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480 [J. Inst. Brew.
BREWING 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
Whereas earlier work on hops has largely
been devoted to the study of the hop oil
and hop resins soluble in organic solvents,
the residue or "hop straw" has received
comparatively little attention. The latter
material contains, however, several con
stituents which are soluble in boiling wort
but whose function in the brewing process
has never been fully ascertained. Prominent
among these is pectin, the presence of which
in hops was first demonstrated by Fink &
Hartmann7 in 1935. Further studies on hop
pectin8*9*10 and its influence on the brewing
process11*12'13 were made by Fink & Just in
the period 1935-39, but difficulties in esti
mating pectin in wort and beer left several
of their results inconclusive in certain
directions.
Chemistry of pectin.—Characteristic of
pectin is its ability to form colloidal solutions
and to produce gels with sugar solutions (see
Kertesz20 and Joslyn & Phaff™ for detailed
reviews). The colloidal nature of pectin
hinders its easy isolation and purification,
especially from other closely-related carbo
hydrates. On hydrolysis it yields D-galact-
uronic 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 poly-
taccharide. Relatively pure fractions rich
in araban, the arabinose-yielding component,
have been prepared by taking advantage of
its preferential solubility in boiling aqueous
70% alcohol.8^1*.17.20.29 Generally, the most
abundant of the three polysaccharides is
pectic acid which is composed of chains of
condensed galacturonic acid units and to
which the gelling power and high viscosity
of pectin are due. Pectic acid usually
occurs in part as its methyl ester. The free
acid forms soluble salts with monovalent
- kations, but yields insoluble salts with poly
valent kations. The solubility properties of
these salts may often explain the isolation
of both soluble and insoluble pectins from
the same plant source. On the other hand,
the insolubility of pectin may sometimes be
due to its combination with other materials
present in the plant tissue. Treatment with
water under pressure,8*29 with dilute am
monium oxalate solution,21*29 or with certain
other reagents,4*24 renders such fractions
soluble.
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 hydro-
lysing 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
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Vol. 50, 1953] macwilliam: chemistry of hop constituents 481
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 developingmethods 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 pectinfrom "hop straw'1 and other plant materials.
They concluded that extraction with diluteammonium 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 fromseveral varieties of hops in as pure a form
as possible and to study the properties of the
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 hydroly-
sates 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
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482 MACWILLIAM! CHEMISTRY OF HOP CONSTITUENTS [J. Inst. Brew.
(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 positivetests 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 below—Table 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 am-
ntonium oxalate solution.—The acetone-
treated "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
volumes of alcohol containing concentrated
hydrochloric acid (1 c.c. per litre). The
precipitates were washed with alcohol until
free from acid. The fractions extractedoriginally by ammonium oxalate were re-
dissolved 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 ofpectinfractions 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. Pectin-
degrading 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)
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)
(II) Fraction extracted by a
Brewer's Gold
Bullion
Early Choice
Eastwell Golding I
(unsulphured)
Eastwell Golding II
FugglesNorthern Brewer ..
Fuggles (green hops)
••
Yield
(% ofdry
hops)
1-50
1-73
1-43
1-52
1*51
1-21
1*28
1*09
N
%
0-53
3*47
204
0-51
2*02
1*50
1*78
2-30
Meth-
°?
1*24
0-73
1*88
312
1-59
1*62000
2*07
mmonium oxalate solution
]
]
:
1
L-54
1-49
1-32
t-461-44
1*23
I 00
1*55
208
1-312-33
317
2*34
2-26
3-45
101
200
0*83
1-27
1*42
1*16
2-08
009
1-88
Pectic
acid %
I
51-3
23*7
48*3
40-8
39-2
414
24*4
340
57*3
51-9
73*2
48*6
47-5
65*4
44*7
70*7
Galac-
tan%
II
40
1*5
21
21
2-6
2-3
2*7
21
3*1
2-7
31
2*1
2-1
2*2
trace
2*3
Araban
%III
6-1
2-2
2-4
4-5
4*3
2-2
3*0
4-5
2*5
2-5
1*9
20
1-7
23
trace
2-3
Pectin
%1 4-II
-Mil
61*4
27-4*
52-8
47*4
46-1*
45*9
301
42-2*
62*9
57-1
78*2
52-7
51*3
70-9
44-7
76-3
Yield of
calcium
pectate
%
71-8
32-6
59*8
52-3
55-1
52-2
30-2
651
69-1
63*9
91*7
59-3
58*7
80-1
50*3
81-6
• Traces of glucose, xylose, and another sugar, possibly rhamnose, were present in these fractions after
enzyme hydrolysis.
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Vol. 59, 1953] MACWILLIAM! CHEMISTRY OF HOP CONSTITUENTS 483
et al.1) was obtained adsorbed on kieselguhr
and was stored at 0° C. When required foruse 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 centri-
fuging 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 hydroly-
sates were chromatographed. The presence of
galacturonic acid, galactose and arabinosewas
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 chromato-
graphically 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 solidwith 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 nin-
hydrin, 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 ofpectinfractions.—The pectin
was dissolved in water (100 parts) and re-
precipitated 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.
Variety
Early Choice
Eastwell Golding
Apple pectin (240
grade—B.D.H.)
TABLE 11
Properties op Purified Hop Pectin
Fraction
extracted by
water
ammonium oxalate solution
water
ammonium oxalate solution
MS*(1%
solutionin
water)
4-215°
+ 223°+ 211°
+ 227°
+ 204°
Relative
viscosity
(20° C.)
1*0
2*0
1*8
2*0
168
Fractions
Pectic
acid
(%)
86-5
87-1
799
831
740
Galac-
tan
(%)
3-8
31
4*6
3*5
2-9
Araban
(%)
3*8
1*7
6-2
2*7
2*5
Pectin
(%)
941
91-9
90*789-3
79-9
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484 macwilliam: chemistry of hop constituents [J. Inst. Brew.
Discussion
As already stated (see Introduction) onlyfragmentary information is available on the
constituents of "hop straw." However, inattempts to isolate pure pectin from thissource it is necessary first to remove protein,
tannin and other water-soluble constituentsas 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 inthe range 20-50° C. The present work has
revealed all these procedures to be somewhatunsatisfactory, resulting in only incompleteisolation of pectin (Table I).
exact relation linking the molecular weights
of pectins with their viscosities, but valuesof 50,000-280,000 have been quoted83 for themolecular weight of apple pectin determinedby this method. On this basis the molecularweight of hop pectin might well be onlyabout 10% of that of apple pectin.
Turning to calculations of the amounts of
pectin isolated as the calcium salts (TableIII), these figures are based on an assumedcontent of calcium. The calcium salt ofpectic acid theoretically contains 10-2% of
calcium. However, several workers3-8 havefound that calcium pectate precipitatesusually 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
Brewer's Gold
Bullion
Early Choice
Eastwell Golding I
(unsulphurcd)
Eastwell Golding II
Fuggles
Northern Brewer
Fuggles (green hops)
Composition of hop pectins(expressed as percentages
of total carbohydrate)
Pectic
acid
83-6
•911
86-5
90-9
91-5
93-6
86-1
92-2
85-1
92-6
90-2
92-3
81-1
1000
820
94-0
Galac-
tan
65
4-9
5-5
4-7
40
40
4-4
40
5-6
4-1
5-0
3-1
90
—
7-3
3-0
Araban
0-9
40
8-0
4-4
4-5
2-4
0-5
3-8
0-3
3-3
4-8
4-6
9-9
10-7
30
Yields of pectin (expressed as percentages of
By estimation
of carbohydrate
002
•0-97
0-48
0-85
0-76
103
0-72
0-77
0-70
0-74
0-56
0-87
0-39
0-74
0-46
1-17
dry!
Totalyield
1-80
1-33
1-79
1-40
1-44
1-43
113
1-03
lops)
By estimation
of calcium pectate
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
Total
yield
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 thefractions extracted by ammonium oxalate solution.
The calculated yields of the pectin fractions
obtained by successive extractions of theprepared "hop straw" by warm water and
by ammonium oxalate are also summarized
in Table I. The products themselvespossessed properties which were generally
similar to those of other pectins.20 It wasnoticeable, however, that solutions of hop
pectins were much less viscous than otherwise comparable solutions of apple pectin
(Table II). Little information exists on any
quantitative estimations of pectins. Thediscrepancy is ascribed to the presence ofaraban and galactan and to incompletecombination of pectic acid with the base.Using this same figure to calculate the yieldsof hop pectin it can be seen that the resultsare not in good agreement with those calculated from the total weights of the threepolysaccharide components. It was, in fact,observed that the degree of co-precipitationbetween calcium pectate on the one hand
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macwiixiam: chemistry of hop constituentsVol. 59, 1953]
and araban and galactan on the other wasvariable. This circumstance together withthe apparently lower molecular weight of hop
pectin as compared with apple pectin makesthe method unreliable as regards pectinsextracted from hops. Accordingly, the resultsobtained chromatographically are regardedas definitely more trustworthy.
Acknowledgements.—The author wishes tothank Professor E. L. Hirst, F.R.S., for muchhelpful 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 withaqueous acetone. Two fractions were ob
tained by using boiling water and subsequently ammonium oxalate solution asextractants.
2. The fractions obtained were impure,since estimation of the galacturonic acid,galactose and arabinose obtained afterhydrolysis of the fractions by enzymes showedthat the pectin contents were between25-80%.
3. The yield of pectin from dry hopswas 1-2% for all varieties examined.
4. Only traces of other polysaccharideswere extracted with the pectin.
5. Purification of selected fractions wascarried out. The resulting materials werefound to resemble other pectins apart fromhaving much lower molecular weights.
References
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2. Campbell, W. C, Hirst, E. L., & Young, G. T.,Nature. Loud., 1038, 142, 012.
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ibid., p. 100-104.
ibid., p. 305.
MacWilliam, I. C, this Journal, 1953, 142.
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