Professional Documents
Culture Documents
by
Dept. of Biochemistry,
Faculty of Biological and Chemical Sciences,
University of Surrey,
Guildford, Surrey. September, 1982.
11
ABSTRACT
samples of roasted coffee. It was found that these Analytical Ratios lacked
presented which suggests that immature beans may be more likely to discolour
in this way than mature ones. The second type has a tendency to retain their
carotenoids, some carotenoids only and others may have lost both by bleaching
reactions.
ACKNOWLEDGEMENTS
the Taste Panel facilities and Dr. M. Crowther for his valued statistical
advice, my fellow research workers and the technical staff of the Department
of Biochemistry especially Mrs. B. Smith for their help and encouragement.
acid (DCQA), Dr. A. Lea of Long Ashton Research Station, Long Ashton,
Bristol, England, for the supply of 3-, 4-, and 5-p-coumarylquinic acid,
Dr. S. Martino of the Department of Plant Biochemistry, University of
Buenos Aires, Buenos Aires, Argentina for the supply of 4,5-DCQA. Also to
Fed. Nat. Cafeteros in Colombia, IFCC in the Ivory Coast, KIRDI in Kenya,
The Royal Botanic Gardens at Kew, for the supply of coffee samples.
support, to Dr. E. Illy and Illycaffe for practical advice and part financial
support.
iv
CONTENT
Title Page i
Abstract ii
Acknowledgements iii
Content iv
Abbreviations vii
CHAPTER 1 INTRODUCTION AND LITERATURE SURVEY
I Introduction 2
II The Composition of Green and Roasted
Coffee Beans 6
a. Moisture content 8
(ii) Polysaccharides 9
e. Nitrogen content 16
(ii) Proteins 16
g. Non-phenolic acids 26
CHAPTER 2 METHODOLOGY
I Introduction 48
II Colorimetric Methods 48
APPENDIX A
C
D
E
F
G
H
vii
ABBREVIATIONS
CA - Caffeic acid
CFQA - Caffeoylferuloulquinic acid
CGA - Chlorogenic acid
cm - Centimetre
°C
- Degree centigrade
CoQA - Coumarylquinic acid
5-CQA - Caffeoylquinic acid
DCoQA Dicoumarylquinic acid
-
DCQA Dicaffeoylquinic acid
-
dmb Dry matter basis
--
Est. - Estimated
FA - Ferulic acid
FQA - Feruloylquinic acid
FDCQA - Feruloyldicaffeoylquinic acid
GC - Gas chromatography
g - Gram
HPLC - High Pressure Liquid Chromatography
HPLC CGA HWLC CQA + HPLC FQA + 1.37 HPLC DCQA
I. D. - Internal diameter
IR - Infra red
L - Length
M. Wt. - Molecular weight
M - Mole
mg - Milligram
mm - Millimole
mm - Millmetre
mL - Millilitre
MV - Molybdate value
Ail, - Microlitre
gum - micron
viii
NCS - N-chlorosuccinimide
NMR - Nuclear magnetic resonance
nm - nanometre
O. D. - Optical density
PC - Paper chromatography
PV - Periodate value
PPO - Polyphenol oxidase
PVP - Poly-N-vinylpyrrolidone
QA - Quinic acid
r- Correlation coefficient
r. p. m. - Revolution per minute
SD - Standard deviation
TBA - Thiobarbituric acid
TBA QA - Thiobarbituric quinic acid
TBA BD QA - Thiobarbituric acid bound quinic acid
TMV - Tobacco mosaic virus
TEA - Triethylamine
- Sigma
Beta
a- Delta
7- Gamma
(max) - Maximum wavelength
var. - Variety
* All data reported in this study are calculated on dry matter basis.
-1 -
CHAPTER ONE
From here it found its way into Western Europe about 1630 A. D.
(Hartman et al., 1981).
Coffee plants were brought to Brazil in 1727 A. D., and after 40 years,
it became, and still is, the world's leading coffee grower and exporter, now
producing one-third of the world's coffee supply - about 1.5 million tons
annually.
Coffee plants are evergreen shrubs within the family Rubiaceae and
in the mountain forests of Ethiopia. There are two varieties, Coffea Arabica
var. Arabica and C. Arabica var. Bourbon, the latter variety being considered
the better. These are cultivated in elevated sites in tropical and subtropical
flavour but the plant is vigorous, with disease resistance (Williams, 197 5).
Assi (1977) there are at least 32 species in Africa. Among them are
Coffee trees are planted about 2.4m (8 ft ) apart and are kept low -
about 1.8m (6 ft ) by pruning to facilitate harvesting. They start bearing
full crops on the lateral branches at about 5 years and reach full production
which is a drupe, remains at its prime for about one week. For this
and Madagascar together produce about 0.8 million tons annually (Ass i, 1977).
In these countries the second species (C. Canephora) is mainly grown. This
yields a cheaper, less flavourful coffee, but it is now in great demand for use
in blending with C. Arabica coffees and in the manufacture of instant coffees.
The coffee fruit contains two seeds, coffee beans (Fig. 2) which are
cherry. After harvesting, the seeds are classified and then separated from
the pulp either by wet or dry processing (Clarke, 1976; Fig. 1).
Classification
Drying Pulping
Mucilage removal
Dehusking Hulling
sold whole to consumers. Ground coffee is used for making instant coffees
the caffeine is selectively extracted from green coffee beans by the use of
water or other solvents.
-6-
Contents (%admb)
Disc
Mesocarp (pulp)
Exocarp (skin-red)
Endocarp (patchment)
Mucilage
Spermoderm (Testa)
-' silverskin' when dry
- 'chaff" when roasted
The quality of coffee beverage depends on the selection of the raw beans
a) Moisture Content
The moisture content of green coffee beans would affect the storage
stability and thus the flavour qualities of the roasted beans (Rao et at., 1969).
A standardised method of moisture determination and the expression of the
analytical data on dry matter basis are essential for accurate comparisons
of samples (Smith, 1965).
The moisture content of green coffee was defined as the loss in mass
undergone by coffee when it is brought to true equilibrium with an atmosphere
having zero water vapour pressure, under conditions such that interfering
reactions are avoided (International Standards Organisation, 1978). However,
most research workers prefer to define it as the weight loss after heating the
Theaker, 1977).
Clifford (197 5) reviewing the data available said that green coffee beans
contain 5 to 8570
sucrose. There was more sucrose in Robusta coffees than
in Arabicas. Small quantities of free glucose (0.5 to 1.0%) are also
present. He indicated that raffinose (1-alpha-6-galactosylsucrose) and
stachyose (1-alpha -6-galactosylraffinose) are also present, these being
higher in Robustas than in A rabicas
.
could not be explained by differences in the sugar content in the green beans.
ii. Polysaccharides
rntive polysaccharides.
The acid hydrolysis of green coffee beans from which the mono- and
oli gosaccharides have been removed yields the constituent sugars of the
polysaccharide, i. e. galactose, glucose, mannose, arabinose, xylose,
rhamnose and at least one uronic acid.
- 10 -
It was not until 1977 that the presence of starch was confirmed
(Dentan, 1977). Previously a galactomannan was considered the major
reserve polysaccharide (Clifford, 1975).
Pectins have been extracted using hot water, dilute acid (primary cell
wall pectins) and ammonium oxalate (middle lamella pectins). Depending
and arabinose were always present - in some cases glucose and mannose
were also present although these are not considered typical components of
pectins. Amorim et at. (1974) reported that the content of oxalate-soluble
pectins bears no relationship to coffee bean quality.
The most recent investigations by Thaler (197 5) and Ara and Thaler
(1976) made extensive use of chlorine dioxide to solubilise lignin. Unfortunately,
this also causes considerable changes in the polysaccharides, and the results
and Robustas.
methods for measuring lignin are designed for the analysis of woody tissues
that have a very low protein content. Coffee has a high protein content and
interference occurs (Clifford, 1972).
Thaler and Arneth (1967) indicated that during roasting cellulose was
very stable, the mannan slightly affected, the galactan partially destroyed,
and the araban was greatly reduced in proportion to the severity of roast.
Ara and Thaler (1976) reported that the hot water-soluble fraction
molecular mass fraction of roasted coffee beans from which sugars and/or
Arabica and 10.0 to 15.0% of similarly roasted Robusta beans, and galactose,
at the effect of bean particle size on the yield and composition of the crude
lipid. They ultimately recommended a particle size of less than 0.5mm
and reported that larger particle sizes led to preferential extraction of
certain components of the oil and thus produced misleading data.
oil extracted from green coffee contains two diterpenoid alcohols, cafestol
and kahweol (Fig. 3). They are present both in the free state and as
with high beverage quality. High kahweol and medium oil content was
- 13 -
:u OH
OH
(i)
(
20H
H
(ii)
Fig. 3
(ii) Cafestol.
(Gibson, 1971)
- 14 -
associated with medium quality and low kahweol and low oil content was
associated with low quality.
The waxes have been studied in some detail, because they are
derivatives of serotonin and have been blamed for digestive disorders
(Van der Stegen, 1979). The waxes are N-beta-alkanoyl-5-hydroxy-
more elements. They noted that Arabicas were richer in bromide and
K 1350-1712 Cr 74-1327
Mg 142-176 V 70-110
Ca 76-120 Ba 100-615
Na 2.3-17 Ni 11-388
Fe 2.1-10.5 Co 10-93
Mn 1.1-9.8 Pb 1827
Rb 0.6-4.2. Mo 11-27
Zn 0.5-3.2 Ti 4-20
Cu 0.5-2.3 Cd 3
Sr 0.4-1.3
- 16 -
acid complex has been reported to be responsible for the colour of good
quality green coffee beans (Northmore, 1965 and 1967). The magnesium
e) Nitrogen content
roasted coffee and coffee brew in small quantities (Pereira and Pereira, 1971).
(ii) Proteins
pH 4.6 to 4.7 indicating that there was a low content of basic amino acids.
- 17 -
Alanine 24 40-80
Arginine 4 0
A sparagine 30 0
Glycine 2 0
Histidine 4 0
Is oleu cine 3 )
3-12
Leucine 3
Lysine 4 4 -1 O*
Methionine 8-28
Phenylalanine 8 2 -10*
Pipecolic acid 3 0
Proline 14 0
Serine 12 18-40
Threonine 0 3-8*
Tyrosine 4 0
Valine 2 14 -22*
component of green coffee of Brazilian origin (Rio and Soft) with different
protein content of Rio or Soft (good quality) coffees. They observed that
and a few in the range of 4.4 to 4.7 using non-urea gel contrary to previous
reports. They were able to show that green coffee contains protein-
coffee observed that when proteins are degraded during roasting, a number
and colour. The degradation of proteins causes loss of protein and the
more severe the roast the lower the protein content. It was reported that
when Santos coffee was roasted to six different degrees of roast, that is,
10,12,14,16,18 and 20 based on percentage loss in weight, there was a
loss in protein of 6% at the lowest weight loss and 14% at the highest (Fobe
et al., 1967).
When extracts of protein from green and roasted coffee beans were
hydrolysed they yielded 18 amino acids (Underwood and Deatherage, 1952;
Clifford (197 5) commented that those amino acids that are destroyed
during roasting do not have simple aliphatic side chains, whereas those that
increase (with the exception of glutamic acid) have an aliphatic side chain.
- 19 -
He suggested that those amino acids that appeared to increase were least
reactive and thus least destroyed. The increase was not necessarily an
indication of synthesis, more likely interference from other roasting
products or a failure to correct the results for dry matter loss. There
is little doubt that protein-bound amino acids with a non-aliphatic side
chain do react and are partially destroyed during roasting.
1980,
with coffee species: wild Coffea from Madagascar does not contain caffeine;
instead cafamarine was isolated (D'ornano et al., 1965). Arabica contains
severe roasts to pyridine (Viani and Horman, 197 5). The decomposition is
responsible for the increase in niacin content of the brew. Other degradation
R3
0
Rl,,, N
o' N N
R2
R1 R2 R3
COON
i(.-H3
Fig. 5. Trigonelline
- 21 -
part in the aroma of coffee. Ammonia, betaine and choline are quite stable
The term chlorogenic acid (CGA) was first introduced in 1846 to describe
a major component of green coffee beans (Payen, 1846). This was later
system (NPAC, 1974), this has become 5-CQA. The literature on CGA
Clifford (1975) and Table 4 has been modified to comply with the latest IUPAC
amended.
It has been confirmed that coffee beans generally contain quinic acid
esterified with p-coumaric, caffeic and ferulic acid (see Fig. 6). Esterifi-
cation occurs via one or more of three vicinal hydroxyl groups and thus gives
rise to at least 13 individual compounds.
sub groups:
- 22 -
Rl
Ho 0
RZ OH
If Rl = R2 = H, p-coumaric acid
4
H0
1 6 IUPAC System
......
6 Old System
GOON
OH
1. caffeoylquinic acids (CQA) which are esters of caffeic acid with quinic
acid;
4. feruloylquinic acids (FQA) which are esters of ferulic acid with quinic acid
flavour, odour (Tressl, 1977) and beverage quality (Amorim et al., 1974).
These hypotheses are discussed further on page. 37. -
usually the major component. Few tissues are as rich as coffee seeds,
where the CQA level may exceed 107 dry matter basis (dmb). In some fruits,
or both) may occur when extracting plant tissues (Nichifores co, 197 0). It has
been said that 5-isomers are normally the 'parent'; it could be so, since to
date no enzyme has been isolated that will synthesise the 3- or 4-isomers.
However, neither has the enzyme responsible for synthesising DCQA been
isolated, but DCQA undoubtedly exist. Indeed the only CGA isomer isolated
et at,, 1979), and since this was under conditions that some workers say would
- 25 -
extraction.
and Theaker, 1977). The caffeoylquinic acids form the major fraction
(Arabicas 5.5 to 7%, Robustas 8%), followed by DCQA (Arabicas 0.6%,
Robustas 1.8%), and feruloylquinic acids (Arabicas 0.3%, Robustas 0.6 to
1.2%).
performance liquid chromatography (HPLC) by Van der Stegen and Van Duijn
(1980). They reported that the total amount of mono-CQA was slightly
reduced without an increase of the fret. caffeic acid and that within the group
of mono-CQA the 5-isomer was clearly reduced and the 3- and 4-isomers were
These observations led these authors to believe that probably under high
fraction of heavily roasted coffee beans (Clifford, 1972; Rees and Theaker, 1977;
Published data suggest that some CGA degradation products are incorporated
into humic acids (see p 11 ). However, some degradation products are not bound
and trihydroxy phenols were Robusta 35. lppm, Arabica 34.6ppm and Arabusta
- 26 -
workers e. g. Rees and Theaker have commented that these three species have
and some of the dihydroxy phenols (e. g. 1,4 -dihydroxy benzene) may be
produced from the quinic acid residue rather than caffeic acid residue, even
the quinic acid residue of FQA.
The major volatile phenols are guaiacol, 4-methoyl guaiacol and 4-vinyl
guaiacol and that these were present above their threshold values. Using
model systems, Clifford (1972) indicated that the guaiacols were almost
certainly derived from the degradation of the ferulic acid residue of the CQA.
volatile phenols (56. Oppm) than Arabusta (38. Oppm) and Arabica (31.1ppm),
FQA precursors.
g) Non-phenolic acids
The non-phenolic acids have received less attention than CGA. Clifford
(1975) reviewed the information available and since there have been no further
malic, oxalic, pyruvic and tartaric acids are between 0.2 and 0.5%
citric,
(dmb) and total about 1.5% (dmb).
Formic and acetic acids increase during roasting, whereas citric and
The volatiles of green coffee have received some attention from Merritt
et El . (1969) and Vitzhum et al. (197 5) . Over 100 volatiles have been
identified and Vitzhum considers methoxy pyrazines primarily to be responsible
for green coffee aroma. Although some green coffee volatiles may contribute
In 1968 Walter and Weidemann reported that 363 volatiles had been
identified in roasted coffees. In his review, Clifford (1975) indicated that the
total had risen above 400 and in 1981 the total exceeds 515 (Smith, 1980; Tressl
et al., 1981). However, not all of these contribute equally to coffee aroma
(Clifford, 1975). The threshold value (the concentration at which it is perceived)
and the odour value (the ratio between its concentration in the food and its threshold
value) are not always available for coffee volatiles. Thus, as Clifford points out,
it is difficult to give the potency of coffee aroma constituents quantitatively.
but by 1981 Tressl and Silwar reported the total exceeded 100. These recent
aroma and coffee staling. Other groups of coffee volatiles to have received
attention are lactones (see Maga, 1976), simple phenols and phenolic
compounds (Tressl, 1977, see Maga, 1978), pyridine (see Maga, 1981a) and
coffee.
Formation of volatiles
coffee variety used for preparation of the beverage and is also determined
essentially by time and temperature of roasting process (Baltes, 1975).
and products as a whole are known, but rarely have the precise pathways been
determined. However, much useful data have been obtained by the use of
roasting-stimulating model systems and one can postulate three major types
of reaction:
- 29 -
that volatile phenols arise from the degradation of CGA. The possible
ii. The interaction of green bean components to yield larger and smaller
molecular mass products, only some of which are volatile, e. g. sugars and
amino acids in the classic Maillard Reaction and Strecker Degradation, both
of which have been recently reviewed by Nursten (1981). The volatile products
include carbonyls and a host of simple heterocyclics such as pyrazines and
furans. Variations in sugar content between Arabicas and Robustas (see
Table 1) could well account for the lower furan content of roasted Robustas
iii.. Reactions involving the degradation products arising from the reactions
mentioned above. It would seem that such reactions are important routes for
the formation of N- and S-containing heterocyclics, e. g. see Schutte (1974),
Tressl et al (1981) and Nursten (1981), Maga (1981b) such as pyrroles, furfuryl
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(i) Pulping removes the outer layer of the cherry from the bean. This
takes place in the presence of water, and involves a mechanical tearing and
squeezing operation. The machines leave a mucilagenous layer which is
,
removed traditionally by fermentation.
are, however, used (Gibson, 1971) to hasten the process and ensure that the
(iv) Hulling : The dry parchment coffee is hulled to remove the dried
parchment layer and also the testa or silverskin layer.
The dry process : The cherry is dried from about 709voto 10% moisture in
Rolz et al. (1969) reported on the use of fluidized beds in the drying of
coffee cherries. They found that the best quality coffee was obtained when
drying was done in two stages, an initial period at a low temperature (20°C)
After drying, the cherry is dehusked to separate the seeds from the
outer layers. With the dry process, particularly applied to Robusta, the
silverskin is difficult to remove and the bean may have a distinct and less
moisture may be used to remove it, hence the expression, 'washed and
cleaned' is used to describe such beans. Green coffee beans from either of
The coffee beans are passed along a rotating horizontal sieve, with
holes or bars varying to permit the recognised sizes of beans to fall through
as they proceed from one end to the other. Defective beans are also removed.
to achieve high quality coffee beans, and is carried out by either mechanical
or optical means.
In the mechanical method, defective beans are hand-picked and fed into
air. The dense beans fall through the air current whilst those that are less
dense are carried upwards to be released when the air speed is reduced.
Alternatively, gravity classifiers may be used, where the beans are passed
over a vibrating table which has a porous woven wire cover. A current of
two separate wavelengths. This is necessary when there are subtle colour
differences in the case of sorting green Arabica coffee (Maughan et al., 1980).
as
of the coffee beans is raised by hot gases, it first dries, then roasts. Beans
At first, free and bound water are driven off. The green coffee bean
colour slowly changes to buff, then light brown as drying continues. As the
bean temperature approaches 200°C, pyrolysis, the second step occurs. The
bean expands due to internal pressure and the chemical changes thus occur
the beans are removed from the heated gases and promptly cooled by ambient
air or a water spray. Most of the water sprayed evaporates off, cooling the
beans, with hardly any water being absorbed by the beans. Cooling of the
In general, light roasting leaves more acidity in the bean and the
roasting weight loss may be only 14%. 'Dark roasting' leaves little acidity
or aroma and has more extensive cell destruction making the extraction of
percentage loss in weight from dry green bean to dry roasted bean.
it gives to the consumer, through its flavour, aroma and desirable physiological
and psychological effects. Good quality is therefore the 'key' to this intangible
experience of pleasure that a cup of coffee arouses (Stirling and Jackson, 1979;
Illy, 1980, Personal Communication). The ultimate beverage quality is
consumer, for assessment of green bean quality varies from one producing
country to another.
In Kenya coffee quality may be associated with the bean size (Munene, 1973).
The following descriptions of coffee standards are used by the Liquoring Department
- 35 -
It has been reported that quality in Kenya coffee is associated also with
the colour of the green bean. The colours normally found in the green beans
are blue, green, yellow and brown (Kulaba, 1978). The best quality beans
are predominantly bluish in colour, whilst the yellow or brown ones have
poor liquoring characteristics. Munene observed that should there be
difficulty in classifying an out turn, the taste assessment by professional
liquorers is considered as important and the coffee classified accordingly.
quality), hard, rioy and rio (poor quality). Rio coffee is less expensive.
However, some consumers in Brazil as well as Latin America, United States
of America (U. S. A. ) and Europe prefer this kind of coffee because of its
strong medicinal or phenolic flavour (Amorim et al., 1974). This
commercial coffee beans of the coffee plant. Bags must be labelled with the
country of origin, grade or type and the species (e. g. Arabica or Robusta).
The composition of the beans must also be given and particularly the moisture
content (12970
maximum). Sometimes the caffeine content is also given
the amount of defective beans and foreign matter present in a sample. Usually
coffees from Kenya and Colombia have a very small amount of defective beans
and French have a similar system with grades known as Extra Prima (15),
Prima (30), Superior (60), Courante (120), and Limite (180). Furthermore,
Clark (1979) indicated that bean size, colour and residue from pesticide also
contribute to the grading of coffee beans.
Ehlers (1980) indicated that when enzymes were used to depulp coffee
there was an improvement in the quality. She produced heavier coffee beans
which were clean, and without the risk of 'tainting' and quality loss through
microbial spoilage.
oxidase activity of green coffee and the quality of the beverage. Sanint and
Valencia (1972) confirmed this observation and explained that the higher the
green bean polyphenol oxidase activity, the higher the beverage quality.
Furthermore it has been suggested that acids such as chlorogenic acids and
caffeic acid act as antioxidants for aldehydes.
The higher the aldehyde content of the green bean, the higher the
beverage quality (Forsyth, 1964). This seems to suggest that when quinones
are formed by enzyme activity in the bean, this renders the aldehydes
unprotected and they are lost causing a reduction in bean quality. The quinones
inhibit the enzyme and cause the low activity associated with low quality beans.
(Amorim et al., 1977)
this enzyme on chlorogenic and caffeic acids react with amino acids (except
lysine and cysteine) primarily through their alpha-amino group to give red or
brown products (Pierpoint, 1969; Synge, 1978).
- 38 -
It has been suspected that high storage temperatures and high bean
moisture contents have been the major factors influencing quality loss in
stored coffee. Stirling (1980) was able to show that for coffee to be preserved,
that membranes must be the first place in the green coffee beans to undergo
chemical and structural changes leading to quality deterioration.
with low quality in green beans. Marigo and Boudet (1979) confirmed that the
acid (CQA). Also, Legrand et al. (1978) reported that tobacco infected with
'Tobacco Mosaic Virus' showed an increase of activity in the phenol methylating
enzymes (i. e. increased potential for the conversion of CQA to FQA) probably as
means of isolating the virus and preventing its spreading through the plant. It
would also increase the potential for the formation of volatile guaicols during
roasting.
Fujimaki et al. (1974) indicated that lightly roasted coffee should not be
Northmore (1965) related the colour of the green bean to the quality of
Amorim et al. (1977) reported that phenolic acids and lipids were
found throughout the bean, but at a greater concentration in the outer layer
of the endosperm. They concluded that these two components were linked
When coffee beans are roasted, gases are formed. These are initially
retained and normally the bean expands, i. e. the roasting reactions can be
thought that the normal reactions do not take place since the normal internal
temperatures and pressure are not achieved. It is also thought that structural
differences at the cellular level may render the Arabica pressure vessel
different from the Robusta's. If this hypothesis is correct, it could, in part,
Villar and Ferreira (1971) found that Brazilian soft coffee (good quality)
had lower percentages of total CGA as compared to other classes of coffee.
However, Amorim et al. (1973) stated that Robusta coffee had more CGA than
the Arabica. This fact alone was not sufficient to account for the difference in
and Hermann, 1980). Paper chromatography lacks the resolution, speed and
accuracy needed for fast reliable analysis of complex mixtures. Thin layer
chromatography (TLC) offers greater resolution and, speed than PC, but lacks
the quantitative accuracy (Harbourne, 1973). Clifford (1974) reported on the
use of TLC with poly-N-vinyl-pyrrolidone (PVP) as the absorbent. He was
able to separate sub groups of CGA, but did not quantify them. Column and
gas chromatography (GC) have also been used (Kung et al., 1967; Andersen
and Vaughan, 1972; Nagels et al., 1979) in the analysis of these complex
molecular mass compounds generally require high injection point and column
temperatures which increase the risk of thermal degradation.
- 41 -
to above.
of CGA was reported by Rees and Theaker, 1977 and Van der Stegen and Van
Duijn, 1980. The individual CQA, DCQA and at least one FQA have been
resolved.
Clifford and Staniforth (1977) discussed the use of six reagents in the
six reagents, three were rejected and periodate (0.25% aqueous sodium
periodate) was most successful. This reagent reacts with the CQA, FQA,
DCQA and caffeoylferuloylquinic acids (CFQA), i. e. some 987 of the , total CGA.
Best results were obtained by controlling the time and temperature of the colour
producing reaction, 10 minutes at 27°C being the optimum.
Clifford and Wight (1973) explained that aqueous periodate oxidises the o-
- 42 -
(2 x 354) - 516
_ 37%
516
caffeic acid and/or the ferulic acid content to individual isomers of the CQA,
FQA, DCQA and CFQA. Free caffeic and ferulic acids are detected by this
reagent, but the molar response is only 25% that of CQA and FQA, and
reagent. This was based upon the reagent reported by Swain and Hillis (1959).
At pH 6.5 molybdate yields a yellow colour (A max 37 0mm) with those CGA
having a caffeic acid residue (i. e. CQA and DCQA). Normally, 5-CQA is used
of the caffeic to quinic acids ratio, this practice also leads to an overestimation
of the DCQA fraction. If caffeic acid is present it also interferes, but since
the molar response is only 60% of that of CGA' and its presence is small in most
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.cl O
Cd
a)
cd
O
a w
0-1
'd
4-1 a)
If) aý
aý .g b
ý. U
Cd
H 04 P64
- 44 -
of analysis allow this problem to be overcome, then this method has considerable
potential because the TBA reagent measures total quinic acid after hydrolysing
the CGA overnight with sodium hydroxide. Since all CGA contain quinic acid
one assumes that all CGA will give an equal response on the basis of their
quinic acid content. Clifford and Staniforth reported that to measure quinic
extracts before and after saponification to correct for the presence of free
quinic acid.
reagents differ in that the periodate reagent detects FQA, whereas the molyb-
date does not. This specificity may be expressed:
This relationship has been borne out by studies using model systems
(Clifford and Wight, 1976), and by comparisons with HPLC methods (Rees
and Theaker, 1977) when analysing coffee beans that are not discoloured.
However, where there is discolouration which might have arisen via polyphenol
similar to, if not identical to, the benzoquinones produced by the periodate
reagent. Thus these quinones might react with periodate but having lost their
the difference between the two reagents may not be just FQA, but FQA and
quinones.
DCQA : Clifford and Staniforth (1977) have reported that the TBA reagent
detects DCQA as one molecule of CQA, whereas periodate reagent detects DCQA
other more accurate and simple methods of estimating these CGA isomers is
appropriate.
It is clear from the literature survey that in the field of coffee chemistry,
many fascinating problems await investigation. However, bearing in mind
the facilities and expertise available, two areas stand out:
Accordingly, these two areas were selected for investigation and it was
proposed to approach these objectives by:
CHAPTER TWO
METHODOLOGY
- 48 -
Introduction
their repeatability and reproducibility were calculated. The methods were not
accepted until these values were equal to or better than those previously
published (Table 5). Any new method would have to perform comparably or
The seeds are dipped in liquid nitrogen until boiling ceased and then
ground to pass through a sieve with a 0.7mm aperture. The ground material
allowed to settle and is decanted onto a Whatman No. 1 filter paper. The
residue is re-extracted five times, the filtered extracts are bulked and diluted
to lOOmL with 70% 2-propanol.
I. Colorimetric Methods
shown in Table 8. They are slightly better than those previously reported.
Periodate reagent
Aqueous sodium metaperiodate (BDH, England) is prepared at 0.25%
(w/v). The stoppered test tubes containing 10. OmL of the reagent are placed
in a waterbath (270C) and the reagent allowed to stabilise at 270 C. This is
The extracts (1. OmL) to be analysed are added to these tubes while
they are still in the waterbath, the contents mixed immediately and thoroughly
by shaking and the test tubes returned to the waterbath. The absorbance
(> 406) is read ten minutes after mixing against a blank of distilled water
max
containing 1. OmL of the coffee extract. All samples were examined in
One millilitre of pH 1.4 buffer (iM toluene sulphonic acid -i- 1M toluene
sulphonic acid sodium salt ; BDH, England) is added, then shaken. Then
1. OmL of 0.8M periodic acid (Sigma, U. S.A. ) in sulphuric acid is added, and
held for 20 minutes at room temperature. This step was later replaced by
holding at 37°C (see p. 54). Sodium arsenite (1. OmL of 47; BDH, England) is
to
ce
1o ý tf)
ö ö 0 0
`d ö ö ö C
to a
W +1 +1 I 1 +1 1 +1 U
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"ýý Ö Ö
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a.+ C14
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to a)
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- 51 -
E370
0.
0.
0. '
0. E
0.5
0.4
0.3
0.2
0.1
25 50 75 100 12 5 175
mg/250mL CQA
- 52 -
E406
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
E549
0.8
0. i
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25 50 7' IO jet- I1 75
When the TBA reagent was used as reported by Clifford and Staniforth
(1977) it was noted that the results were not reproducible. They had indicated
that the TBA reagent method needed improvement. The cause of this variation
An experiment to find the optimum conditions for periodic acid oxidation step
Method
The TB.A reagent method was carried out using a synthetic quinic acid
the oxidation step with periodic acid. This step was held at room temperature
37°C for various times (20,30,40,50,60,120 minutes and overnight).
and
The temperature of 37°C was chosen, because it was easy to operate at this
temperature.
This experiment was repeated a week later to see if the results could
be reproduced.
Results
In the first experiment (Table 6) at room temperature almost half of
the quinic acid was not accounted for, compared with the recovery of quinic
acid oxidised with periodic acid at 37°C. This was so at each time studied.
the recovery of the product of oxidation of quinic acid over that at approximately
19°C. The data from both experiments show that the oxidation product of
quinic acid recovered at room temperature increased with time while that at
37°C remained constant. This implies that oxidation of periodic acid is
temperature dependent and 37°C for 20 minutes was chosen, because this
curves.
Moisture Content
The moisture content was taken to be the weight loss after heating the
Rees and Zheaker (1977) and Van der Stegen and Van Duijn (1980)
demonstrated the value of HPLC for the analysis of CGA in coffee. Accordingly
the latter method, with minor modifications, was adopted as an approach that
Instrumentation
440 used at 313nm, with a solvent programmer Model 660 equipped with a U6K
minute(tor41).
All organic solvents, standard solutions and distilled water used for
the HPLC were filtered through 0.22um millipore filter paper (Millipore Ltd.,
England).
Reagents
Methanol (AR, BDH, England)
5-CQA (Sigma Chemical Inc., U. S. A. )
- 56 -
Several standards were injected into the HPLC in order that their tR
values could be determined. These standards and their rR values are listed
in Table 7 (page 60).
the amount of material available. The results are shown graphically in Fig.
12,13 and 14. Most standards proved to be almost homogeneous (CQA, FA).
The crude DCQA (Roth, W. Germany) was recrystallised until the three DCQA
isomers (3,4-, 3,5-, and 4,5-DCQA) accounted for some 95% of the material
detected by HPLC. The peak heights of the three isomers were summed and
related to the total amount injected. This is possible since the three isomers
have virtually identical molar absorbtion coefficients (Rubach, 1969).
Calculations
Peak retention time (tR) is measured as the time from the injection of
the sample until the time at which the elution peak is at its maximum. Peak
height measurements are used to quantify the standard solutions and components
Peak
Height
(cm)
1.4
1.2
1.0
0.8
0.6
0.4
().'D2
0.35
0.30
Peak
Height
(Cm)
0.20
0.10
Q. O2
Peak
Heigh-
( cm)
1.5
1.0
0`. 5
0.1
t
Standards tR values (minutes)
Gallic acid 2.5
3-CQA 3.0
3-CoQA 5.0
4-CoQA' 7.5
5-CQA 8.6
4-CoQA 11.0
CA 13.5
5-CoQA 14.0
5-FQA 16.0
FA 16.0
Caffeoyl glucose +
16.5
Coumaric acid 21.0
5-CQA isomers (a) 3.0,6.5 and 9.0
(b)
5-FQA isomers 9.0,12.5,16.0 and 16.5
3,4-DCQA 24.5
3,5-DCQA 26.0
4,5-DCQA 32.0
* Impure standard
(a) (b)
and - isomerised by heating with ammonia
+- recovered after ion-exchange purification method
(Griffiths, 1982)
Coff ee extract
The green and roasted coffee extracts used for colorimetry were
filtered through a 0.22pm millipore filter, and diluted whenever necessary.
HPLC Data
The method of Van der Stegen and Van Duijn (1980) with sample
detection at 313nm, was used. Though the HPLC was equipped with a high
resolution reversed phase system, it could still be limited with respect to peak
identification and peak purity.
Peak Identification
In an extract up to 20 peaks have been separated (see Chapter 3, Fig. 16, ?,'IS)
Peaks identified with a high degree of certainty:
These are peaks matching a homogeneous commercial standard or
homogeneous sample provided as a gift and matching closely with the behaviour
Peak Assignment
1 3-CQA
3 5-CQA
6 5-FQA
8 p-coumaric acid
10 3,4 -DCQA
12 3,5-DCQA
16 4,5-DCQA
and Van Duijn or which match a less well defined standard, e. g. isomerised
FQA, CQA or 4-p-CoQA.
Peak Assigment
24 -CQA Van der Stegen & Van Duijn;
is omeris ed 5-CQA
4 4-FQA/4 -CcQA Van der Stegen & Van Duijn;
isomerised 5-FQA
By comparing the relative retention times with those of Van der Stegen it
- 62 -
acid and after 5-CQA. However 4-CoQA gave two peaks, one at 7.5 and the
Unknowns:
Peaks nos. 7,9,11,13,14,15,17,18,19 and 20.
When the total peak heights of the unknowns of Tanzanian Arabica
sample was measured and compared to 5-CQA standard, it was shown that
those peaks contributed 0.064 mg/mL. However when the unknown peaks for
Peak Purity
certain that all peaks in all samples are homogeneous. This problem is
highlighted by considering ferulic acid (tR =16 minutes) and 5-FQA (tR = 16
minutes); these do not resolve when present in mixtures. Van der Stegen and
Van Duijn found that their peak 4 (tR = 12.5 minutes) was impure and could,
using acetonitrile, be resolved into 4 -FQA and what may have been 4 -CoQA .
One can never completely rule out the possibility of co-chromatography and
simultaneous detection, but for simplicity, it has been assumed that the peaks
possibly greater with immature or peculiarly coloured beans than with typical
commercial beans for which the system was developed and of which the
are not detected at 313nm. Similarly one can hypothesise about substances
which might be present and thus which might account for one of the unknowns
or which might co-chromatograph.
- 63 -
Ulbrich and Zenk (1980) reported that a coffee cell culture contained
the enzyme p-coumaroyl shikimate transferase which implies that coffee beans
could contain p-coumaroyl shikimic acid (CSA) and possibly other shikimic
acid esters. Even if such esters do occur they may be only non-accumulating
intermediates.
4-4
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- 65 -
Notes to Table 8.
,ý222
(J1 -+..... (In
TOTAL=
discussed on p. 61 All methods are of high precision (see Table 8). Thus
.
one would expect that:
Pv
HPLC CGA = 1.0 Ratio 1
and HPLC CGA = HPLC CQA + HPLC FQA + 1.37 I[PLC DMA
HPLC BDQA
_ 1.0 Ratio 2
TBA BDQA
,
only. from the quantitatively major CGA such that: HPLC BDQA = HPLC ODA + 1PLC FQA +
unknowns all contain quinic acid the ratio would fall to approximately 0.90 for
Arabicas and approximately 0.80 for Robustas. It is unlikely that all the
unknownswill contain quinic acid, thus one can predict that this ratio will fall
ratio will not approximate to unity because of the different responses to DCQA.
This may be explained as follows. Assume a typical green bean has the
composition:
CQA 57, FQA 1%, DCQA 1.597o
These ratios are based upon certain assumptions about the nature of
the phenols in typical green coffee beans. If these ratios can be confirmed for
such beans, then it may be deduced that for any sample which yields peculiar
ratios there must be a peculiar composition. The manner in which the ratio
changes could give some pointers to the nature of this change in composition.
In the following chapter these hypotheses are tested.
- 68 -
EXPERIMENTAL,
CHAPTER THREE
Introduction
green and some roasted coffee beans were analyzed. The samples were
supplied by Lyons and Illycaffe. The data from these samples served as a
baseline for the interpretation of all other analyses carried out in the course
of the study.
made, and the residue after extraction dried and packed for analysis.
Method
as previously described.
Table 9 shows that for Robusta coffees the periodate value, molybdate
value, and TBA BDQA value are highest, Arabusta as intermediate, and
Arabicas the lowest. The estimated FQA follow a similar trend although the
Arabusta has a lower estimated DCQA content than some of the Arabicas. The
Indonesia.
The HPLC data (Table 10)essentially confirm these trends but total
HPLC CGAwere lower than the periodate values indicating that at least some of
the HPLC unknowns contributed to the colorimetric data. Rees and Theaker
(1977) reported that higher CGA in Robusta coffee arose primarily from higher
FQA and DCQA contents rather than differences in CQA content. However for
the samples analysed during this study the Robustas also had higher CQA
contents.
b
a)
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- 73 -
obtained experimentally.
Hypothetical Experimental
Mean + SD
Conclusion
Introduction
progressive and ultimately severe loss of the mono- and dicaffeoylquinic acids.
This observation was supported by the results obtained with the molybdate and
- 74 -
b
G)
U,
E U Ö
A
0 C ON N N N
C)
C O O O O O
0 v r4
U
V 00 11 N %O
to if)
Ch. t- %0 Lo Cf)
ä12 O O O O O
U v) CO tr) 0
r-+
O O C%
4
O
1 . --, . -4
O
0
U 14
.O'
W
0 0 Ö 0
O
cad Ö Ö
O O O
-9
to
4
A
G)
Co
u r-I oo O so as.
r-1 'D tf) ßi4 i-4
ct! O O O Ö
0 . -i
ci Iti
U
A
ctS
H
cd a)
Cd cat
2
tti
-
. -1
N
IN
0%
to
co
to
'0
N
H
cd a
v O O O O
b
O
Cý
a
w
0
Co
U
U)
Wý
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N w
ul 1 O tN O . -4 5O
. ta 11
0
E4 P4
- 75 -
Rees and Theaker (1977), and Van der Stegen and Van Duijn (1980)
using HPLC methods reported that roasting caused a loss in the CQA, FQA
and DCQA. 'Also, Rees and Theaker observed that with increased roasting
there appeared two other peaks in the chromatogram which increased in height
with roasting.
roasting might arise from the use of different methods of analysis by these
three groups. To clarify this point, and to provide a base-line in this study, a
high quality Tanzanian Arabica was roasted.
Method
previously described. Also the HPLC chromatograms are presented. (Fig 16 to 20)
disappearance of CGA and the HPLC data (Table 13; Fig. 15) showed that the
relative rates of disappearance were CQA 1.0, DCQA 0.56 and FQA 0.09 as
judged by the relevant slopes in Fig. 15. These data essentially confirm
Clifford's (1972) previous observation of the relative stability of the small
amount of FQA.
The CGA are not volatile and these substantial losses imply the
formation of new products. One of these appear to be quinic acid (QA) which
increased progressively during roasting. Up to the third degree of roast the
increase in free QA could have accounted for much of the BDQA that had been
destroyed. Beyond this stage the loss of BD QA far exceeds the production of
free QA which suggested that the BD QA was rapidly converted to a 713A-insensitive
product(s).
Cf) rn to rn N
M 4
4 ö ö ö Ö
0
ca
a
O oo m r- 'I'D
00 N 144 14 M
N Ö Ö Ö Ö
O
cý
a
6
U, C% IN "3+ M
A 110 t- O
"-i v
N
A
U
a
cU IP) %0 M 00 Cl)
00 di M N . -1
O O O O O
0) co
ÄH
rj)
Cd
U ö, N
0 O O O
ýU Ö
oA O O O O
Cd
U
cý
H
Ö Ö Ö Ö O
Ca
'C Ö Ö Ö Ö
w O
q 00 ei N co N
to to Cl) N - -
O Ö Ö Ö Ö
U
O
P4
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0
to
U
to
to
0
0-4
to O
M ... i lý O --i '.O
"-ý . -1
1>1
O
H
- 77 -
Figure 15: Effect of Roasting Tanzania Arabica on CGA
as Determined by HPLC (g/100 beans)
U)
cd
1.0
O.S
o.F
0.7
0.6
0.5
0.4
0.3
0,2
0,1
02468 10 12 14 16
p 3o Mý ýUi%% Ko
to ao
1.
3 to
6 12
2. to
it 1113
RNI
O to so 0,0-h, U 40
Fig. 18 HPLC separation of phenolic constituents of the 2nd Roast
of Tanzania Arabica Coffee
11
IIt
0iD 10 3o -'
'to
Mºýuýas
- 79 -
Fig. 19 HPLC separation of phenolic constituents of the 3rd Roast
of Tanzanian Arabica Coffee
),
0 10 3.0 10 10
1.1iw4tts
- 80 -
HPLC CGA rose. This observation may be explained by the production during
The guaiacols are volatile and may be lost during roasting, but the
di- and trihydroxy phenols are unlikely to be lost in this way. The majority of
these react with the molybdate reagent and/or the periodate reagent and could
thus account for the observed changes in Ratios 1 and 3, but few have significant
roasting. This peak is almost certainly a mixture but could contain 4-FQAk12.5m')
and caffeic acid (tß = 13m) particularly in view of the propable release of
Ratio 2 monitors the relative changes in the HPLC and TBA estimates
pyrolysis loss, ' and indicated greater loss of BDQA as judged by HPLC (free QA
and/or ferulic acid residue of the CGA such that the new product(s) chromato-
still released a substance that reacted with TBA in such a way that it was
indistinguishable from QA.
Ratio 3 compares the PV and TBA estimates of the CGA. This ratio
declined at 7970
pyrolysis loss, remained essentially constant until 117 pyrolysis
loss, and then declined rapidly at 167 pyrolysis loss. Such a decline may be
sensitivity compared to the site reacting with TBA. This is consistent with the
preceding statement.
Chlorpgenic acid and/or its roasting degradation products are also found
- 81 -
M M N
O O O O
4,,;
U)
C) CV N er) N
1 Ö 0
H O 0
U
H
'Lb
+.+
Q)
.'Q "0
M M M M
U
H O O O O
O
O
CY
0
't7 CCýý O O O O
aý
A
a
(ý N N Cl) Cl)
O O O
Co
R7
0
0
H Cl) Cl) N r-+
C7 0 Ö O 0
aý
aý
w
0 11) 1! ) t1)
U
a ä ö 0 0
10
aý
Ha3i
Cý
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w
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44 "-+
O
Cob - - -
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C)
H
_82_
Conclusion
then the quinic acid residue, although some quinic acid appears to be released
by hydrolysis. Later in roasting quinic acid may also be destroyed.
coffee graders. The highest quality have the highest PPO activity.
In general terms it would seem that high PPO activity indicates high
quality in the final beverage, but the inherently higher PPO activity in Robusta
It was this possible connection with quality that led to the selection
of quinones for priority of attention rather than the other potential interfering
substances.
Quinones
Introduction
Quinones are widespread in the plant kingdom (Thomson, 1971). In
general they are yellow, red or brown in colour, but salts of hydroxyquinones are
purple, blue or green (Ikan, 1969). The naturally occurring quinones may be
1.1,2-benzoquinones, e. g. chlorogenoquinone;
2.1,4 -benz oquinones ;
3. naphthoquinones;
4. anthraquinones;
5. quinones with larger fused rings.
Thomson states that anthraquinones are found in Rubiaccae, the family which
includes the genus Coffea. Accordingly this investigation concerned itself
primarily with 1,2-benzoquinones while recognising the possibility that anthra-
enzymic browning quinones. Whatever the reason, it is clear that the 1,2-
benzoquinones have been studied relatively little and this fact hampered this
investigation.
Many texts and original papers, e. g. Shibata et al, (1950); Fiegl and
Anger (1966) ; Simatupang and Hausen (1970); Thomson (1976); Robinson (1980),
and Durst et al, 1975). However the identification of the quinones has proved
difficult because of their high reactivity and instability which led to their
incorporation into complex, high molecular mass polymers (Minard et al, 1981).
catechol, catechol, ferulic acid and caffeic acid and 5-C A, using the method
O Polymer
Reaction:
.... _.. _ ... ý (brown)
NO
0.2. OH 1. NCS -TEA
1,2-quinone
Catechol oxidase
H2O
1,2 dihydroxyphenol
Fig. 21: Basic Reaction of Quinone Synthesis
Chemical Synthesis
Materials
3,5-di-tert-butyl. 1,2-benzoquinone
(Aldrich Ltd., England)
3,5-di-tert-butyl- catechol
(Aldrich Ltd. England)
,
1,4-benzoquinone - technical grade
(Sigma Chem. Inc., USA)
Methods
A solution of NCS (400mg) in fifteen mL methylene chloride was made
with stirring and allowed to cool at -10°C. The 3,5-di-tert-butyl -
catechol (445mg, 2mM) or the molar equivalent of the acids was added. This
mixture was held in the cold for 10 minutes, then 0.3mL TEA added dropwise.
Stirring was resumed for another 10 minutes, the solution filtered and evaporated
to dryness. The red residue was dissolved in hot hexane and then filtered,
- 85 -
examined. In addition IR spectra were made for hexane and nujol, so that
their absorbance peaks could be eliminated from the test substances' own peaks.
starting materials, then the products and the commercially available reference
compounds.
from catechol and the acids would not crystallise so the whole residue was
dissolved in 5OmL chloroform. These solutions were used in the W and
Results
other quinones were very labile. Any attempt to concentrate the chloroformic
solutions, ' or even storage in the dark at low temperature, resulted in, a
darkening of colour and reduced solubility. This instability hindered precise
Nevertheless a body of data was collected that strongly imply that the
products contained the desired quinones along with more complex, possibly
as follows :
1. IR Spectra
at 1680 cm-1. This peak was absent from the starting materials and from the
quinones synthesized from catechol, caffeic and ferulic acids and 5-CQA.
However these quinones showed peaks near 1700 cm-1, presumably due to the
2. NMR
The NMR spectra (see Appendix B) show that the synthesized 3,5-
di-tert-butyl 1,2-benzoquinone was very similar to the standard, thus
The products derived from the acids yielded NMR spectra that defied
interpretation, even after attempts to purify the products by TLC. This
difficulty was ascribed to the impurity of the products arising from rapid
polymerisation after the initial purification.
3. UV Visible Spectra
The relevant data are summarised in Table 15. In all cases the
products had a spectrum that was distinct from that of the starting material.
This is evidence of reaction.
quinone were close and showed two UV and one visible peak. This is in
keeping with the data of Berger and Rieker (1974) who reported that 1,2-
benzoquinones had three ranges of absorbance with strongest max in the
250 to 290nm, an intermediate in 37 0 to 47 Onm and a weak one in the 500
to 580mm range.
However, the product from catechol showed only two peaks and the
products from the acids only one diffuse peak which suggests the presence of
impurities.
4. Reaction with Molybdate
cases the response is low, indicating almost complete loss of the 1,2-dihydroxy
3,5-di-tert-butyl'
1,2-benzocatechol 280 260-280 450-470 560-580
Quinones Molybdate
(mg/lOOmL as
(CQA)
3,5-di-tert 1,2-
benzoquinone 15.0
Catechol 12.0
5-CQA 0.0
Ferulic acid (FA) 3.2
cannot be so certain that the other synthesis were successful, but there is no
doubt that a reaction occurred. The overall impression is that the products
were a mixture in which the desired quinones were present at least transiently.
Because of the difficulty of synthesis and purification of the products, further
investigation was considered unprofitable. Accordingly, no further attempt was
made to:
(a) examine 5-CQA quinone for interference in the analytical methods;
(b) isolate such quinones from green coffee beans.
Conclusion
3
3
II
1I 10
. n. -I ;'a
2 to M.
ction mixture of
and CQA at 1 hour.
3 4. Reaction mixture
of PPO and CQA
at 2 hours.
H 8 lo Mo
6 ,0M.
Enzyme Synthesis
Material
Mushroom tyrosinase (EC No. 1.14.18.1, Sigma Chem. Inc. , U. S. A. )
Activity - 800 units/mg (1 unit = 0.001 absorbancy increase per
Method
A mixture of enzyme, phosphate buffer and CQA or DCQA was prepared
and allowed to react for different times (0,60 and 120 minutes). One millilitre
added. The SDSwas added to enhance activity of the enzyme with CGA (Walker
At 0,60 and 120 minutes the reaction was stopped with 17mL of 99%
nearly 9070(Fig. 22). In contrast the DCQA vanished completely from the
chromatogram within one hour, and the 5-CQA impurity declined nearly 10°70
(Fig. 23) over two hours. This implies that the DCQA bound to the PPO, inhibited
it, and thus effectively protected the 5-CQA from attack. The ability of DCQA
to bind to proteins is discussed further in Chapter 6.
and at least in the case of the two hour samples, this cannot be attributed to
co-chromatography.
-91-
reagent and periodate reagent (Table 17) indicated progressive loss of 5-CQA,
compounds are and it is thus unlikely that such compounds would be extracted
this source would be small and any attempt to continue this investigation by
Conclusions
1. In vitro 5-CQA is a substrate for PPO, but DCQA cause rapid
inactivation of the enzyme.
2. With 5-CQA as substrate PPOproduces a transient oxidation product(s),
CHAPTER FOUR
I Introduction
time of year at which they were formed. Late seeding by one month resulted in
Similar changes have been reported in other tissues, Hillis and Swain
(1959) reported that there was a correlation between the accumulation of CGA
and maturity of fruit and the ultimate seed development. They found that there
in age.
In ara s'colymus, Lattanzio and Morone (1979) showed that the CGA
content decreased progressively as the plant grew. They observed that the
CGA content decreased from 4.4 to 1.670' as the plant gradually developed to the
at a constant level.
and Wanner (1964). They reported that the highest CGA content was found in
immature coffee beans. This fell to 0.17% in half-ripe fruits, and then rose to
0.37% in seeds from the ripe fruit. These values seem extremely low compared
to, for example, the value 7.67 for mature Arabica beans from Kenya and 10.1%
for mature Robusta coffee beans from Indonesia as reported by Rees and Theaker
(1977). The difference could be explained by differences in the methods of
analysis used.
- 96 -
are set out in the format (Format 1). However, not all participating bodies
kept precisely to the guideline. In addition, many samples contained discoloured
beans. The beans were examined by Illycaffe using reflectance spectrophotometry
and were converted to homogeneous samples on the basis of this criterion.
typical reflectance spectra are drawn in Appendix C, D and E.
Kew, England
coffee discovered near Bahia, Brazil, and has very large cherries of heavy
flavour. This variety is mostly sold in Europe at a premium price (Sivetz and
Desrosier, 1979).
Collection of fruit samples started twelve weeks after flowering and continued at
regular intervals up to the stage at which ripe fruit were falling. Normal
commercial maturity occurs just prior to this stage for wet processing, and at
fruit fall for dry processing.
These were two Robusta clones (IVC 182 and IVC 503) and one of
FORMAT I
after harvesting and subjected to wet and dry processing, and possibly further
All the coffee cherries were sun dried and dehusked. The samples
Samples of Arabica green coffee beans were sent from the Federacion
National de Caf eteros, Bogota, Colombia.
ripening through to normal commercial maturity. They were dried using two
methods -wet, and dry (60°C) processing. The cherries were then packed in
removed. In this form the green beans were examined by reflectance spectro-
Three samples of Arabica green coffee beans were sent from Kenya
Industrial Research and Development Institute, Nairobi. These cultivars are
French Mission (FM), SL28 and SL34, which are widely grown in Kenya.
respectively and collection started twelve weeks later and continued at regular
intervals.
The cherries were either wet processed and mechanically dried (30°C)
The flow diagram (Fig. 25) shows the relationship between samples,
method of processing and maturity of bean. Each coffee bean was examined for
homogeneity of colour by Illycaffe (Appendix E)
.
- 100 -
Immature
Mature
Immature
SL28 dry
'-', Mature
Immature
dry Under ripe
SL34 ý ýý Mature
wet .
afore
a
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r-1 O O O
Cd
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- 102 -
N :° ö ° ö
c'
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cd
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C t rn rn 00 N
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Qi
O r+ N ri O '-+ --ý
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x
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cd
aý
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aý 0
'C
ý, ö ö ö ö ö 0 ö 0
A
ö ö ö ö ö ö ö W
1) P4 U
x
aý
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Q cd
Cý1 0
+
0)
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ö 00 00
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cd c ö ö ö ö ö ö
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rn -4
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( 1 1 1 1
0)
H
- 103 -
The most striking feature of this set of data is the apparent absence
of TBA BDQA in the -19 and -16 week samples. The small amounts of CQA
FQA and DCQA detected by HPLC (Table 19) at -19 weeks are consistent with
this within the limits of experimental error, which are larger than usual because
of the unusually dilute extract. The low PV and MV are similarly consistent.
However the very low TBA BDQA at -16 weeks is not in such good agreement
normal experimental errors for the data from -16 weeks onwards.
(Table 19) at -16 and -11 weeks. There is clear evidence of periodate-inflation
Ivory Coast
(a) Robustas
These IVC Robusta cultivars have a significant bound quinic acid
content, even when immature (Table 20). But in neither case are they sufficient
to account for the periodate value or the total HPLC CGA value (Table 21).
This is illustrated by the extreme Analytical Ratios in Table 21, and is evidence
U
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- 105 -
N CO ti) M -4 CO N N M 01 0 00
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-106 -
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- 108 -
IVC 182, these substances may well cochromatograph with the DCQA.
By -12 weeks IVC 182 has lost 72% of its initial HPLC DCQA and the
PV has fallen by some 45%. Thereafter MV, PV, TBA BDQA, HPLC CQA and
HPLC DCQA show progressive increases to full maturity. Both measures of
FQA are low and steady. The -12 week dip is not observed in IVC 503. Instead
there is a steady rise in MV, PV, estimated FQA, TBA BDQA and HPLC CQA
and a steady decline in HPLC DCQA. In neither case do the Analytical Ratios
Indenie
Table 23). This observation in part can be accounted for by experimental errors
associated with the unusually dilute extracts. Ratio 2 at -12 weeks suggests the
otherwise the Analytical Ratios approach the provisional norms set for mature
beans.
Kent'
For two cultivars the beans were supplied at immature, under ripe
and mature stages, and for the third cultivar they were supplied only as
immature and mature (Table 24). Processing was typical of commercial practice -
for most samples dry processing was used.
ä
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- 110 -
defined stages of maturity might have provided data that were more amenable
to discussion.
Reflectance spectra for all immature and the SL 34 under ripe samples
removal of the pulp, the beans are left in water at ambient temperatures. In
this state soluble components diffuse out of the bean into the surrounding water
(Wootton, 1971). This effect can contribute to the loss of CGA from the bean.
Chlorogenic acid and other phenols are located throughout the whole bean but at
a greater concentration near the surface (Amorim -et al, 1977). Since CGA are
relatively soluble in water (Hamidi and Wanner, 1964; Van der Stegen and Van
Duijn, 1980), it is possible that some CGA diffused out during wet processing,
thus resulting in a lower CGA content for beans processed in this manner.
Assuming that the CGA content in these samples was identical to start
'
with, there would be a significant loss of dry matter and some CGA loss not
substantial peak of caffeic acid in the HPLC chromatogram of samples that were
Colombia
of maturity defined as green, yellow, semi ripe and mature. The last three
There are three striking features about these beans (Table 26 and Table
O
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- 112 -
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- 113 -
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- 114 -
acid free substance that is periodate-inflating; (2) the mature samples do not
achieve the provisional norms set out in Chapter 2; (3) when the mature beans
were roasted, brewed and tasted by Illycaffe, it was found that the beans were
of low beverage quality. Illycaffe suggested this may reflect the beans having
been transported in the cherry. Amorim et al, (1977) commented that such a
practice adversely affected cup quality.
CQA and HPLC DCQA. In contrast, PV, MV, TBA BDQA and HPLC FQA showed
maturity all analytical values and average bean weight were lower for the wet
processed samples.
presence of chlorophyll in the silverskin, but this had disappeared by the yellow
"The presence of chlorophyll is indicated by the dip at 650-700 nm.
stage.
Summa
errors, the values for Ratio 3 were substantially higher than the norms.
The C. Arabica cultivars from Kenya and those from Colombia were
quite different. The most immature samples had higher compositional values
and near normal values for Ratio 3. This observation could indicate that these
samples were more mature than the least mature Indenie and Kew Arabicas.
In contrast, the most immature IVC Robusta samples had a higher PV.
MV, TBA BDQA and total HPLC CGA.
For both Arabicas and Robustas the Analytical Ratios suggest the
yield quinic acid. Apparently the Robustas have a much higher content than the
Arabicas, ' but this is the normal pattern for phenols in these species. It is not
- 115 -
CGA. It should be noted that these substances are also detected by the
caffeoylglucose was present and that the level rose from approximately 0.5mg/
100 beans at -19 weeks to a maximum of 11mg/100 beans at -2, and then declined
to approximately 6mg/100 beans at full maturity. This substance cannot
account for all the interference reported here, since it did not cochromatograph,
and the quantity is far too small.
and this would have been detected as quinic acid. Accordingly, neither could
these substances have been responsible, and for the present the precise nature
The CGA content generally rose in both Arabicas and Robustas as the
bean increased in development until full maturity.
- 116 -
CHAPTER FIVE
I Introduction
quality. Such an operation can only be efficient if the relevant pigments have
been characterised.
(2) Green and blue-green pigments -oxidation of kahweol esters Gibson (1971).
(3) Yellow - brown - black pigments - enzymic oxidation of CGA. Luh and
Phithakpol (1972).
Gibson has reported also that the silverskin of some coffees may
(a) these beans turned brown in acid, consistent with the acid catalysed
These results were accepted as prima facie evidence for the presence
.
of a chlorophyll in the peculiar beans. However, chlorophyll is not commonly
found in plant tissues that are not exposed to light, and since the nature of green
coffee pigments is controversial and such pigments are commonly linked with
- 118 -
pronounced absorbance peaks in the blue-green and red regions of the visible
spectrum. All exhibit similar, though not identical, solubility in various
solvents.
Fig. 26. Protochlorophylls are of interest since they have been found in seed
coats of Cucurbitaceae (Jones, 1966) and are identical to bacterial chlorophylls
extraction solvent but other workers have suggested the use of liquid nitrogen
at -20 to -30°C (Wickliff and Aronoff (1962).
of chlorophylls. These include (1) the ease with which magnesium is lost by
the action of dilute acids or replaced by other divalent metals; (2) the ease with
Chlorophyll a.
Phaeophorbide a Protochlorophyllide a
(vinyl Mg phaeoporphyrin a5)
-carbomethoxy +H20
of C-6e hydrolytic
cleavage between
C-6d, e
Pyrophaeophorbide a Chlorin
ebmonomethyl ester
However, a trial showed that the chlorophyll was lost during the
cleaning up procedure and the method was simplified to an extraction of
Method
Samples of ten whole beans were extracted twice with 5OmL portions of
80% acetone. The extracts were immediately pooled, concentrated under
vacuum and the absorbance spectra re corded from 350 to 700nm. The four
types of coffee beans used were normal beans (A), beans with the dark green
silverskin (B), light green (C), and 'foxy' red silverskin (D). For comparison
a standard was prepared using chlorophyll extracted from fresh grass (E).
and then streaked on a 500.µn layer of silica gel (Merck, Germany) prepared
using a Shandon TLC spreader. The plates were air dried at room temperature
- 121 -
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- 122 -
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- 123 -
and activated at 110°C for two hours. The plates were developed in hexane/
Two peaks are distinct in the spectra, one in the region 420 to 440nm
and the second at 664 to 666nm (Figs. 27, '28 and 30). These peaks are
typical of chlorophyll (Robinson, 1980), thus suggesting that for samples B and
C the pigments extracted included chlorophyll and/or related compounds.
These peaks were not detected in the spectrum obtained for good quality beans
It was shown that coffee with dark green and light green silverskins
gave almost identical chromatograms. The beans with a 'foxy' red silverskin
gave only two components. Identifications are based primarily upon the data of
Gibson (1971) and cannot be taken as unequivocal. However, it would appear
that both green silverskin samples contain chlorophyll and/or related pigments,
whereas 'foxy' red silverskin coffee is quite different. It had been thought that
the pigment of 'foxy' coffees might be phaeophytin', a brown haem produced from
extracts in Fig. does not support this; one band from 'foxy' coffee appears
to be lutein (yellow-orange) and the other may well be another carotenoid.
One assumes that the pigments extracted from the silverskin have been
synthesised within the coffee cherry in the absence of light. Accordingly they
may resemble the protochlorophylls that Jones (1965, '1966) found in the seed
chlorophyll can be converted to chlorophyll by light and heat. The rate and
Such conversions may occur during sun drying or mechanical drying of coffee,
11 00000
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the chlorophylls, especially since they differ in their polarity. Nor is there any
Conclusions
(1) Kenya Arabica beans supplied by Illycaffe which had been sorted by two
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- 127 -
0-
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ABCDE
similar to 1(a).
groups was examined on the Perkin Elmer Model 554 spectrophotometer, and
any beans not typical of the group were discarded. Thus these groups were homo-
geneous.
Methods
The six batches were extracted and analysed with periodate, molybdate
The discoloured beans from Kenya (Batch 1) are illustrated in Figure 32.
The data are presented on Table 28. The higher moisture content of
Sample 5 (Batch 1 and 2) is noteworthy. The presence of water could suggest
- 129 -
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-130-
that there had been a favourable environment for enzymatic activity during
storage. However, ' the higher moisture content could have resulted from
All samples show variable bean weight, but there is no doubt that
sample 5 (Batch 1) is significantly lighter. This could suggest that these beans
same time.
The selected. bean sample had a higher PV, MV and TBA BDQA value
than the rejected bean 'sample. Estimated FQA and DCQA are higher in the
rejects than in the selected samples. These results are consistent with those
for 1a.
The colorimetric data for Batch 2 (Table 29) is essentially the same.
The HPLC data (Batch la and lb, Table 30) was in agreement with that
from colorimetric analysis. There was a progressive fall in CQA and DCQA
and a rise in FQA (Batch la). In Batch lb the selected samples had a higher
CQA and lower FQA and DCQA than the rejected sample.
Unexpectedly the best sample from Batch 1 had abnormal values for
Ratios 1 and 2, and the worst sample had apparently normal values for all three
Analytical Ratios (Table 30). For Batch 2 only Ratio 3 could be calculated.
Again there was no significant change in this value from the best to the worst
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- 133 -
tasted.
- 134 -
reference to their reflectance spectra. In practice this was not achieved, due
The results showed that the discoloured samples fall into three groups:
(i) A/B, C and D/E;
(ii) F and G;
(iii) H and I.
Within the groups the samples are similar, but PV, MV and TBA BD QA decline
progressively from group (i) to group (iii). Estimated FQA and estimated
DCQA are little different in groups (i) and (ii), but higher in most discoloured
group. Group (iii) was distinctly smaller and had a slightly higher moisture
content. Ratio 3 was able to discriminate only the darkest beans (Sample I) and
not sample H from group (iii). This suggests that Ratio 3 lacks discrimination.
PV, ° MV, TBA BDQA and HPLC CQA, I but higher FQA and DCQA by both methods
variation. The occasional spurious bean was found and removed from the sample
On the basis of these descriptions these six samples fall into three
groups.
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- 137 -
Samples 7 and 8, the dark green-brown and black beans, have poor
liquoring characteristics. These samples had extreme values for Ratios 2
and 3, and sample 8 had the significantly lower average bean weight (Table 34).
Periodate Value, MV, TBA BDQA and HPLC CQA typical of the blackest
Samples 3,5 and 6 are low quality beans of types which are either not
present in, or not resolved from, previous batches. Sample 3 was pale,
bleached, ' and samples 5 and 6 had spectral properties characteristic
possibly
of chlorophyll-pigmented silverskins. The values for Ratio 1 were within the
provisional norms, although samples 3 and 5 are borderline. The values for
In both cases the discoloured beans had lower MV, PV, BDQA and
HPLC CQA, and in the case of batch 6, lower average bean weight (Tables 35
and 36). The samples in batch 5 had identical average bean weights. This
tends to suggest that discoloration does not cause a particular weight loss, and
that the lower average bean weight associated with the darker beans in batch 5
The Analytical Ratio (Table 36) for batch 5 were extreme, but this was
not surprising since these are immature beans and must be treated as a special
case.
The seco nd sample from batch 6 had Analytical Ratios within the
then fell to an extreme value, presumably passing through the normal range.
Ratio 3 rose to an extreme value and then fell towards the norm, whereas Ratio
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- 140 -
because these ratios approach the norm once more at extreme discoloration,
Summary
Integrating the results from the six batches one finds that there are
two types of peculiarly coloured beans: (a) those that fall in the sequence
Type (a), which accounts for the majority of samples used in this
investigation were characterised by progressive decline in PV, MV, TBA
.a
BDQA, HPLC CQA and usually lower average bean weight and higher moisture
content.
In many cases (e. g. Batches 1,2 and 4) the most discoloured beans
had a higher estimated FQA content. This could reflect the presence of
showed similar changes in PV, MV and HPLC CQA but not in TBA BDQA.
The Data from batches 5 and 6 suggested that the discoloured beans
might have been immature. Immature beans would have a higher moisture
content and higher physiological activity, and thus be more prone to mechanical
damage and subsequent enzymic and chemical changes.
and this is consistent with the rise and fall observed in batch 6 as discoloration
progressed. Ratios 2 and 3 were significantly higher than the provisional norms
in all reject or peculiarly low in batches 4 to 6. (The
coloured quality samples
partially foxy sample fell within the norm and had acceptable liquoring
Loss by hydrolysis is possible but there was never a marked rise in free QA
by the production of a compound(s) which does not contain QA but which co-
chromatographed with the major CGA and which reacted with periodate.
to 3 could be due to the relatively poor sorting and thus non-homogeneous nature
of the peculiarly coloured beans, since Ratios 2 and 3 perform much more
satisfactorily in the better defined samples of batches 4 to 6.
norms could be reduced. This may be possible and would in turn be favoured
by the use of samples of guaranteed homogeneity.
of the most immature sample of beans from Kenya and Colombia (see Chapter 4,
However the beans in samples 5 and 6 (batch 4) are too large to be more than
slightly immature and possibly the fundamental peculiarity is the failure to shed
other seed coats (Jones 1966) and Sheen (1973) recorded that tobacco with
chlorophyll content also has a high CQA content. Of the two current theories
coffee beans, then the more chlorophyll synthesized in the beans, the higher
foxy beans can be formed from the greens by destruction of chlorophyll which
Conclusions
(1) The yellow, brown and black discolourations may arise by an enzymic
browning mechanism and may be associated with small immature beans with
CHAPTER SIX
COFFEE ASTRINGENCY
- 144 -
I Introduction
It has been said that coffee brew occasionally may be astringent (Illy
tara tannin from Caesalpinia spinosa quinic acid replaces the glucose.
the screening tests developed by Haslam (1974). These tests were negative.
It is generally accepted that the structural feature responsible for the astringent
description. This group is the DCQA (Fig. 34). It must be noted that some
workers,. e. g. Haslam (1973), have said that CGA are not astringent, but it is
OH
Figure 33:
Tara Quinic Acid Tannin (n = 0,1,2). (Haslam, E. 1981)
Hooc OH
Hö CH
Figure 34:
3,4 Dicaffeoylquinic Acid. (Waiss et al, 1964)
- 146 -
astringent.
contrast DCQA had a lingering astringent taste that was quite distinct from
CQA. It was felt that these results justified a more thorough investigation of
coffee astringency.
II Organoleptic Investigation
properties which can be perceived by the human senses (1FT, 1975). Although
sensory investigations are subjective and imperfect and efforts to replace them
with more reliable objective chemical and physical tests have been made
(Reudnitz, 1980), subjective methods are still necessary. However, if possible
b. Sensitivity.
Threshold
Dilution
II. Descriptive:
Attribute rating
Category scaling
Ratio scaling
(magnitude estimation)
Descriptive analysis
Flavour profile analysis
Texture profile analysis
Quantitative descriptive analysis
- 148 -
The triangle test consists of three coded samples, two identical and
one different, presented at the same time. None of the samples are identified
as the control. The judge determines which of the three samples presented
differs from the other two.
choosing the different or odd sample by chance alone is one-third (Smith, 1981).
Responses, measure like and dislike on a rating scale. This method is more
representing the population can assess the food or drink under a given set of
conditions.
The rating scale is simple and flexible; judges do not need previous
experience and the data can be analysed by analysis of variance (Amerine et al,
1965; IFT, 1981). Usually a nine-point structural scale is used, although
several variations of this have been reported (IFT, 1981). These include:
(1) a reduced number of ratings, although not fewer than five; (2) a greater
number of like ratings; (3) omission of the neutral rating category; (4) verbal
ratings from the judges, and limited application unless large populations are
used.
Method
The threshold for CQA and DCQA were defined by using ten volunteer
DCQA was more variable, some detecting at 0.05%, others requiring 0.1%.
These data were utilised in preparing test materials for all future investigations.
- 149 -
Triangle Tests
Home Economics using their specialist taste panel facilities. The panel
members were drawn from the University community using the Home
Economics register of volunteers. First, fifteen people tasted water with
Twenty-nine people took part in the second testing experiment with 0.1 0
DCQA added to coffee brew. The taste panel were informed of the purposes of
the test and this helped to increase tasters' confidence.
The coffee was made at three grams of instant coffee (Nescafe Gold
All test sampies were served at 60°C. The panellists were asked to
pick out by taste the single different sample among the three cups presented.
Rinsing agents such as water, milk or unsalted crackers were used to remove
traces of sample.
Rating Test
The third taste assessment took the form of a rating test. This method
was used to find out the effect of CQA on the perception of DCQA. The
concentration of the acids were 0.1% CQA, 0.1% DCQA and 0.1% CQA plus
DCQA, i. e. 0. lg DCQA added to 0. lg CQA then dissolved in 100. OmL of instant
coffee.
The HPLC analysis of the instant coffee is shown in Table 38. The
- 150 -
actual test concentrations were 1.45,1.08 and 0.08mg/mL of CQA, DCQA and
FQA respectively. The corresponding data for the original green beans were
not known.
At each session individual judges were placed in single booths and were
presented with the three different randomly coded samples for evaluation. All
samples were served at 60°C. Judges were asked to follow the instruction sheet
provided (Appendix F ). The scale was a seven-point scoresheet which was
clearly explained as having the stronger flavour at point 7 and the milder and
the beverages were tested using a multiple-range test (Amerine et al, 1965).
Results
Triangle Tests
Distilled water with added 0.1% DCQA
Out of the fifteen people who took part in the taste assessment, only
eight selected the odd sample correctly. Using the significant table for triangular
tests (Amerine et al, 1965) these data were found not significant at 5% level
of significance. (One needs at least nine people to make the correct selection
before significance can be recorded at 5% level. )
- 151 -
panellists were able to correctly identify the odd sample. Only nineteen are
required to record 0.1% level of significance. Therefore the panellists were
able to select the odd sample and the taste of instant coffee with added 0.1970
the odd sample, they chose unprompted descriptions like: bitter lingering
after-taste, metallic bitter taste, bitter and sour at first on top front of tongue
I
Since, for products and judges, ' the calculated values of F (Table 39)
exceed the tabulated values the statistical analysis indicates, that differences,
significant at 0.1% level existed between mean scores of products and mean
scores of judges. Arnold and Noble (1978) reported that even when the judges
were rigorously selected and trained, the judges were significantly different.
Thus such results with an untrained panel are not unexpected. However, the
products were found to be different and therefore the judges were using the scale
correctly.
Ideally the data from the Rating Scale should have been analysed by a
non-parametric method, but this method would not show which of the beverages
the use of a parametric method the data for the different beverages had an
different, the tabulated values of Qp for 124 degrees of freedom, arc each
multiplied by the standard error 1.0/68 = 0.015, ' to form the shortest
Table 40: Multiple Range Test for the Effect of CQA on DCQA
At 17 level the mean score of product CQA, DCQA and their mixtures
was higher for DCQA > mixtures (1 : 1) > CQA respectively. Therefore the
objectionable? " Everyone in the panel stated instant coffee with added 0.1%
DCQA.
Discussion
sensation in the mouth which affected the whole of the tongue uniformly. One
- 153 -
cannot expect someone who is not in the coffee trade or experienced in tasting
to be able to use accurately a technical word such as astringent. Whatever the
different groups called the sensation, it is obvious that the effect would
There has been little previous work upon the organoleptaic properties
of the CGA: Lee et al, (1975) reported that salts of CQA, caffeic acid and
cynarine (1.4 DCQA) have a sweet taste. Whiting and Coggins (1975) studied
the astringency content of ciders and found that a high 5-CQA content mellowed
the astringent notes associated with condensed tannins.
methods have been reported, based upon the ability of such compounds to
Method
Rat blood was haemolysed by diluting with distilled water at a ratio of
1: 50 immediately after collection. From this, 1. OmL was taken and added to
1. OmL of tannic acid, 5-CQA, DCQA or green bean extract at various concentrations
during mixing (Bate-Smith, 1973). After shaking for 15 seconds, the mixture
colour; aqueous DCQA left a red supernatant. These observations led to the
39). According to Kisugawa et al, (1981) the haemoglobin may have been
acid.
These results fitted a logistic curve (Causton, 1977) which had a linear
y= sample DA
t= final concentration of test substance that would
have occurred in the reaction mixture in the absence of precipitation.
- 155 -
astringency shown by tannic acid, thus confirming the hypothesis that DCQA
are astringent.
Bate-Smith (Colorimetric)
Tannic acid 107 -12.90 0.36 1
DCQA 23.1 - 1.78 1.76 0.21
Gravimetric
It was thought that the failure to correlate might be due to the poor
solubility of DCQA in water.
Tannic acid and DCQA behaved as expected (see Table 41), but the data
-157-
It would seem that other substances in the coffee extracts are able to
interfere in the DCQA haemoglobin interaction. Among the substances known
to be present in the extract are caffeine and CQA.
caffeic acid residue, and thus possibly with DCQA (Hormann and Viani, 1972).
It was anticipated that caffeine might interfere in the DCQA-haem interaction.
Non-astringent phenols such as CQA are relatively poor protein precipitators..
One must assume that this is because they do not cross-link, rather than
because they do not associate with the protein, since they offer a virtually
identical shape. Indeed, their smaller size might be expected to favour
and reduce the DCQA threshold for precipitation. However the astringent phenols
to which Bate-Smith refers do not, in general, contain carboxylic acid residues
and alternatively one can postulate antagonism using the following model:
Protein with n readily available binding sites may bind (1) n molecules
n/2
of CQA yielding n carboxylic acid groups; (2) molecules of DCQA yielding
n/2 (3) some combination yielding intermediate
carboxylic acid groups; number
of carboxylic acid groups. The pka values for 5-CQA and that of DCQA is 4.25
value, (approximately pH 6.5) and one can envisage that the accumulation of
sufficient charges would significantly impede precipitation. Such a mechanism
CHAPTER SEVEN
considered critically. The more promising were selected and much time was
and TBA
spent in gaining expertise with HPLC and the molybdate, periodate
In all cases the precision was determined and in the case of the TBA
reagents.
Pv
1. J
2. HPLC BDQA
TBA BDQA
3. Pv
TBA BDQ A
commercial green beans and led to the further hypothesis that these ratios
- 160 -
early could not. It is these nearly mature samples that arc more likely to
enter commercial batches and thus for which some discriminating method is
desirable.
Thus one must conclude that these Analytical Ratios are likely to
distinguish only very good and very bad batches and to be of little help at
intermediate quality levels unless further work permits the width of the norms
to be reduced. However, these Analytical Ratios have some merit as an
analytical tool and clearly indicated the presence of unusual phenolic compounds
in the immature beans. The presence of these substances would not necessarily
have been obvious if only one method of analysis had been employed. The
HPLC. In part this may be a reflection of the relative precision of the two
methods, but this observation does tend to suggest that some CQA oxidation
- 161 -
products are present in many of the samples, particularly the darker ones.
Arising incidentally from this section of the investigation was the interesting
with blackening, it declined much more slowly than CQA, and in the darkest
beans accounted for approximately one-third of the CGA. This is consistent
with CQA also being the preferred substrate in-vivo. The possible organoleptic
implications of an increased proportion of DCQA were discussed in Chapter 6.
this study. Immature beans were detected if they retained their chlorophyll-
pigmented silverskin, although usually this was easily shed during processing
peculiar beans which retain their silverskin during green bean processing.
The reason for this is not known. Some of these beans, characterised by
The CGA content of the yellow, brown and black beans is consistent
whereas CQA do not. This astringency can be detected in instant coffee brew at
1.08mg/mL if the CQA content is low. However, raising the proportion of
CQA from 1: 2.5 to 1: 1 and finally 18 :1 progressively reduces the intensity.
this is linked
avoid green beans with high DCQA content, particularly where
with a relatively low CQA content. Both CQA and DCQA are destroyed
progressively during roasting, but CQA is destroyed more rapidly and thus the
- 162 -
contain 0.8' to 1.5% dmb DCQA with a CQA : DCQA ratio of 4: 1 and Robustas
contain 1.8 to 2.2% dmb DCQA with a CQA : DCQA ratio of 3: 1.
beans, e. g. 1.9% dmb IVC 503 with a CQA : DCQA ratio of *: 1, and as
Unfortunately these were unsuccessful (Chapter 6). The DCQA content was
correlated poorly and since the HPLC method was more precise and simpler
for this purpose, that would be the preferred quality control method.
Future Work
1. Me development
of a rapid method for determining the CQA:DCrA ratio
the testing of the hypothesis and
that this ratio is related to beverage
acceptability.
2. Characterisation
of unknowns on the chromatograms and development the
existing analytical of
methods for use with roasted beans.
3. The analysis of samples similar to those used in this investigation
other components that might influence for
the quality, e. g. diterpenes.
- 163 -
FINAL CONCLUSIONS
1. The CGA can influence beverage quality via the astringent property of
the DCQA. To produce high quality beverage green beans of high DCQA
5. High pressure liquid chromatography was the most precise and most
informative single analytical method, but is expensive.
BIBLIOGRAPHY
-165, -
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APPENDIX
- 176 -
IR Spectra of:
1
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- 182 -
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- 185 -
Appendix C
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lit
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-187 -
{: 1
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"Al
+-
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Reflectance spectra of ý.ý' 11cwl )cpari.
.
it t: ,'E
fj, I
=t.
190
------------- -- f
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111
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Reflectance spectra c.L u nCel -1:ßp;,
ITF
-Olp es '-_r t-
71 lrA
f. _ .. 1 z
-47 -
0 - __:
i
-- ý\ X
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Reflectance spectra of nuLu u Iw uls.
4 t_.
I t
_L1i. i_. _
t Tý :a'
<r
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411-
}ý
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- 193 -
77W':
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1: Iii
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.. -i
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.
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i
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kýflectante spectra of under-ripe beans.
! ýý
-- ;
41 TT
%y 71-
Reflectance spectra beans.
of mztum
i1 IT i
I` ; fir
1--;
T-` ý, f4
1 ,ý. - r<"-I
77. -ET ci
I(ý i
-_ -_- T. to
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Reflectance
tiýxýcýtr"ýýv[' i11ffl tu ly,
ý;I( fl hýý,It
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1 ti 197
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Til
T T-
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(- ; 1+ -"--- a"º
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Reflectance spectra of matum, b aliti,
:i:..: 198
_f:..
....
E
....
O
-law
0
do
_,
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O
TTT IAW v
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17
Reflectance spectra cf' irnnaturc: e grctýn
Ff
k-
I
I. I.. 199
y-k
Ji
10
'1I:
I: -
T_1
4-1 14
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Reflectance spectra of under-ri ixv b; ', uls.
ý.; iý ý,
ý! ?ý!
9ý ýyp"ýr
ýý1ý 200-
y'"ý
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-ý-
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op
IMF--
-2 02 -
Appendix F
RATING SCALE
higher end of the scale (5,6,7) and the milder at the lower
Beverage A1234567
B1234567
C. 1234567
COFFEE ASTRINGENCY
COFFEE ASTRINGENCY
Introduction
soluble phenolic compounds having molecular weight between 500 and 3000, and
besides giving the usual phenolic reactions they have special properties such as
the ability to precipitate alkaloids, gelatin and other proteins". On structural
or o-trihydroxy orientation within a phenyl ring" and that at least two such
sites are required for an astringent cross links to form. Some data, although
- 205 -
not all support the hypothesis that binding is via hydrogen bonding and Van
Sumere (1975) has suggested that unionised hydroxyls and unionised carboxyls
may act as binding sites. Carboxyl groups are found in some ellagitannins and
the quinic acid tannins.
0.56 effective sites per 100 Daltons (effective site = 0-dihydroxy or o-trihydroxy
pnehyl group). An increase in this ratio from 0.41 to 0.47 caused a marked
increase in precipitation.
There were data indicating the presence of dicaffeoyl quinic acids (DCQA) which
effective site mass ratio for the DCQA is 0.38 if only the two caffcoyl residues
et al (1965) reported for DCQA a pKa of 4.25 (at 20°) indicating extensive
Experimental
Green coffee beans were ground and extracted as previously described
(Clifford,1979) and analysed by reversed phase high pressure liquid
chromatography using the method of Van der Stegen and Van Dui jn (1980).
Very crude DCQA was obtained from Roth GmbH, Halsruhe and purified
by repeated recrystallisation from water until the three DCQA peaks separated
the species Coffee arabica, C. canephora var robusta and C. arabusta. It was
concluded that neither condensed nor hydrolysable tannins were present In these
commercial green beans.
2. DCQA Content
and the arabusta hybrid is intermediate (1.477). Two arabica Santos samples
had exceptional DCQA contents of 2.1 and 2.47. The reason(s) for this are not
observed that beans from slightly immature fruit may contain markedly higher
Roasting causes a progressive loss such that the content in the roasted
bean is a function of the amount originally present and the severity of the roast.
In severe roasts there may be complete destruction. A brew prepared from
Haemoglobin precipitation
Preliminary investigations using tannic acid showed that: -
1) haemoglobin precipitation is a function of haemoglobin concentration
These data fit a logistic curve (Causton, 1977) which has a linear form
Loge (y - 1) = Loge b- kt
y= sample A A,
line.
relative astringency is not only a function of the phenol but also of the protein
used in assessment. These methods are not entirely satisfactory for application
simple correlation between DCQA content and the extent of precipitation possibly
because extraneous substances are entrained in the precipitate.
investigations are continuing but our provisional conclusions arc that DCQA
has an easily detected and peculiar lingering metallic taste which can influence
Acknowledgements
The authors wish to thank the following for supplying coffee samples :-
Fed Cafe in Colombia
IFCC in the Ivory Coast
KIRDI in Kenya
References
Inoue, Y., Aoyagi S. and Nakanishi, K (1965) Chem. Pharm. Bull. 13,101-104.
Kisugawa, K., Arai, S. and Kurechi, T. (1981) Chem. Pharm. Bull. 29, '1694-1701.
Van der Stegen, G. H. D. and Van Duijn, J. (1981) 9th International Scientific
Colloquium on Coffee, 1,107-122, London, 1980, Association
Scientifique Internationale du Cafe, Paris.
Van Sumere, C. F. (1975) in The Chemistry and Biochemistry of Plant Proteins,
Phytochemical Society Symposium, Series No. 11, Edited Harborne, J. B.
and Van Sumere, C. F.
- 209-
Table 1:
Analytical Method
t50J Relative
and Test Substance bk
mg/mL Astringency
Bate-Smith
Tannic Acid 107.7 -12.90 0.36 11
Computer-linked sorting
samples a- 28.2
1 10.6 0.1
2 11.3 -0.03
3 12.9 0.2
4 13.1 0.6
5 15.0 0.7
6 15.0 0.3
7 14.0 -0.3
8 9.2 -0.8
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