J. Sci. Food Agric.

1986, 37, 297-309

Analysis of Cocoa Pulp and the Formulation of
a Standardised Artificial Cocoa Pulp Medium
Graham L. Pettipher
Cadbury Schweppes PLC, Group Research, The Lord Zuckerman Research Centre,
The University, Whiteknights, Reading RG6 2 LA
(Manuscript received 4 June 1985)
Results of analyses of cocoa pulp from the Ivory Coast, Nigeria and Malaysia are
reported. These include pH, viscosity and a, measurements and concentration of
soluble sugars, vitamins and anions. Pulp from Malaysian cocoa pods had a higher
water content, lower citrate, hemicellulose, lignin and pectin concentration and a
higher pH than Ivorian or Nigerian cocoa pods. The composition of a defined
cocoa pulp medium which has the same overall composition, pH, a, and viscosity
as cocoa pulp is described. The defined cocoa pulp medium supported the growth
of yeasts, lactic acid bacteria and acetic acid bacteria.
Keywords: Cocoa pulp; analysis; cocoa pulp medium.
1. Introduction
Cocoa fermentation is essential for the formation of chocolate flavour precursors. True chocolate
flavour is only developed during roasting. Cocoa fermentation is a natural complex process
involving a mixture of external microbiological processes occurring in the pulp surrounding the
bean and internal structural changes and enzymic reactions. A succession of microorganisms, in
particular yeasts, lactic acid bacteria and acetic acid bacteria, grow in the pulp producing ethanol,
lactic acid and acetic acid as metabolic products.'
The exothermic formation of acetic acid causes the temperature of the fermenting mass to rise,
often to >45"C. Bean death is caused by the ingress of acids and the increased temperature.
These factors prevent germination and cause structural changes which remove the compartmen-
talation of enzymes and substrates, thereby permitting the activity of enzymes such as
glycosidases and proteases.* During the subsequent aerobic conditions polyphenol oxidase
activity gives rise to the characteristic brown coloration of well-fermented cocoa.
I n view of the fact that both the initial colonisation and subsequent microbial activity essential
to the fermentation of cocoa is largely dependent upon the composition of the pulp surrounding
the bean, it is surprising that no detailed analysis of cocoa pulp has been reported. Published
results state that cocoa pulp contains 8&90% water, 1&13% simple sugars, ca 1% pectin,
>0.2% non-volatile acids and, more recently, up to 2% citric acid.s6 The concentration of
glucose, fructose and sucrose is known to vary with the age of the pod.' The amino acids,
aspartic acid, glutamic acid and asparagine, have been reported to account for the nitrogen
content of the pulp.'
The formulation of a defined cocoa pulp medium will facilitate both microbiological and
biochemical laboratory studies on cocoa bean fermentation. A detailed analysis of cocoa pulp has
been completed and the results used to formulate a defined cocoa pulp medium. The results of
the analyses and the formulation of the medium are given in this report.
291
298
G. L. Pettipher
2. Materials and methods
2.1. Samples of cocoa pulp
Amelonado cocoa pods (three to five) were obtained from the Ivory Coast and Nigeria, and
hybrid cocoa pods from Malaysia, usually within 5 days of harvesting. Disease-free pods were
opened and samples of cocoa pulp taken using the following methods:
1. Cocoa pulp was removed from the beans by scraping with a sharp scalpel.
2. Cocoa beans surrounded with pulp were deep frozen and then freeze-dried using an
Edwards DO1 freeze drier (Edwards, Crawley, UK) at a shelf temperature of 20C. After drying
the pulp was easily removed as a powder from the outside of the beans by scraping with a scalpel.
Freeze-dried samples were kept in a desiccator and both types of samples of cocoa pulp were
stored deep frozen until required for analysis.
2.2. Analyses
2.2.1. Measurement of p H
Samples of fresh cocoa pulp were suspended in distilled water as a 1 in 10 dilution and the pH
measured using an EIL 704S/46 pH meter and 1180 series pH electrode (Kent Industrial
Measurements, Chertsey, UK).
2.2.2. Measurement of water content
Weighed samples of fresh cocoa pulp were dried to constant weight at 80C on pre-weighed
planchets and the percentage water content was calculated.
2.2.3. Measurement of water activity (a,)
The a, of fresh Amelonado cocoa pulp and the defined cocoa pulp medium was measured by
hydrometry using a Humiditat TH2 (Novasina AG, Zurich) at 25C.
2.2.4. Measurement of viscosity
The viscosity of decimal dilutions (w/v) of fresh and freeze-dried Amelonado cocoa pulp and
defined cocoa pulp medium prepared with low or high viscosity carboxymethyl cellulose was
measured by means of a U-tube viscometer (Poulton, Selfe and Lee Ltd, Wickford, Essex-BS/
U size A) at 25C.
2.2.5. Mono- and disaccharide analyses
A weighed quantity of fresh pulp (ca 1 g) was ground in a pestle and mortar with a small quantity
of distilled water and then made up to 10ml total volume. One millilitre of the suspension was
added to 4ml acetonitrile. After shaking, the mixture was centrifuged at 15OOXg for 20 min and
then the supernatant was analysed using a Waters HPLC system (model 720 system controller,
model 730 data module, M6000A pump, U6K injector and an R401 differential refractometer;
Waters Associates, Cheshire, UK). Supernatants were filtered through a 0.45pm pore size
membrane filter before injecting 1Spl on to the column. Separation of the sugars was achieved
on a Waters carbohydrate analysis column using acetonitri1e:water (80:20, v/v) at a flow rate of
2mlmin-'. The concentration of each sugar in the samples was determined after calibration of
the instrument with standard solutions of known concentrations. Samples were analysed in
duplicate.
2.2.6. Hemicellulose, cellulose, lignin and pectin analyses
The hemicellulose, cellulose and lignin content of pulp was measured using the methods
described by McAllan and Smith.' Freeze-dried material was hydrolysed in O.SMH~SO~ for 4h at
100C which releases mannose, galactose, arabinose and xylose from hemicellulose. These sugars
were then quantified by high pressure anion-exchange chromatography of their borate-
complexes. The amount of these sugars was taken as a measure of the hemicellulose content in
the sample.
Analysis of cocoa pulp 299
Cellulose -was estimated by automated enzymic analysis of glucose after more stringent acid
hydrolysis of the residue from the first acid hydrolysis.
A crude measure of lignin content was obtained by weight loss following overnight incineration
at 550C of the residue from the cellulose hydrolysis.
The extraction of pectic substances from freeze-dried cocoa pulp, divided into two groups on
the basis of their solubility in water and sodium hydroxide, followed the procedure of Atkins and
Rouse. lo The estimation of pectic substances was determined by the colorimetric method of
Dische" as applied to citrus juices by Dietz and Rouse."
2.2.7. Organic acid and ethanol analyses
A 1 in 10 dilution of fresh cocoa pulp was prepared by grinding a weighed amount of material in
a pestle and mortar with the appropriate volume of distilled water. The suspension was
centrifuged for 20 min at 15OOxg and the supernatant filtered through a 0.45pm pore size
membrane filter. The samples were then analysed for organic acids by replicate injections on to
an h.p.1.c. column.
The h.p.1.c. system consisted of an Altex model llOA HPLC pump, an Altex sample valve
(5Opl) and a Waters differential refractometer. Organic acids were separated on an Aminex
HPX-87H column (Bio Rad Laboratories) fitted with an Aminex HPX-85H guard column using
0. 004~H~SO~ as the eluent at a flow rate of 0.6mlmin-'. The concentrations of individual
organic acids and ethanol were determined from linear calibration graphs obtained from
analysing levels of each (0.5-1.5 mgg-l) in the presence of the internal standard (propionic acid).
2.2.8. Measurement of total nitrogen
The total nitrogen content of freeeze-dried samples of cocoa pulp was determined by
conventional Kjeldahl digestion using a copper catalyst. The ammonia was distilled and collected
into a solution of boric acid which was then titrated with standard acid.
2.2.9. Free and total amino acid analyses
Free amino acids were determined in freeze-dried pulp by making a 100 mg ml-' suspension in
0.2 mM lithium citrate buffer (pH 2.0) and adding 1 ml norleucine (10 mM) as internal standard.
The suspension was mixed in a blender for 5 min. The blended mixture, plus the lithium citrate
rinses used to remove solid material adhering to the blades of the blender, was centrifuged for 30
min at 20OOxg and the supernatant containing the free amino acids was removed. The solid pellet
was resuspended twice more in lithium citrate buffer and the centrifuge procedure repeated until
4C50 ml of supernatant had been collected. The supernatant was then deproteinised with
5-sulphosalicyclic acid (20 mg ml-' supernatant) and left to stand overnight at 4C. The
supernatant was then centrifuged to remove any precipitated protein and the clear supernatant
adjusted to pH 2.0 using 6 N lithium hydroxide. Samples were filtered through 0.45pm pore size
membrane filters before being analysed by an automatic Biotronik LC 2000 amino acid analyser
(Biotronik, Frankfurt). The concentration of each amino acid in the sample was determined after
calibration of the instrument with a solution containing known concentrations of individual
amino acids.
Total amino acids in freeze-dried pulp were estimated by oxidising with performic acid before
acid hydrolysis to give stable analogues of methionine and cystine. l 3 This procedure gives good
recovery of all amino acids except tyrosine and tryptophan. Values obtained for tyrosine and
tryptophan are likely to be underestimates of the true concentration.
2.2.10. Measurement of fat
A weighed quantity of freeze-dried pulp was placed in a Soxhlet thimble. The thimble was then
placed in a Soxhlet apparatus, above a pre-weighed flask containing 40/60 petroleum ether (by vol)
on a water bath, with a water condenser in the top. The apparatus was left syphoning overnight and
then the petroleum ether was evaporated off. The flask was dried to constant weight in an oven and
the fat content of the freeze-dried pulp was calculated.
300 G. L. Pettipher
2.2.11. Trace metal analyses
Sample preparation and analyses were carried out using the methods described by Anon.14 A
weighed quantity of freeze-dried cocoa pulp (ca 1 g) was dry ashed. The ash was dissolved in HC1
and made up to 10ml with distilled water. Appropriate dilutions were made for each trace metal.
Sodium and potassium were analysed by flame photometry and other trace metals by atomic
absorption.
2.2.12. Vitamin analyses
The concentration of individual vitamins in freeze-dried cocoa pulp was estimated by microbiolo-
gical assay using known strains of vitamin-requiring microorganisms. Due to the limited amount
of sample available, only a composite of Ivorian and Nigerian cocoa pulp was analysed.
2.2.13. Ani on analysis
Fresh cocoa pulp was diluted 1 in 5 with distilled water and the anion content was estimated by
h.p.1.c. after filtration through a 0.45pm pore size membrane filter. Samples were injected on to
an ionosphere column (25 cmX4.6 mm; Chrompck, London) using sodium salicylate (25 mM, pH 5)
at 1 mlmin-' as eluent. Anions were detected by changes in refractive index.
2.3. Formulation of a defined cocoa pulp medium
The results of the analyses of I vorian, Nigerian and Malaysian cocoa pulp were used as a basis
for devising the formulation of a defined cocoa pulp medium. The formulation was selected to
give similar overall concentrations of individual components and to have a similar viscosity, a,
and pH as cocoa pulp.
2.3.1. Preparation of a cocoa pul p medi um
The polymeric materials, carboxymethyl cellulose, locust bean gum and pectin, were best
dispersed by adding to 500ml warm distilled water and blending in a Waring blender for 2 min.
To this suspension most of the remaining water and a11of the sugars, casein, casein hydrolysate,
glycerol monostearate, salts and mineral solution were added and then the medium was stirred
until the contents dissolved. The pH of the medium was adjusted to 3.6. The medium was then
made up to volume and autoclaved. To prevent caramelisation of the sugars an autoclave
temperature of 115C was used. Before use, the vitamin mixture and ascorbic acid solution were
added aseptically to the cooled medium.
2.4. Microbiological studies
The ability of the defined cocoa pulp medium to support the growth of microorganisms important in
the fermentation of cocoa was assessed by incubating with pure cultures of yeasts, lactic acid bacteria
and acetic acid bacteria and examining for an increase in the plate colony count after static
incubation for 2 days at 30C. The cultures used were Saccharomyces chevulieri C26 isolated from
fermenting cocoa, Lactobacillusplantarum C22, L. mali C34 (NCI B 10560) and Acetobacter aceti C3
(NCI B 8941).
3. Results
3.1. Analytical results
A summary of the results for the analysis of cocoa pulp from Ivorian, Nigerian and Malaysian
cocoa pods is given in Table 1. Detailed results for individual components are given in Tables
2-8. Due to the limited number of cocoa pods available some analyses were not made on pulps
from all three origins. Using averaged results for missing values, recoveries of 99.4, 100.2 and
100.2% were obtained.
3.1.1. p H
The pH values of I vorian, Nigerian and Malaysian cocoa pulp were 3.3, 3.6 and 3.9, respectively.
Analysis of coeoa pulp 30 1
3.1.2. Water content
The water content of the West African cocoa pulps were similar, ca83%, whilst that of the
Malaysian cocoa pulp was ca 86% (Table 1).
3.1.3. Water activity
The a , of Amelonado cocoa pulp and the defined cocoa pulp medium was 0.97 and 0.98,
respectively.
3. 1. 4. Viscosity
The viscosity of decimal dilutions of fresh and freeze-dried Amelonado cocoa pulp and defined
cocoa pulp medium made using low or high viscosity carboxymethyl cellulose is given in Table 10.
The reason why the viscosity of the fresh pulp was lower than that of the freeze-dried pulp
was probably due to the method of sample preparation. Both samples were centrifuged to
remove insoluble material but, in the case of fresh pulp, this was insufficient and filtration
through a 0.45pm pore size membrane filter was also used.
Table 1. Composition of Ivorian, Nigerian and Malaysian cocoa pulp
Ivorian Nigerian Malaysian
g1OOg-' fresh weight pulp
Water
Mono- and disaccharides
Plant cell wall polymers
Citrate
Protein, peptides, amino acids
Fat
Metals
Vitamins (composite sample)
Ethanol
82.60 82.50
11.15 13.05
2.81 ND
1.31 0.79
0.74 0.64
0.45 0.75
0.24 0.22
0.05
0 0.1
Recovery (%) 99.4 98.1
Recovery (70) with average - 100.2
results for missing values)
ND. not determined.
85.90
11.06
1.48
0.29
0.65
0.35
ND
ND
0.2
99.9
100.2
Table 2. Concentration of mono- and disaccharides in fresh Ivorian,
Nigerian and Malaysian cocoa pulp
Ivorian Nigerian Malaysian
g 100g-' fresh weight
Sucrose 4.35 1.92 1.35
Glucose 3.00 5.06 4.90
Fructose 3.80 6.07 5.35
Total 11.15 13.05 11.60
Analyses were made in duplicate, coefficient of variation ca 5%.
Table 3. Concentration of cellulose, hemicellulose, pectin and lignin
in freeze-dried Ivorian, Nigerian and Malaysian cocoa pulp
lvorian Nigerian Malaysian
g kg-' dry weight
Cellulose 51.8 ND 47.3
Hemicellulose 28.5 ND 15.8
Pectin 66.1 59.1 37.5
Lignin 15.0 ND 5.0
Total 161.4 105.6
302 G. L. Pettipher
The viscosity of the defined cocoa pulp medium can be varied over a wide range by using
different proportions of low and high viscosity carboxymethyl cellulose. It should be noted that
excessive blending of the polymeric constituents during the preparation of the media leads to a
reduction in the final viscosity.
3.1.5. Mono- and disaccharides
While the total amounts of mono- and disaccharides in pulps of different origins were similar,
ranging from 11 to 13% (Table l), the concentrations of the individual sugars, sucrose, glucose
and fructose, varied considerably, from 3 to 6% (Table 2) . The ratio of disaccharides to
monosaccharides was highest in the Ivorian pulp. As this ratio is known to decrease with
increasing age of the pod, it is likely that the pods from Nigeria and Malaysia were older than the
Ivorian pods.
Table 4. Concentration of total nitrogen, amino acids, protein,
peptides, and ammonia in Ivorian. Nigerian and Malaysian cocoa
Pulp"
lvorian Nigerian Malaysian
g1OOg-I fresh weight
Total nitrogen 0.11 0.11 ND
Free amino acids 0.15 0.11 0.21
Proteidpeptides 0.57 0.51 0.43
Ammonia 0.02 0.02 0.01
"Analyses made in duplicate on freeze-dried material, results
calculated as percentage fresh weight
Table 5. Concentration of free amino acids and free ammonia in freeze-
dried Ivorian, Nigerian and Malaysian cocoa pulp
Ivorian Nigerian Malaysian
Amino acid mgg-l dry weight
~_... . ~
Aspartic acid 1.06 0.73 1.56
Threonine 0.16 0.11 0.20
Serine 0.23 0.14 0.36
Asparagine 1.29 0.67 2.84
Glutamic acid 1.29 0.73 1.89
Glutamine 2.19 1.56 5.40
Proline 0.22 0.16 0.23
Glycine 0.03 0.02 0.07
Alanine 0.13 0.09 0.50
Valine 0.08 0.06 0.14
Cystine 0.01 ND ND
Methionine 0.01 0.04 0.01
Isoleucine 0.03 0.02 0.02
Leucinc 0.04 0.02 0.25
Tyrosine 0.12 0.07 0.02
Phenylalanine 0.06 0.05 0.05
Lysine 0.24 0.31 0.10
Histidine 0.14 0.11 0.06
Arginine 0.42 0.34 0.12
Hydroxyproline ND ND ND
a-Amino adipic acid ND ND ND
y-Amino butyric acid 0.44 0.63 0.62
Citrulline 0.09 0.03 0.01
Ornithine 0.17 0.07 0.10
Ammonium ions 0.12 0.17 0.09
Total 8.57 6.13 14.64
ND, not determined.
Analysis of cocoa pulp 303
3.1.6. Hemicellulose, cellulose, lignin and pectin
Only the Ivorian and Malaysian cocoa pulps were analysed for hemicellulose, cellulose, lignin
and pectin. Of these, the Ivorian pulp contained ca two times more hemicellulose, 1.8 times
more pectin and three times more lignin than the Malaysian pulp; the cellulose content of the
two pulps was similar (Table 3). The pectin content of the Nigerian pulp was higher than that of
the Malaysian pulp and similar to that of the Ivorian pulp. The ratios of water-soluble to
alkali-soluble pectin for the Ivorian, Nigerian and Malaysian pulps were 1.6, 1.4 and 1.1,
respectively.
The starch content of the Ivorian and Malaysian pulp was very low, as the starch plus glucose
fraction after the acid hydrolysis of hemicellulose could be accounted for by the free glucose plus
the glucose present as sucrose as determined by h.p.1.c.
3.1.7. Organic acids
Citrate was the major organic acid in the cocoa pulps from all three origins. Ivorian pulp
contained ca 4.5 times more citrate than Malaysian pulp and ca 1.7 times more than Nigerian
pulp (Table 1). The citrate concentration in the cocoa pulps was inversely and linearly related to
the pH. Other non-volatile organic acids, such as malic, tartaric and oxalic, occurred in
concentrations of (0.1%.
Ethanol was present in Malaysian pulp at a level of ~ ~ 0 . 2 % and in Nigerian pulp at caO.l%.
Ethanol was not detected in the Ivorian cocoa pulp.
3.1.8. Nitrogen
The total nitrogen content of Ivorian and Malaysian cocoa pulp was the same (Table 4). The
Table 6. Concentration of total amino acids and ammonia in freeze-dried
Ivorian, Nigerian and Malaysian cocoa pulp
Amino acid
Ivorian Nigerian Malaysian
mgg-' dry weight
Aspartic acid
Threonine
Serine
Asparagine
Glutamic acid
Glutamine
Proline
Glycine
Alanine
Valine
Cystine
Met hionine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Lysine
Histidine
Arginine
Hydroxyproline
a-Amino adipic acid
y-Amino butyric acid
Citrulline
Ornithine
Ammonium ions
Total
ND. not determined.
8.22
1.54
2.06
ND
8.65
ND
2.02
1.63
3.12
1.15
1.35
0.71
0.92
2.46
0.51
1.62
2.92
0.66
2.09
ND
ND
ND
ND
ND
1.41
43.04
3.52
2.13
1.97
ND
6.76
ND
2.02
1.89
1.61
1.21
1.51
0.48
0.90
2.31
1.08
1.67
2.57
0.90
1.62
0.78
ND
0.39
ND
0.09
0.96
36.37
8.21
1.51
1 .IS
ND
11.46
ND
1.50
2.39
2.55
1.31
1.60
0.96
0.96
2.57
1.04
1.60
2.70
0.82
1.53
ND
ND
0.89
ND
0.11
0.95
46.41
21
304 G. L. Pettipher
total concentration of amino acids, either free or incorporated into peptides and proteins, in
Ivorian, Nigerian and Malaysian cocoa pulp was 0.72, 0.62 and 0.64glOOg-' fresh weight,
respectively. More than 90% of the nitrogen content of the pulps could be accounted for by
amino acids, Using a factor of 6.25 to convert nitrogen into crude protein. the total nitrogen
measured is equivalent to 0.69g protein 1OOg-' fresh pulp. All three types of cocoa pulp
contained only a low concentration of ammonium ions. Samples were not analysed for nitrate
content.
Of the individual free amino acids determined, glutamate, asparagine, glutamic acid and
aspartic acid occurred in high concentrations in Ivorian, Nigerian and Malaysian pulps (Table 5) .
Of the individual total amino acids determined, glutamic acid and aspartic acid were present in
high concentrations (Table 6).
3. I . 9. Fat
The concentration of fat in Ivorian, Nigerian and Malaysian pulp was 2.6, 4.3 and 2.6glOOg-'
freeze-dried pulp, respectively. This is equivalent to less than 0.8 g 100 g-' fresh weight (Table 1).
The concentration of fat in Nigerian pulp was 2.1 times more than in Malaysian pulp and 1.7
times more than in Ivorian pulp.
3.1.10. Trace metals
The trace metal content of West African cocoa pulp was 0.22-0.24% (Table 1). Potassium
accounted for the majority of the trace metals detected in both Ivorian and Nigerian pulp,
forming 87 and 89% respectively (Table 7). Magnesium, calcium and iron were present in higher
Table 7. Concentration of metal ions in freeze-
dried Ivorian and Nigerian cocoa pulp
Metal
Ivorian Nigerian
mg kg-l dry weight
Potassium
Sodium
Copper
Calcium
Iron
Magnesium
Zinc
Manganese
Nickel
Total
12000
40
6
730
230
800
15
5
L
13828
11000
60
12
460
80
730
3.5
5
3
12385
Table 8. Concentration of vitamins in a
composite sample of freeze-dried Ivorian
and Nigerian cocoa pulp
Vitamin M 100g-I
Thiamine (B, )
Riboflavin (B,)
Pyridoxine (B,)
Cyanocobalamin (BIZ)
Niacin
Pantothenate
Folie acid
Biotin
Ascorbic acid (C)
Total
220
250
30
4.0X16
2.6~10'
2.3 x 10'
95
63
3.0X1@
3.1X1@
Analysis of cocoa pulp 305
concentrations than sodium, copper, zinc, magnesium or nickel. The iron content of the Ivorian
pulp was ca three times that of the Nigerian pulp.
3.1. I I. Vitamins
Ascorbic acid (vitamin C) accounted for nearly 97% of the vitamins included in the analysis of a
composite sample of freeze-dried lvorian and Nigerian cocoa pulp (Table 8). All the vitamins
Table 9. Composition of a defined cocoa pulp medium
Fructose 50 g
Glucose 40 g
Sucrose 25 g
Locust bean gum (Sigma GO783) 5g
Casein, vitamin free (Difco 0579-17-2)
5.5 g
Casein hydrolysate, vitamin free (Difco 0646-15-3) 1.5g
Glycerol monostearate (BDH 26105) 5g
Carboxymethyl cellulose (high viscosity; BDH 27929)
Carboxymethyl cellulose (low viscosity; BDH 27649)
Pectin (Sigma P9135) 10.9g
1.35g
7.65g
Salts
Potassium dihydrogen citrate
Magnesium citrate .I 4 H20
Citric acid .1 H2 0
Ferric citrate .5 H20
Calcium citrate .4 H2 0
Calcium sulphate
Mineral solution
Distilled water added to give a final volume of 980 ml pH adjusted to 3.6 with H2SOn
Added after autoclaving:
Vitamin mixture
Ascorbic acid solution
Mineral solution
Disodium hydrogen phosphate
Sodium chloride
Zinc sulphate 0.7H20
Copper sulphate 0.5 H20
Manganese sulphate 0.4H20
Nickel chloride 0.6 HzO
Distilled water added to give a final.-volume of 1 litre
Vitamin mixture
Thiamine (B,)
Riboflavin (B2)
Pyridoxine (B,)
Cyanocobalamin (Bi2)
Niacin
Pantothenic acid
Folk acid
Biotin
Distilled water added to give a final volume of 1 litre, filter sterilised
Ascorbi c acid solution
Ascorbic acid
11.8g
1.44g
1.9g
0.16g
0.8Bg
0.09g
lOml
10 ml
lOml
1.4g
1.2g
1.2g
0.55 g
0.4 g
0.2g
39 mg
44 mg
700 mg
5 mg
455 mg
403 mg
17 rng
11 mg
0.53 g
Distilled water added to give a final volume of 10ml. filter sterilised
306 G. L. Pettipher
analysed for were present. Vitamins B6, niacin and pantothenate were present in higher
concentrations than vitamins B1, B2, BI2, folic acid and biotin.
3.1.12. Anions
The h.p.1.c. system used enabled the quantitation of nitrate and sulphate; phosphate and chloride
co-eluted. The quantity of these anions in Ivorian and Nigerian pulp averaged 0.05%. Of this,
sulphate comprised ca 88% and chloride plus phosphate ca 12%.
3.2. Formulation of a defined cocoa pulp medium
The composition of the defined cocoa pulp medium given in Table 9 has the same overall
concentrations of sugars, plant ceil wall polymers, citrate, protein, amino acids, fat, trace metals
and vitamins as those found in cocoa pulp. The medium does not contain lignin as this material is
not readily available commercially. The viscosity of the medium can be varied between 1.07 and
2.37cSt by using either low or high carboxymethyl cellulose (Table 10). Different ratios of the
two polymers can be used to obtain intermediate values. The a, of the medium is 0.98, which is
similar to that measured for cocoa pulp, namely 0.97. The pH of the medium is ca3.8 but it can
be adjusted to the required value by the addition of acid. Like cocoa pulp, the medium has a
higher concentration of sulphate than chloride or phosphate.
3.3. Microbiological studies
The defined cocoa pulp medium was able to support the growth of yeast, lactic acid bacteria and
Table 10. The viscosity of decimal dilutions of fresh and freeze-dried
Amelonado cocoa pulp and defined cocoa pulp medium prepared with high
or low viscosity carboxymethyl cellulose (CMC)
Viscosity (cst)
Fresh pulp 1.18
Freeze-dried pulp 1.29
Cocoa pulp medium with low viscosity CMC 1.07
Cocoa DUID medium with high viscositv CMC 2.37
Table 11. Growth of yeast, lactic acid bacteria and acetic acid bacteria in a defined
cocoa pulp medium as assessed by the plate colony count
Plate colony countlmY
After 2 days
pH of incubation
Organism medium Initial at 30C
Saccharomyces chevalieri C26 3.6 LOX 103 1. 5~10~
Lactobacillus plantarum C22 3.6 8.2Xl P 7. 8~10~
4.0 73x10 4.2X10
5.0 7.5~10 7. 7~10~
Lactobacillus mali C34 3.6 1. 4~10~ 1. 4~10~
4.0 1.8X105 1.1 x lo4
5.0 1. 4~10~ 2.5~10
Acetobacter aceti C3 3.6 2. 0~10~ 4.4x 106
Plate colony count methods:
Yeast-pour plates using Malt Extract Agar (Oxoid)
Lactobacilli-pour plates using MRS Agar (Oxoid), incubated anaerobically
Acetobacter-spread plates using WL Agar (Oxoid).
All plates were incubated for 2 days at 30C before counting colonies.
Analysis of coeoa pulp 307
acetic acid bacteria (Table 11). The increase in the plate colony count during the 2 days
incubation involved in the test was >2 log cycles for the yeast, lactic acid bacteria and acetic acid
bacteria.
The yeast, S. chevalieri, grew well and gassing of the medium was observed. The two species of
lactobacilli showed different growth responses. Whereas L. plantarum could grow in the
medium at pH4.0, L. mali could only grow if the pH was increased to 5.0 or higher. Neither
organism grew in cocoa pulp medium at pH3.6.
Preliminary results have shown that the cocoa pulp medium can be used as an isolation
medium if it is first solidified with 1.5% agar. The solid medium supports the growth of yeast and
acetic acid bacteria.
4. Discussion
4.1. Composition of Ivorian, Nigerian and Malaysian cocoa pulp
The analyses of the cocoa pulps from three origins gave similar results for water content, soluble
sugars, pectin and citrate concentrations to those reported previously.s6 The analyses provided
useful data on the cellulose, hemicellulose, lignin, amino acid, trace metal, vitamin and anion
concentrations of cocoa pulp, information which is lacking in the literature. In general, higher
recoveries of monosaccharides, disaccharides and organic acids were obtained from fresh as
opposed to freeze-dried samples of cocoa pulp. This may have been due to difficulties in
rehydrating the freeze-dried material. It is therefore recommended that when required for
analysis fresh cocoa pulp is preserved by freezing rather than by freeze-drying.
Major differences between the Ivorian, Nigerian and Malaysian cocoa pulp samples were the
pH and citrate levels. The Ivorian pulp which was probably obtained from the youngest pods, as
indicated by the high disaccharide:monosaccharide ratio, had the lowest pH and highest citrate
content. The Malaysian pulp had the lowest citrate content and the highest pH. Nigerian pulp
was intermediate in both pH and citrate level. Both the presence of citrate as the major organic
acid in unfermented cocoa pulp and the inverse and linear relationship between citrate content
and pH, suggests that the concentration of citrate is mainly responsible for the pH of the
unfermented pulp. The presence of ethanol in the Malaysian and Nigerian pulps suggests that
fermentation may have commenced, either in the pod or during sample preparation. It would
therefore be advisable to obtain further samples of Malaysian cocoa pods to establish whether or
not the differences between Malaysian and West African pulp pH and citrate levels are
significant.
The cocoa pods obtained from the Ivory Coast, Nigeria and Malaysia were received within 3-5
days of harvesting, although the exact time ,interval was unknown. Dehydration of the pulp
occurs progressively after harvesting and the extent of this will inevitably affect the water content
and hence the absolute concentrations of all the other pulp components. When the pods from the
different origins were received their pulps appeared visually similar. The sample of Malaysian
cocoa pulp contained more water and less of certain plant cell wall polymers than the samples of
West African cocoa pulps. The water content of the Malaysian pulp was >3% higher and,
although the cellulose levels were similar, Malaysian pulp contained only 53% of the
hemicellulose/pectin/lignin content of the Ivorian pulp. In addition to the increased pulp to bean
ratio of Malaysian cocoa beans,15 a higher water content and lower concentration of plant
cell-wall polymers could affect the cocoa fermentation by excluding air, thereby creating more
anaerobic conditions than those attained with Amelonado cocoa beans. This may partially
account for the higher levels of lactate observed in Malaysian beans compared with West African
beans. Lactic acid bacteria grow better anaerobically or in the presence of increased CO2
concentrations. l6
Although the levels of total amino acids in all three types of pulp were similar, the free amino
acid:peptide plus protein amino acid ratio in Malaysian cocoa pulp was approximately twice that
of the West African pulps. This may have been partially due to age differences between the pods
308 G. L. Pettipher
of different origins. Aspartic acid, glutamic acid and asparagine were present in cocoa pulp at
high levels compared with other amino acids but they do not, as Forsyth8 suggests, account for
the total nitrogen content of the pulp.
The concentration of trace metals in Ivorian and Nigerian cocoa pulp was similar and
approximately 50% of that reported by Ni ~hol l s.~ Potassium accounted for the majority of the
metal ions in both pulps. Other than citrate, sulphate comprised the major anion, with lower
levels of chloride andlor phosphate.
The composite sample of Ivorian and Nigerian cocoa pulp contained all of the vitamins
included in the analysis. These vitamins were at levels suitable for microbial growth, with the
possible exception of strains requiring high levels of vitamins. Vitamin C accounted for ca 97% of
the total vitamin content of the pulp. The quantification of the vitamins may be an underestimate
as the effect of freeze drying on the stability of vitamins in cocoa pulp is unknown.
4.2. Defined cocoa pulp medium
The defined cocoa pulp medium has the same overall composition, pH, a, and viscosity as cocoa
pulp. The limited microbiological studies undertaken indicate that the defined cocoa pulp
medium is capable of supporting the growth of the micro-organisms important in cocoa
fermentations, namely yeasts, lactic acid bacteria and acetic acid bacteria. The composition of
the medium may need minor modifications to obtain good growth of some species. e.g. an
increased pH for certain lactobacilli. Improved growth of some species especially lactobacilli may be
expected if anaerobic or enriched C02 atmospheres are used.
The cocoa pulp medium contains some insoluble components, such as the locust bean gum and
glycerol monostearate. If the medium is not shaken continuously these ingredients form a bottom
and surface layer respectively. It is likely that these components could be omitted from the
medium to improve handling without having a detrimental effect on the growth of micro-
organisms. For detailed microbiological studies on nitrogen metabolism, it would be advisable to
replace the casein hydrolysate content of the medium with a defined mixture of amino acids
based on the analytical results obtained.
The formulation of the cocoa pulp medium given in Table 9 is based on the analysis of
unfermented cocoa pulp. It can be considered as the starting point for model systems for studying
the microbiological and biochemical aspects of cocoa fermentation. Microbiological studies may
for example, include the effect of temperature and oxygen on the growth of microorganisms,
competition experiments, succession of microorganisms, and the utilisation of fermentation
products. By varying the composition of the medium to mimic the pulp at various stages of cocoa
fermentation, e.g. reducing the concentration of sugars and citrate and adding lactic acid and
ethanol, the different stages of cocoa fermentation can be modelled. Biochemical studies may
entail the incubation of fresh cocoa beans in the cocoa pulp medium which, by means of a
fermenter type system, is progressively altered in composition to mimic the pulp at different
stages of fermentation. During this incubation the diffusion of the fermentation products into the
bean, changes in enzyme levels and activities and the development of chocolate flavour
precursors could be monitored. The development of the defined cocoa pulp medium described in
this report should facilitate both microbiological and biochemical laboratory studies of cocoa
fermentation.
Acknowledgements
The author wishes to acknowledge the help of the following: M. D. Conisbee for skilled technical
assistance; Cadbury Schweppes: Cocoa Buying Department, Bournville, for arranging the supply
of cocoa pods; Technical Services, Dollis Hill, for trace metal analyses; Carbohydrates
Department, Group Research, for mono- and disaccharide analyses; Biochemistry Department,
Group Research, for pectin analyses; Flavour Chemistry Department, Group Research, for
organic acid and anion analyses; Lipids Department, Group Research, for fat analyses. National
Analysis of cocoa pulp 309
Institute for Research in Dairying: Basic Ruminant Nutrition Department for carbohydrate and
amino acid analyses; Analytical Services for total nitrogen analyses. Harry Pritchard Laborator-
ies, Birkenhead, for vitamin analyses.
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