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

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

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.

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

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

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.

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