480

BREWING

[J. Inst. Brew.

INDUSTRY RESEARCH

FOUNDATION

CHEMISTRY OF HOP CONSTITUENTS

V.

HOP PECTIN

By I. C. MacWilliam, B.Sc., Ph.D.

{Breunng Industry Research Foundation, Nutfield, Surrey)
Received \llh July, 1953

After preliminary extraction of hops with organic solvents and with cold
aqueous acetone, pectin was extracted from the residue in two portions, using

first boiling water and then dilute ammonium oxalate solution. The purity of
the fractions was determined by estimating the sugars produced after enzymic
hydrolysis. Different varieties of hops were found to contain between I and 2%
of pectin.

Introduction
in araban, the arabinose-yielding component,
Whereas earlier work on hops has largely have been prepared by taking advantage of
been devoted to the study of the hop oil its preferential solubility in boiling aqueous
and hop resins soluble in organic solvents,
70% alcohol.8^1*.17.20.29 Generally, the most
the residue or "hop straw" has received abundant of the three polysaccharides is
comparatively little attention. The latter pectic acid which is composed of chains of
material contains, however, several con
condensed galacturonic acid units and to
stituents which are soluble in boiling wort which the gelling power and high viscosity
but whose function in the brewing process of pectin are due. Pectic acid usually
has never been fully ascertained. Prominent occurs in part as its methyl ester. The free
among these is pectin, the presence of which acid forms soluble salts with monovalent
in hops was first demonstrated by Fink & - kations, but yields insoluble salts with poly
Hartmann7 in 1935. Further studies on hop valent kations. The solubility properties of
pectin8*9*10 and its influence on the brewing these salts may often explain the isolation
process11*12'13 were made by Fink & Just in of both soluble and insoluble pectins from
the period 1935-39, but difficulties in esti
the same plant source. On the other hand,
mating pectin in wort and beer left several the insolubility of pectin may sometimes be
of their results inconclusive in certain
due to its combination with other materials
directions.
present in the plant tissue. Treatment with
Chemistry of pectin.Characteristic of water under pressure,8*29 with dilute am
pectin is its ability to form colloidal solutions monium oxalate solution,21*29 or with certain
and to produce gels with sugar solutions (see other reagents,4*24 renders such fractions
Kertesz20 and Joslyn & Phaff for detailed soluble.
reviews). The colloidal nature of pectin
hinders its easy isolation and purification,

especially from other closely-related carbo

hydrates.
On hydrolysis it yields D-galacturonic acid, D-galactose, L-arabinose and

methyl alcohol in varying proportions. On

present evidence18*17 it appears probable that
pectin is itself a mixture of three different
polysaccharides, each a polymer of one of
the above-mentioned components. As yet
no method has been developed which leads
so the quantitative isolation of each polytaccharide. Relatively pure fractions rich

The methods of estimating pectin have,

in many cases, been complicated and un
certain. Thus, the galacturonic acid released
from pectic acid during acid hydrolysis is
itself subject to decomposition by the hydrolysing agent and so cannot be determined
quantitatively.18*21
The pectic acid com
ponent has, therefore, been estimated in other
cases by measuring the amount of carbon
dioxide produced when pectin is refluxed
with 12% hydrochloric acid.22*27 Even under
these conditions, however, a small error still
arises because hexoses yield small quantities

Vol. 50, 1953]

macwilliam: chemistry of hop constituents

of carbon dioxide.2*28 Similarly, uncertainty

exists in the method of estimation of pectin
by means of its insoluble calcium salt, since
it appears that araban and galactan are
precipitated with the calcium pectate to
varying extents.
Recent advances in two fields have together
led, however, to a more satisfactory method
of analysing pectin.
First, highly active
enzyme preparations which are capable of
decomposing pectin completely1*18*81*30 can
now be readily prepared. Secondly, the new
technique of partition chromatography on

paper,5*14 has provided a basis for developing

methods capable of determining the com
ponents of a complex sugar mixture both
qualitatively and quantitatively. Through
the kindness of Mr. W. W. Reid (of Messrs.
H. W. Carter & Co., Coleford, Glos.), a highly
active pectin-degrading enzyme complex
was available in the present work; sugars
resulting from its use were estimated by the
new chromatographic techniques.14*26
Hop pectin.Fink & Just8*9*10 extracted
the pectin from "hop straw" after treatments
with water at 25 and 40 C. to remove
simple sugars, tannins, nitrogenous sub
stances and other materials. Pectin was
then removed by repeated treatment with
water, first at the boiling point and then
at 125 C. under pressure, the total yield
being 12% of the weight of ''straw/' After
removal of some of the araban present by
extraction with 70% alcohol, the remaining
material was hydrolysed yielding the follow
ing mixture: D-galacturonic acid (62%),
i>-galactose (25%), L-arabinose (22%). The
methoxyl content of the original pectin
was 1-7%.
Norris & Resch89 carried out a more
systematic study of the extraction of pectin
from "hop straw'1 and other plant materials.
They concluded that extraction with dilute
ammonium oxalate solution (0-5%) afforded
materials of more constant carbohydrate
composition than those obtained by using
other extractants such as water at 110 C.
under pressure.

These authors did not quote

their yields of pectin, but Kertesz26 found

that four samples of hops contained amounts
of pectin (estimated as its calcium salt) vary
ing between 0-60% and 1-84%.
The aim of the present work was, therefore,
to extract hop pectin quantitatively from
several varieties of hops in as pure a form
as possible and to study the properties of the

481

materials obtained. The pectin was isolated

in two fractions from the "hop straw" after
preliminary extraction of the latter with
aqueous acetone (1:1, v/v).. A part was
removed with boiling water and presumably
represents the fraction dissolved from hops
during brewing, whilst the remainder of the
pectin was extracted with dilute ammonium
oxalate solution. Although the fractions
contained impurity, the yields of pectin,
calculated from the amounts of individual
sugar constituents found after hydrolysis by
enzymes (see above), were shown to be
between 1% and 2% (Table III) and agreed
with the results of Kertesz25 (see above).
Experimental

All evaporations were carried out at 50 C.

under reduced pressure.
Chromatography.This was carried out by
the methods described in Part IV.2a All
chromatograms were prepared using as
solvent ethyl acetate: pyridine: water (2:1:2
top layer). Ribose was added to hydrolysates in the quantitative estimations (see
below) as a reference sugar.
Preparation of "Jtop straw"The hop
samples used in this work were kindly
supplied by Dr. A. H. Burgess, Wye College,
and (unless stated otherwise) had been
kilned with sulphur under normal conditions.
They were macerated in alcohol in a Waring
blender, and heated under reflux first with
alcohol until the extracts were colourless and
then with ether. The resulting "hop straw"
was air-dried at room temperature.

Preliminary extraction of '*hop straw"

Extraction of "hop straw" (50 g.) with water
(2 litres) at 17 C. was found to remove a
small amount of pectin (ca. 50 mg.), contrary
to the findings of Fink & Just.9 On the
other hand, exhaustive treatment of the
"straw" with water at this temperature and
even at 50 C. failed to remove nitrogenous
and other undesired constituents completely,
since the pectin fractions subsequently
extracted from the "straw" were still
impure.

Extraction of "hop straw" (100 g. portions)

at 17 C. was carried out using (a) 0-5%
ammonium sulphate solution (2 litres twice);

(b) 10% sodium chloride solution (2 litres

twice); and (c) 50% (v/v) aqueous acetone
(2 litres twice).
Extractants (a) and (b)
removed only small quantities of material

MACWILLIAM! CHEMISTRY OF HOP CONSTITUENTS

482

(1% of "hop straw"), but (c) removed a con

siderable amount (5% of "hop straw"). The
material removed by aqueous acetone was
obtained as a brown solid which gave positive
tests for the presence of tannin, nitrogenous
substances and inorganic materials. Hydroly
sis of the brown solid with 2n acid followed
by paper chromatography indicated that a
trace of arabinose, probably derived from
araban, was present. All "hop straw"
samples (see belowTable I) were ex
haustively extracted with 50% aqueous
acetone at room temperature, four extrac
tions (2 litres of solvent per 100 g. of "straw")
generally being necessary.
Extraction of pectin with water and amntonium oxalate solution.The acetonetreated "hop straw" was heated with water
(3x1-5 litres) at 100 C. for 1 hr. This
removed the bulk of the water-soluble
fraction. The remainder of the pectin was
extracted by subsequent boiling with 0*5%
ammonium oxalate solution (3 x 1-5 litres).
The respective solutions were combined and
concentrated to ca. one-quarter of their
original volume and then poured into four

[J. Inst. Brew.

volumes of alcohol containing concentrated

hydrochloric acid (1 c.c. per litre). The
precipitates were washed with alcohol until
free from acid. The fractions extracted
originally by ammonium oxalate were redissolved in water, the solutions dialysed
(3 days) against distilled water, and the
pectin reprecipitated in alcohol as described
above. When free from acid, the precipitates
were washed with ether and finally dried in a
vacuum desiccator. The yields of impure
pectin obtained from different varieties of
hops are given in Table I. The products
varied in colour from a light buff to dark
brown. They were readily soluble in water
to give turbid solutions.
Hydrolysis ofpectin fractions and estimation
of constituent sugars.The pectin samples
(0*1 g.) were dissolved in (Mn sodium
hydroxide solution (10 c.c.) and the mixtures
shaken for 30 min. to effect hydrolysis of the
methoxyl groups. The alkali was neutralized
with 0'1n hydrochloric acid solution. Pectindegrading enzyme complex (20 mg.) in water
(10 c.c.) was then added. The complex
(similar to the S-type preparation of Ayres

TABLE I

Yield and Composition op Crude Hop Pectin Fractions

(All hop samples were 1051 crop except the "green" Fuggles sample, which was 1052)
Yield

(% of
Variety

(I) Fraction extracted by water

Brewer's Gold
Bullion
Early Choice
Eastwell Golding I
(unsulphured)
Eastwell Golding II
Fuggles
Northern Brewer
..
Fuggles (green hops)

Meth-

27-4*
52-8

0-53
3*47
204

1*24
0-73
1*88

51-3
23*7
48*3

40

0-51
2*02
1*50
1*78
2-30

312

40-8

21
2-6

2*7
21

3*0
4-5

57*3
51-9
73*2

3*1
2-7
31

2*5
2-5
1*9

62*9
57-1
78*2

69-1
63*9
91*7

2*1

20
1-7
23
trace

52-7
51*3
70-9
44-7

2-3

59-3
58*7
80-1
50*3

76-3

81-6

24*4

2*07

(II) Fraction extracted by a mmonium oxalate solution

200
]L-54
208
Brewer's Gold
]1-49
0*83
1-31
Bullion
2-33
1-27
1-32
Early Choice

Northern Brewer
..
Fuggles (green hops)

: 1*23
1I 00
1*55

317
2*34
2-26
3-45

101

1*42
1*16
2-08
009
1-88

52-3

340

1*62

1-44

21

2-3

000

t-46

1*5

pectate

4-5
4*3
2-2

39-2
414

Eastwell Golding I
(unsulphured)
Eastwell Golding II
Fuggles

71-8
32-6
59*8

6-1
2-2
2-4

1-50
1-73

1-21

61*4

1*28
1*09

III

1 4-II

II

1-59

-Mil

Araban

dry

1-52
1*51

Yield of
calcium

Galactan%

hops)

1-43

Pectin

Pectic
acid %
I

48*6
47-5
65*4
44*7

70*7

2-1

2*2
trace

2*3

47*4
46-1*
45*9
301
42-2*

55-1
52-2
30-2

651

Traces of glucose, xylose, and another sugar, possibly rhamnose, were present in these fractions after
enzyme hydrolysis.

Vol. 59, 1953]

483

MAC WILLIAM! CHEMISTRY OF HOP CONSTITUENTS

et al.1) was obtained adsorbed on kieselguhr

and was stored at 0 C. When required for
use the active material was dissolved from
the adsorbent, which was filtered of!. The
pectin-enzyme solution was covered with a
layer of toluene and incubated at 40 C. for
2 days, blank experiments being carried out
simultaneously. Examination of the solu
tions by paper chromatography indicated
(i) that hydrolysis was complete within
2 days, and (ii) that no sugars were present
in the blanks. With all fractions, precipi
tates were noted on completion of the
hydrolysis. These were removed by centrifuging and were dried off, but in no case did
they contain carbohydrates (see also below).
The solutions containing the sugars were
concentrated to ca> 0*5 ex., and the hydrolysates were chromatographed. The presence of
galacturonic acid, galactose and arabinose was
invariably indicated, while traces of other
sugars (see Table I) were detected in certain
fractions. Estimations of the three main sugar
components were carried out chromatographically as described previously.28 The
results, calculated as anhydro sugar, are
included in Table I.
Analysis of pectin samples by precipitation
with calcium chloride solution.This was
carried out by the procedure described by
Coxa based on the method of Carr & Haynes,3
the results being expressed as the percentage
weights of calcium pectate derived from the
different pectin fractions (Table I). These
results are discussed later.
Examination of insoluble residues obtained
after enzymic hydrolysis.Impure pectin
(water-soluble fraction from Fuggles, 1951,
1*0 g.) was incubated with enzyme as above.
After hydrolysis was complete, the residue
was removed by centrifuging and dried to

a brown solid (0*115 g.). The latter (nitro

gen 3*3%; ash, as sulphate, 29%) was
insoluble in cold water, but was soluble in
boiling water and in sodium hydroxide
(0-5n) giving turbid solutions.
Neutral
solutions gave a blue-green coloration with
ferric chloride solution, indicating the pre
sence of tannin. Hydrolysis of the solid
with sulphuric acid (2n) followed by neutrali
zation and chromatography failed to reveal

the presence of any sugar. The hydrolysate,

however, gave a purple coloration with ninhydrin, indicating the presence of amino
acids. Residues obtained from Bullion and
Brewer's Gold hops were of similar nature.
The fractions extracted by water (Table I)
yielded 10-15% of insoluble material, whilst
those removed by ammonium oxalate solu
tion gave 3-11%.

Purification of pectin fractions.The pectin

was dissolved in water (100 parts) and reprecipitated in alcohol as above. The pro
cedure was repeated a further 5 times. The
nitrogen contents of the samples (cf. Table I)
were reduced to 0*5-0*9% by this procedure,
but were not further reduced by continued
treatment. Subsequent precipitation of the
samples as the calcium salt was followed by
regeneration of pectin by shaking the acidified
alcoholic suspension for 6 hr., then washing
the solid first with alcohol until it was free
from acid and then with ether (Hirst &
Jones16).
This procedure reduced the
nitrogen contents to ca. 0*2%.

Examination of purified fractions.The

buff-coloured solids dissolved readily in water
to give clear solutions which were not viscous.
Observations on certain properties of the
solutions are summarized in Table II.
Hydrolyses of the fractions (0*1 g.) were
carried out using enzymes as described above.

TABLE 11
Properties op Purified Hop Pectin Fractions

MS*
(1%

Variety

Early Choice
Eastwell Golding

Apple pectin (240

gradeB.D.H.)

Fraction

extracted by
water

ammonium oxalate solution

water

ammonium oxalate solution

solution
in
water)

Relative
viscosity

(20 C.)

Pectic
acid

Galac-

(%)

tan

Araban

(%)

(%)

(%)

3*8

941

4-215
+ 223
+ 211
+ 227

1*0
2*0
1*8
2*0

86-5
87-1
799
831

3-8
31
4*6

+ 204

168

740

Pectin

1*7
6-2

91-9

3*5

2*7

89-3

2-9

2*5

90*7

79-9

484

macwilliam: chemistry of hop constituents

Discussion

As already stated (see Introduction) only

fragmentary information is available on the
constituents of "hop straw." However, in
attempts to isolate pure pectin from this
source it is necessary first to remove protein,
tannin and other water-soluble constituents

as completely as possible, because subsequent

purification of any pectin is limited by its
physical properties. Previous workers in
this field8-10-92 attempted to extract undesired
constituents with water at temperatures in
the range 20-50 C. The present work has
revealed all these procedures to be somewhat
unsatisfactory, resulting in only incomplete
isolation of pectin (Table I).

[J. Inst. Brew.

exact relation linking the molecular weights

of pectins with their viscosities, but values

of 50,000-280,000 have been quoted83 for the
molecular weight of apple pectin determined
by this method. On this basis the molecular
weight of hop pectin might well be only

about 10% of that of apple pectin.

Turning to calculations of the amounts of

pectin isolated as the calcium salts (Table

III), these figures are based on an assumed
content of calcium. The calcium salt of
pectic acid theoretically contains 10-2% of
calcium. However, several workers3-8 have
found that calcium pectate precipitates
usually contain only 7-7% of calcium and
this figure has commonly been used in

TABLE III

Estimated Yields and Compositions of Pork Hop Pectin Fractions

(from the results in Table I)

Variety

Composition of hop pectins

(expressed as percentages
of total carbohydrate)
Pectic
acid

Brewer's Gold
Bullion
Early Choice
Eastwell Golding I
(unsulphurcd)
Eastwell Golding II
Fuggles
Northern Brewer
Fuggles (green hops)

Galactan

83-6
911
86-5
90-9
91-5
93-6

65
4-9
5-5
4-7

86-1
92-2
85-1
92-6
90-2
92-3
81-1
1000
820
94-0

4-4
40
5-6
4-1

40
40

5-0

3-1
90

Araban

7-3

dry!lops)

By estimation
of carbohydrate

Total
yield

0-9
40
8-0
4-4
4-5
2-4

002
0-97
0-48
0-85
0-76

0-5
3-8

0-72
0-77

1-40

0-70
0-74
0-56
0-87
0-39
0-74
0-46
1-17

1-44

0-3
3-3

4-8
4-6
9-9

3-0

Yields of pectin (expressed as percentages of

10-7
30

103

1-80
1-33

1-79

1-43
113

1-03

By estimation
Total
of calcium pectate yield
0-09
*0-08
0-52
0-77
0-79
1-12

0-77
0-80
0-73
0-78
0-58
0-91
0-43
0-77
0-40

1-22

1-97
1-29
1-91

1-87
1-51
1-49
1-20
1-71

The upper figures in each case are for the fractions extracted by water, the lower figures for the

fractions extracted by ammonium oxalate solution.

The calculated yields of the pectin fractions

obtained by successive extractions of the

prepared "hop straw" by warm water and
by ammonium oxalate are also summarized
in Table I.
The products themselves
possessed properties which were generally

similar to those of other pectins.20 It was

noticeable, however, that solutions of hop
pectins were much less viscous than other
wise comparable solutions of apple pectin
(Table II).

Little information exists on any

quantitative estimations of pectins. The

discrepancy is ascribed to the presence of
araban and galactan and to incomplete
combination of pectic acid with the base.
Using this same figure to calculate the yields
of hop pectin it can be seen that the results
are not in good agreement with those calcu
lated from the total weights of the three
polysaccharide components. It was, in fact,
observed that the degree of co-precipitation
between calcium pectate on the one hand

Vol. 59, 1953]

macwiixiam: chemistry of hop constituents

and araban and galactan on the other was

variable. This circumstance together with
the apparently lower molecular weight of hop
pectin as compared with apple pectin makes
the method unreliable as regards pectins
extracted from hops. Accordingly, the results
obtained chromatographically are regarded
as definitely more trustworthy.

485

Carre", M. H., & Haynes, D., Biochem. J., 1922,

16, 60.
4.

Charley, V. L. S., Chem. & Ind., 1051, 394.

5.

Consden. R., Gordon. A. H., & Martin, A. J. P.,

Biochem. J:. 1044, 38, 224.

e.

Cox, H. E., The Chemical Analysis of Foods,

4th Edn. London: Churchill,
and 101.

1050, pp. 44

7.

Fink, H., & Hartmann, J., Woch Brau., 1935,

8.

Fink, H.. & Just, F., Woch. Brau., 1935, 52.

9.

Fink, H., & Just, F., Woch. Brau., 1935, 52,

10.

Fink, H., & Just, F., Woch. Brau., 1035, 52,

11.

Fink, H., & Just, F., Woch. Brau., 1036, 53,

12.

Fink, H., & Just, F., Woch. Brau., 1937, 54,

13.

Fink. H., & Just, F., Woch. Brau., 1938, 55,

14.

Flood. A. E.. Hirst, E. L., & Jones, J. K. N.,

/. chem. Soc, 1948, 1679.
Hirst, E. L., & Jones. J. K. N., Adv. Carbo
hydrate Chem., 1046, 2, 235.
Hirst, E. L., & Jones, J. K. N.. /. chem. Soc.

52,221.

Acknowledgements.The author wishes to

thank Professor E. L. Hirst, F.R.S., for much
helpful discussion, and Sir Ian Heilbron,
D.S.O., F.R.S., and Dr. A. H. Cook, F.R.S.,
for their advice and constant encouragement.
Summary

1. Pectin was extracted from "hop straw"

after preliminary extraction of the latter with
aqueous acetone.

Two fractions were ob

tained by using boiling water and subse

quently ammonium oxalate solution as
extractants.

2.

The fractions obtained were impure,

since estimation of the galacturonic acid,

galactose and arabinose obtained after

hydrolysis of the fractions by enzymes showed
that the pectin contents were between
25-80%.

3. The yield of pectin from dry hops

was 1-2% for all varieties examined.

4.

Only traces of other polysaccharides

were extracted with the pectin.

5.

Purification of selected fractions was

carried out. The resulting materials were

found to resemble other pectins apart from
having much lower molecular weights.

341.
349.

362.
33.

281.
17.

15.
16.

1939, 454.

17.

18.
10.

Ayres, A., Dingle, J., Phipps, A., Reid, W. W.,

& Solomons, G. L., Nature, Land., 1062,

2.

Campbell, W. C, Hirst, E. L., & Young, G. T.,

170, 834.

Nature. Loud., 1038, 142, 012.

1050, 47. 437.

Joslyn, M. A., & Phaff, H. J..
Lab. Commun., 1047, 10, 30.

Wallerstein

20.

Kertesz, Z. I., The Pectic Substances. New

York: Interscience Publishers, 1951.

21.

ibid., p. 10.

22.

ibid., p. 36.

23.

ibid., p. 61-66.

24.

ibid., p. 100-104.

25.

ibid., p. 305.

26.
27.

MacWilliam, I. C, this Journal, 1953, 142.

McCready, R. M., Swenson, H. A., & Maday,
W. D., Industr. Engng Chem., Anal. Ed..

28.

Norman, A. G., Nature, Lond., 1930, 143, 284.

20.

Norris, F. W., & Resch. C. E., Biochem. J..

30.

Reid, W. W., /. Sci. Food Agric, 1950, 1, 234.

References
1.

Hirst, E. L., Jones, J. K. N.. & Walder,

W. O., J. chem. Soc, 1047, 1228.
Jermyn, M. A., & Tomkins. R. G., Biochem. J..

1946, 18, 290.

1937, 31. 1945.