ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

69,

Pectic Acid from the Mucilage

157-164 (19%)

of Coffee Cherries1

Richard J. Coleman, James F. Lenney, Anthony T. Coscia and

Frederick J. DiCarlo
From the Fleischmann

Laboratories,
Received

Standard
March

Brands,

Inc., Stamford,

Connecticut

21, 1955

INTRODUCTION

When the outer pulp is removed from ripe coffee cherries, the mucilaginous layer covering the green bean is exposed. In the commerical
wet processing of coffee, this mucilage is removed in order to facilitate
drying. For this purpose, there are three processes in current use. In
the oldest method, the pulped beans are allowed to ferment naturally.
Sodium carbonate (1) or sodium hydroxide (2) also removes the mucilage.
A rapid and mild process for mucilage removal was provided by the
discovery of Johnston and Foote (3-5) that pectic enzyme preparations
may be employed for this purpose. Carbonell and Vilanova (2) interpreted the solubility properties of crude mucilage as further evidence
for the presence of pectin in coffee cherry mucilage. In the absence of
rigorous chemical evidence, it was considered of interest to ascertain
the presence or absence of polymerized galacturonic acid in coffee
mucilage.
For the present investigation, crude coffee cherry mucilage was obtained from a Guatemalan coffee plantation where it had been removed
from the cherries with dilute alkali. In the laboratory, a polysaccharide
fraction was extracted from the crude material. The main component
of this fraction was found to be galacturonic acid. Arabinose, galactose,
xylose, and rhamnose were present in small quantities. Comparative
hydrolytic work involving catalysis by acid and by a variety of enzyme
preparations demonstrated that the coffee mucilage polysaccharide
behaves like pectic acid.
1 Presented before the Division
of Carbohydrate
Chemistry
Chemical Society at New York City, September, 1954.
157

of the American

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COLEMAN, LENNEY, COSCIA AND DICARLO

EXPERIMENTAL

Preparation of Crude Mucilage

At an experimental station in Guatemala,
ripe coffee cherries were pulped,
washed with water, and immersed for a few minutes in a solution containing 0.5yo
sodium hydroxide and 0.1% Petrowet R.* The viscous opaque liquid was removed
from the cleaned beans by passage through a lo-mesh screen and adjusted to the
phenolphthalein end point with acetic acid. Ethanol was added to a concentration
of 20%, and the residue was collected on filter cloth in a hydraulic press. After
drying at 45C. the residue represented about 0.5% of the wet weight of the original pulped beans. Analysis of the mucilaginous residue showed 9.0% uronide CO* ,
which corresponds to 36.070 anhydrogalacturonic
acid.

PuriJication

of Mucilage Polysaccharide

Crude mucilage (100 g.) was suspended in 2 1. of 1% sodium hydroxide and

clarified by centrifugation at 30,000 r.p.m. in a Sharples supercentrifuge. The supernatant liquid was acidified to Congo red with hydrochloric acid. The gelatinous
precipitate which formed was collected by centrifugation at 40,000 r.p.m., transferred to a Biichner funnel, and drawn as dry as possible. At this stage the mucilage had a dry weight of about 13 g. This material was extracted repeatedly with
300-ml. portions of 55% ethanol to remove lower molecular weight carbohydrates.
The residue was dried by successive treatments in a Waring blendor with hot 95yo
ethanol, hot absolute ethanol, and anhydrous ether. This mucilage polysaccharide
(Fraction 1) contained 19.4% uronide CO* , which corresponds to 77.6% anhydrogalacturonic acid.
A sample of Fraction 1 (10 g.) was refluxed with 335 ml. of 0.5 N H2S01 for 2%
hr. The mixture was brought to room temperature and diluted with 560 ml. of
absolute ethanol. The solid material was collected by filtration, washed with cold
60% ethanol and dried in vacua. The dried product (Fraction 2) was found to be
free of simple sugars. Fraction 2 contained 20.9yo uronide carbon dioxide, corresponding to 33.6% anhydrogalacturonic
acid.

Peck Acid from Citrus Pectin

A suspension of N.F. citrus pectin (20 g., methoxyl content, 8.5yo) in 95% ethanol (40 ml.) was added slowly and with vigorous mechanical stirring to 3 1. of
distilled water. After stirring for 1 hr., 11. of 2 N sodium hydroxide was added and
the mixture was stirred for 20 min. Concentrated hydrochloric acid was used to
make the solution acidic to Congo red. The white precipitate was collected and
washed thoroughly on the funnel with 55% ethanol followed by hot absolute
ethanol. The residue was suspended in 1 1. of 55% ethanol and stirred for 1 hr.
The mixture was filtered and the residue was washed with hot aqueous alcohol of
increasing concentration @O-95%). Further washing was performed in a Waring
blendor with hot absolute ethanol, and finally with ether. The white powder
weighed 17.5 g. after drying in vacua over calcium chloride. It was found to con2 E. I. du Pont de Nemours and Co.

PECTIC

ACID

159

tain 20.5yc carboxyl carbon dioxide or 32.0y0 polygalacturonic acid on an ash-free

basis. The methoxyl content was 0.2oJ,.
Uronide carboxyl carbon dioxide was determined by a modification of the procedure of McCready et al. (6). The hydrochloric acid concentration was reduced
from 19% to 11-1270 in order to eliminate charring. Results within 0.2y0 of the
theoretical values were obtained by this method for pure galacturonic acid and
synthetic mixtures of galacturonic acid, arabinose, and xylose.
The methoxyl analyses were performed by the procedure of Joseph (7).
For the determination of sugars, the metaperiodate method of Hirst and Jones
(8) was applied to solutions prepared as follows: Fraction 1 (2.0 g.) was refluxed
for 2% hr. with 67 ml. of 0.5 N H&SO, . The mixture was cooled and 106 ml. of
absolute alcohol was added. After standing for 20 min., the mixture was filtered
and the residue (Fraction 2) was collected for further work. After neutralizing the
filtrate with BaCOl , the BeSO, and excess BaCOa were removed by filtration.
The filtrate was concentrated in uacuo to a small volume, and the resultant cloudy
solution was clarified by the use of a filter aid. The final volume was 5.0 ml.; the
solution of sugars was neutral and practically free of inorganic ions. Three aliquots were applied to a sheet of Whatman No. 1 filter paper (14.5 X 37 cm.) in
such a manner that a narrow zone remained clear for obtaining blanks.
The chromatograms were run by the descending technique for 48 hours using
n-butanol, ethanol, and 1% aqueous NHdOH (4: 1:5). They were dried overnight
in a forced-draft oven operating at 60-70C. Aniline oxalate (9) was the spray
reagent employed to locate the sugars on the center strip clipped from the chromatogram. Development of color was effected by heating in an oven at 100C.
Each sugar was eluted from the side strips with 5.0 ml. of distilled water by heating for 7-8 min. on a water bath maintained at 80C. Four milliliter aliquots of
the cooled extracts were then transferred to Wassermann tubes and analyzed by
oxidation to formic acid with sodium metaperiodate. Adjacent areas were clipped
from the sugar-free zone and employed as blanks.

Galacturonic Acid from Fraction 2

Fraction 2 (300 mg.) was suspended in 15 ml. of water. There were added 30
mg. of Benefax concentrate, a fungal pectic enzyme preparation, and a few drops
of toluene. The suspension was allowed to stand for 64 hr. at pH 3.9-3.5 and 23C.
After clarification with Celite, an excess of BaC03 and a small quantity of decolorizing carbon were added to the solution. The mixture was kept at 65C. for 1 hr.,
cooled, and filtered. The filtrate was added dropwise to 4 vol. of absolute alcohol.
A white flocculent precipitate formed immediately. After cooling, the solid was
collected by filtration. It was washed with hot 99% alcohol, hot absolute alcohol,
and ether. The yield was 268 mg.; [ol]? +29.1 [c, 0.98 in water].
Anal. Calcd. for (C~H~Or)~Ba.HzO: C, 26.60; H, 3.70; Ba, 25.37; neutr. equiv.
270.7; found C, 26.37; H, 3.90; Ba, 25.33; neutr. equiv. 264.
Mucic acid was prepared from a 50-mg. sample of the isolated barium uronate.
A solution of the salt in 3 ml. of water was treated for 1 hr. with 100 mg. of Amberlite lR-120 (H) to remove the barium ions. After filtering the exchange resin, the
galacturonic acid solution was saturated with bromine water and stored at room
temperature for 7 days. Excess bromine was removed by flushing the solution with

160

COLEMAN,

LENNEY,

COSCIA

AND

DICARLO

TABLE I
The Composition

of Coffee Mucilage

Component
Polygalacturonic acid
Arabinose
Galactose
Xylose
Rhamnose
Methoxyl
Ash
Nitrogen
Acetyl groups

Fraction 1
Percentage

(dry basis)

77.6
3.1
0.8
1.3
0.7
0.4
6.5
1.0
Nil

nitrogen. The product was filtered and dried; it weighed 17.5 mg. The melting
point was 225-7 (dec.) ; there was no depression in a mixed melting point with an
authentic sample of mucic acid.
The p-bromophenylhydrazine p-bromophenylhydrazone of the isolated uronio
acid was prepared by the procedure of Niemann et al. (10). The product melted at
141-3O(dec.) and did not depress the melting point of an authentic sample of
p-bromophenylhydraeine p-bromophenylhydrazone d-galacturonate.
Anal. Calcd. for ClsH2r06NdBr2: C, 39.35; H, 3.85; N, 10.19; found C, 39.98;
H, 4.11; N, 10.16.
RESULTS

AND

DISCUSSION

The mucilage of ripe coffee fruit represents the inner layers of the
mesocarp (2).3 In agreement with Carbonell and Vilanova (2), we found
no evidence of cellular structure in the mucilage of freshly pulped
mature cherries [cf. (1, ll)], Since the preparations used in this investigation were exposed to alkali, esterified uronide carboxyl groups would
have been hydrolyzed to the free-acid form. The purification procedure
as described yielded 10-20 % of a nondialyzable polysaccharide (Fraction 1) which could not be partitioned by paper chromatography.
(Additional pectic acid was found to be present in the supernatant
fluid after the precipitation of Fraction 1 with HCl.) Citrus pectic acid
was prepared from N.F. pectin by a similar procedure so that its properties could be compared with those of Fraction 1.
It was found that Fraction 1 could be freed of non-uronide residues
by hydrolysis with 0.5 N H&304 for 2-s hr. By paper chromatography,
the hydrolyzates were found to contain galactose, arabinose, xylose,
rhamnose, and uranic acid. A chromatogram of such a hydrolyzate was
compared with chromatograms of (a) a similar hydrolyzate of citrus
pectic acid. and (b) a mixture of sugars of known composition. The
3Also, personal communication from W. H. Cowgill.

PECTIC

161

ACID

citrus pectic acid contained the same sugars as Fraction 1, although the
former showed only traces of xylose and rhamnose. The quantities of
these sugars found in Fraction 1 are listed in Table I. The uronate
isolated in good yield (about 70%) by the enzymatic hydrolysis of
Fraction 2 was identified as barium galacturonate. Elementary analyses,
optical rotation, and derivatives (mucic acid and p-bromophenylhydrazine p-bromophenylhydrazone)
substantiated this conclusion.
The Rate of HY~TO~YS~S of Fraction 2 and
Citrus Pectic Acid by Hd504
Samples of Fraction 2 and the polyuronide from citrus pectic acid
were hydrolyzed with acid under identical conditions. Pectic acid
(1.0 g.) was freed of sugars by the acid treatment described for sugar
analyses. The polyuronide residue (500 mg.) and the same quantity of
Fraction 2 were treated as described below.
The solid was suspended in 35 ml. of 1.0 N HzSO* and heated at gentle
reflux. After specific time intervals, the heating was discontinued. The
mixtures were cooled rapidly and centrifuged. Small aliquots were
withdrawn from the supernatant fluids for chromatography and the
determination of total reducing matter by the ferricyanide procedure
of Hagedorn and Jensen (12). After 16 hr. of hydrolysis, paper chromatograms showed galacturonic acid only. Curves depicting the rates of
hydrolysis of pectic acid and of Fraction 2 are presented in Figure 1.
9060co 70cii
3 600
g 50= 40+
z 30%
a 20if

IO0

M6E

IN8 OUkS

12

14

16

l-l
FIG.

1. Rates of hydrolyses
of pectic acid (A) and Fraction
1.0 N H#O, at reflux temperature.

2 (B) by

162

COLEMAN,

LENNEY,

COSCIA

AND

DICARLO

The evident similarity in hydrolysis rates led to the conclusion that

Fraction 2 contained a polygalacturonide chain.
Enzymatic Attackability

of Pectic Acid and Fraction 1

Nine different fungal enzyme preparations were employed to hydrolyze

Fraction 1. Their activities against this substrate were compared with
their activities against the above-mentioned preparation of citrus pectic
acid. The substrate solutions contained 0.04% of either pectic acid or
Fraction 1 in 0.05 M acetate buffered at pH 4.6. The enzyme being
tested was dissolved or suspended in distilled water. One milliliter
was added to 5.0 ml. of substrate, and the mixture was incubated at
30C. for 20 min. The reaction was stopped by the addition of 4.0 ml.
of 0.175% K3Fe(CN)G in 1.06% Na2C03 . Blanks were made up by
adding 1.0 ml. of enzyme to the mixture containing 5.0 ml. of substrate
and 4.0 ml. of alkaline ferricyanide solution.
The digest and blank solutions were analyzed for galacturonic acid
by the micromethod of Hagedorn and Jensen (12) using double quantities
of all reagents. Calibration curves were constructed by plotting the
amounts of Benefax concentrate versus the micrograms of galacturonic
acid produced. The curve obtained with Fraction 1 as substrate was
almost linear and superimposable upon the curve obtained with pectic
acid as substrate, up to 30% hydrolysis (O-100 pg. of Benefax concentrate). The activities of other enzymes, relative to that of Benefax
concentrate, were found by reference to these calibration curves; comparative data for nine enzyme preparations are presented in Table II.
Each preparation displayed the same relative activity against both
substrates. Seventeen additional crude enzyme preparations (including
a variety of commercial products) were assayed with both substrates
and these showed similar correlations. Several attempts were made to
separate the activity against Fraction 1 from the activity against pectic
acid. Benefax concentrate was fractionated by means of alcohol solubility, heating at various pH values, and by treatment with inhibitors.
In every case, the resulting fraction showed the same activity against
both substrates, indicating that the same pectic enzyme was attacking
both substrates. Considering the specificity of enzyme reactions and
the diverse origins of these preparations, the close agreement of the
results in every comparison was taken as evidence for the presence of
1,4-linkages in the polygalacturonic acid chain of Fraction 1.
The preceding observations demonstrate that Fraction 1 is primarily

PECTIC ACID
TABLE
Relative

Activity

of Fungal

Enzymes

163

II

in the Hydrolysis
Fraction 1

Benefax concentrate*
Purified polygalscturonasec
Aspergillus
Aspergillus
Aspergillus
Aapergillus
Penicillium
Penicillium

Pectic Acid

and

Relative activity0 against

Fraction 1
Pectic acid

Enzyme preparation

Rhizopua

of Citrus

199

oryzae

oryzae

199
1450
8
13

oryzae

niger

3
85
109
10

niger

sp.
sp.

0 A value of 199 was assigned to Benefax concentrate.

* Standard Brands, Inc.
c Obtained from Dr. E. F. Jansen.

pectic acid. The arabinose and galactose in Fraction 1 are presumably

present as an araban and galactan, which usually accompany the
polygalacturonide backbone of pectin. Xylose and rhamnose have been
reported as constituents of crude pectic preparations, but are usually
regarded as impurities rather than part of the pectin molecule (13).
ACKNOWLEDGMENTS
Samples of crude coffee mucilage were received from Mr. Robert J. Carbonell
of these Laboratories. They were prepared by Mr. Carbonell and by Dr. M. A.
Jones, Foreign Agricultural
Service, Guatemala City, Guatemala. Purified polygalacturonase was supplied by Dr. E. F. Jansen, Western Regional Research
Laboratory, Albany, Calif. Elementary analyses were performed by Mr. Joseph
F. Alicino,Metuchen,
N. J. Technical assistance was received fromMr. A. Vanderborg and Mr. Ralph J. Addesso of these Laboratories.
SUMMARY

1. A polysaccharide fraction was isolated from the mucilaginous

layer of coffee cherries.
2. This purified fraction was found to contain 77.6% anhydrogalacturonic acid residues, and small amounts of arabinose, xylose
galactose, and rhamnose.
3. The sugars were removed by hydrolysis with dilute acid, and
barium galacturonate was isolated in good yield from the sugar-free
residue.

164

COLEMAN, LENNEY, COSCIA AND DICARLO

4. Coffee mucilage fractions responded to acid and enzymatic

drolyses in a manner very similar to citrus pectic acid.

hy-

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Agronomia (El Salvador, C. A.), Nov., 1952.
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Anal. Ed. 18, 290 (1946).
7. JOSEPH, G. H., Bull. Nat.!. Formulary Comm. 9,21 (1940).
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