chemistry 50: 177–184. High-performance liquid chromatography was used for estimation of amino acids in polyphenol-free

extracts of cotyledons, lyophilized after treatments.

a^d

1442 COCOA/Chemistry of Processing

enzymatic browning requires high oxygen concentra-

tions, browning does not take place before drying. A

consequence of polyphenol oxidation and quinone

polymerization is the reduction of the astringent

flavor, which is due to the tanning property of cate-

chins and procyanidins, especially the oligomeric

forms. Oxidation and polymerization transfer these

mono- and oligomeric flavonoids to nonastringent

brown polymers. (See Browning: Enzymatic –

Biochemical Aspects.)

0036 Thus, the extent of these polymerizations is con-

trolled by the nib pH, the permeability of the shell to

oxygen, and the destruction of subcellular compart-

mentation. Roughly, the internal color of raw cocoa

beans is related to astringency. Slaty beans reveal a

strongly astringent taste while entirely brown beans

and, especially, overfermented beans give an insipid

taste.

0037 The extent of the browning reaction also affects the

residual seed proteins. Free amino- and sulfhydryl

groups of proteins readily react with quinones and

thus participate in the production of brown polymers.

Amino acids and oligopeptides are less reactive.

However, they participate in the formation of Mail-

lard products during drying. The spontaneous reac-

tion of proteins and quinones is also responsible for

the toxic effects of quinones on bacteria and fungi.

Therefore, overfermentation is suppressed during

browning reactions in the course of drying. However,

the resulting polymers are not toxic and would not

impair microbial growth on brown raw cocoa beans

during storage and on fully brown beans in particular.

Microbiology of Pulp Fermentation

0038 The events taking place during pulp fermentation

correspond to a succession of microorganisms meta-

bolizing the pulp. The wide range of organisms in the

wild inoculum is similar in different cocoa-growing

countries. When starting fermentation, the low pH

value and the high sugar content of the pulp allow

anaerobic fermentation by yeasts and lactic acid bac-

teria. Ethanol is produced, and after exhaustion of

pulp sugars, acetic acid bacteria predominate. Acetic

acid and lactic acid are the most significant metabol-

ites penetrating the beans. Several authors have isol-

ated more than 30 species from 13 genera of yeasts.

The size of the population of lactic acid bacteria is

subordinate, and is further repressed when acetic acid

bacteria subsequently become active after exhaustion

of pulp sugars. Several homofermentative and hetero-

fermentative lactobacilli have been isolated. (See

Lactic Acid Bacteria.)

0039 A number of species of the genus Acetobacter

which have been found in fermented cocoa not only

oxidize ethanol to form acetic acid but also oxidize

this acid, causing a decrease in acetic acid during late

stages of fermentation. Additionally, Gluconobacter

spp. were found.

0040The sizes of these populations correlate with the

amount of products formed (ethanol, lactic acid,

acetic acid). However, there is no information avail-

able about the more detailed competition and signifi-

cance of all these individual species.

0041More recent experiments have been described

studying the effect during usual fermentation of pure

cultures of selected microorganisms either in addition

to or in the absence of wild inoculum. In total, from

different observations with respect to changes in the

pulp, temperature increase, acid production, and final

bean quality, it can be deduced that a limited number

of species, yeasts, lactic acid, and acetic acid bacteria,

which are adapted to the conditions in the cocoa

pulp, can completely substitute for the numerous or-

ganisms in fermenting cocoa.

0042Besides pulp sugar and acid metabolism, pectino-

lytic activity is of interest: this assists in drainage of

pulp in the early stage of fermentation. Kluyvero-

myces marxianus was found to have highest pectoly-

tic (endopolygalacturonase, EC 3.2.1.15) activity but

additionally Saccharomyces chevalieri, Torulopsis

candida, Candida norvegensis,andKluyveromyces

fragilis were reported to reveal significant endopoly-

galacturonase activity.

0043The last stage of fermentation is characterized by

the development and dominance of aerophilic bac-

teria, depending on aeration. Up to 14 species of the

genus Bacillus have been isolated which, in part, are

facultative anaerobes. Further species of other genera

have been reported. There is no direct evidence as to

how they contribute to cocoa fermentation in detail.

Most probably they participate by increasing the shell

permeability and later on in overfermentation. Bacil-

lus stearothermophilus, B. subtilis, B. circulans, B.

licheniformis and Streptococcus thermophilus have

been found as dominating organisms. Species like

B. megaterium, B. subtilis, B. coagulans, B. cereus, B.

polymyxa or Enterobacter aerogenes have been isol-

ated, which metabolize carbonic acids, proteins, and

amino acids, producing low-molecular-weight fatty

acids. Propionic, butyric, and isovaleric acids are in-

creased to 0.1–1.0% of dry weight each in overfer-

mented cocoa beans, compared to 0–0.02% in

controls. These findings strongly indicate bacterial

degradation of amino acids (and flavor precursors)

during overfermentation. (See Bacillus: Occurrence.)

0044A large number of different filamentous fungi de-

velop on the seeds in well-aerated niches, especially

when less acidic and strongly aerated during the later

stages after a steep pH increase. They may develop

COCOA/Chemistry of Processing 1443

further during drying when the humidity is high.

Xerophilic fungi like Aspergillus fumigatus, as well

as A. glaucus, Penicillium spp., Mucor sp., Paecilo-

myces sp. and Geotrichum sp., which have been

found to develop during late stages of fermentation,

have also been reported to occur on stored raw cocoa

beans.

Flavor Compounds and Flavor Precursors

Formed during Fermentation and Drying

0045 Some characteristic flavor compounds in cocoa prod-

ucts are original constituents of the unfermented

seeds but they are changed in amount during fermen-

tation and drying: theobromine tastes bitter. Astrin-

gency is due to tanning flavonoids, and their

oligomeres. Further oligomerization during fermenta-

tion increases. Oxidation and polymerization during

drying decrease their taste intensity. Acidic taste is

due to lactic and acetic acid, which diffuse into the

beans during fermentation and persist in variable

amounts after drying and roasting. When present in

high concentrations, acetic acid may mask the im-

pression of cocoa aroma.

0046 Cocoa flavor precursors comprise compounds in

fermented beans which react during drying and

manufacturing, especially roasting, to give flavor.

Specific cocoa aroma precursors are not original com-

ponents of the seed but are formed during fermenta-

tion and drying: reducing sugars, free amino acids,

and oligopeptides result from enzymatic hydrolysis.

Their formation and significance is described above,

in the section on changes during fermentation. They

undergo Maillard reactions during drying and

roasting in order to generate cocoa aroma.

0047 After in vitro proteolysis under suitable conditions

of cocoa (7S) globulin in the presence of cocoa seed

proteases, the methanol extract of aroma precursors

gives a typical cocoa aroma when roasted in the pres-

ence of reducing sugars. Synthetic mixtures of amino

acids, adapted to the composition of free amino acids

in fermented cocoa beans in the presence of reducing

sugars but in the absence of the fraction of hydro-

philic oligopeptides, do not generate cocoa aroma on

roasting.

0048 Intermediate Amadori products and several pyra-

zines have been found in fermented and dried beans

before roasting; pyrazines (mono-, di-, tri-, and tetra-

methyl-pyrazines) in part are already generated during

fermentation. Polyphenols (flavonoids), although im-

portant for the final astringent taste, do not contri-

bute to the cocoa aroma. Some authors found that

they enhance cocoa aroma.

0049 In addition to cocoa aroma precursors, oligopep-

tides and theobromine are flavor precursors: during

roasting, N-terminal amino acids of several hydro-

phobic peptides produce diketopiperazines which

give the characteristic cocoa bitter principle in a 1:2

complex with theobromine on roasting.

Chemistry of Manufacturing Processes

0050Cocoa flavor is chemically very complex because it

arises from fermentation and roasting and is influ-

enced by further processing steps. The flavor of

plain chocolate is modified but chemically very simi-

lar to cocoa. The flavor sensations of cocoa products

and chocolates are based on a great number of vola-

tile aroma compounds and their interaction with non-

volatile constituents which affect taste and tactile

characteristics.

Cocoa Flavor Development

0051Flavor of unroasted cocoa Raw cocoa has an acid

taste and a flat but characteristic aroma. As a result of

the fermentation process it contains aroma precur-

sors, mainly free amino acids, monosaccharides, and

their first reaction products (Amadori compounds),

peptides, monomeric flavonoids, and methyl-

xanthines. The first aroma compounds were de-

veloped in an early Maillard reaction during the

drying of the fermented seeds or absorbed from the

fruit pulp. The major volatiles in the concentration

range 0.1–1mg kg

1

are aldehydes (produced via

Strecker degradation of amino acids), alcohols, acet-

ates, and acids, which derive from valine, leucine,

isoleucine, and phenylalanine (Tables 4 and 5). Tetra-

methylpyrazine is formed by microorganisms. Special

flavor-grade cocoas, mainly harvested in Venezuela,

Trinidad, and Ecuador (Arriba), reveal a flowery and

tea-like aroma and contain significant concentrations

(0.5–2mgkg

1

) of linalool and further terpenoids

which contribute to this valuable note. On the other

hand, basic cocoas from West Africa, Malaysia, or

Brazil (Bahia) carry a fairly strong inherent flavor and

have very low concentrations of linalool. The sharp

acidity of raw cocoas arises from acetic acid, the

astringency and bitterness from soluble polyphenols

(mainly epicatechin), tannins, theobromine, and caf-

feine. A smoky flavor, which is a flavor defect of some

cocoas, is produced by the absorption of volatile

phenols from the smoke of the firewood used for

drying.

0052Roasting and Aroma Formation Aroma is formed

from its precursors in raw cocoa beans. In contrast to

amino acids, glucose and fructose are consumed to a

large extent during roasting (up to 90%). While the

composition of triacylglycerols in cocoa butter

remains unchanged during roasting and further

1444 COCOA/Chemistry of Processing

processing steps, the entire cocoa aroma is formed by

careful roasting of fermented and dried seeds at tem-

peratures between 120 and 140



C. Using modern

technology, cocoas can be roasted at different particle

sizes, such as whole beans, as nibs, which are coarsely

ground and broken beans, or as liquid cocoa mass,

which is produced by a fine grinding of cocoa and

liquefying within its own fat. The roasting of smaller

particles has the advantages of better controlled

roasting, partial exhaustion of abundant acetic acid,

and reduced roasting time (beans about 30 min, nibs

12 min, mass 2 min).

0053 About 600 compounds have been identified in

cocoa aroma. In common with all roasting aromas

there is a large number of carbonyl and heterocyclic

compounds. The main carbonyl groups are acids and

esters (each about 50 compounds), alcohols, alde-

hydes, and ketones (each between 30 and 40 deriva-

tives). The main heterocyclic compounds are pyrazines

and chinoxalines (together about 80 compounds), fur-

ans, pyrones, and lactones (together 40 compounds),

diketopiperazines, phenols, pyrroles, and oxazoles

(each group about 10 compounds). Derivatives of

phenylalanine are prominent volatiles after roasting.

As a result of the Maillard reaction, pyrazines,

pyrroles, phenylacetaldehyde, phenylalk-2-enals,

pyrones, furanones, and furans increase during

cocoa roasting, whereas alcohols, esters, and acids

remain more or less unchanged.

0054Simple aldehydes arise by Strecker degradation of

free amino acids. Their immediate flavor characteris-

tics are not very striking, but they act as most import-

ant reactants. Some phenylalk-2-enals are generated

via the aldol condensation, for example, which carry

a typical flowery odor fairly reminiscent of chocolate

or cocoa. Many pyrazines with different substitutes

and chinoxalines with desirable flavor properties con-

tribute to the roasted note. It was found that methyl-

pyrazines are formed during roasting according to a

specific rate depending on substitution. Some pyrones

and furanones, as dihydrohydroxymaltol, hydroxy-

maltol, and furaneol, arise from degradation of

monosaccharides and reach remarkable concentra-

tions in roasted cocoas. Characteristic of cocoa

aroma are abundant phenyl derivatives which arise

from phenylalanine and contribute to the intensive

sweet aromatic note. The typical intense bitterness

of roasted cocoa is to a great extent induced by

adducts of such diketopiperazines with theobromine

and caffeine. In the course of roasting, monosacchar-

ides react to the extent of about 60–90%, while free

amino acids only react to about 20%. Therefore, in

several roasters the unroasted nibs or masses are

treated with aqueous sugar solutions to increase the

concentration of the carbohydrate precursors and to

improve the aroma yield. In addition, during the

moist thermal treatment of cocoa, the level of Ama-

dori compounds may be raised and the aroma may be

intensified. (See Caffeine.)

0055Technologically Influenced Flavor Changes Roasted

cocoa nibs and mass are important semimanufac-

tured goods and are used for the production of

cocoa powder, cocoa butter, or chocolate mass. The

cocoa flavor is modified and improved by the subse-

quent processing, mainly by alkalization of nibs, de-

gassing of cocoa mass, or conching of chocolate mass.

0056Conching of chocolate The conversion of the bitter

cocoa flavor to the finer chocolate flavor occurs in the

conche. There, a mixture of cocoa mass, sugar, cocoa

butter, and milk powder (for milk chocolate produc-

tion), fine ground in rolling mills, is stirred vigorously

and treated for about 8–24 h at 50–60



C. In addition

to the aroma improvement, this process gives the

desired consistency to the chocolate by covering all

the solid particles with fat. Aroma and consistency

are important for the flavor of the finished product.

There are two main functions of the conche: degas-

sing and mechanical–thermal treatment of the

chocolate mass. Low-boiling volatiles are removed,

together with steam. Acetic, propionic, isobutyric,

and isovaleric acids are reduced by about 30%.

About 20–40% of the unspecified aroma compounds,

tbl0005 Table 5 Typical aroma compounds in unroasted cocoas and

some of their precursors

Acids Carbonyls

Acetic Acetone

2-Methylpropanoic Val 2,3-Butandione

3-Methylbutanoic Leu Acetophenone

2-Methylbutanoic Ile Dihydrohydroxymaltol

Benzoic Phe

Phenylacetic Phe

Aldehydes Heterocyclic compounds

2-Methylpropanal Val Tetramethylpyrazine

3-Methylbutanal Leu 2-Acetylpyrrole

2-Methylbutanal Ile

Benzaldehyde Phe

Phenylacetaldehyde Phe

Alcohols Terpenes

2-Methylpropanol Val Linalool

3-Methylbutanol Leu Linalool oxides

2-Methylbutanol Ile

Benzyl alcohol Phe

2-Phenylethanol Phe

Acetates

2-Methylpropyl acetate Val

3-Methylbutyl acetate Leu

2-Methylbutyl acetate Ile

Benzyl acetate Phe

2-Phenethyl acetate Phe

Val, valine; Leu, leucine; Ile, isoleucine; Phe, phenylalanine.

COCOA/Chemistry of Processing 1445