FLAVOUR AND FRAGRANCE JOURNAL

Flavour Fra`r[ J[ 1999^ 04] 089Ð083

Essential oil and glycosidically bound volatiles of

Origanum vulgare L. ssp. hirtum (Link) Ietswaart
J. Mastelić, M. Milos̆ and I. Jerković
Department of Organic Chemistry, Faculty of Chemical Technology, N. Tesle 10/V, 21000 Split, Croatia

Received 12 June 0888

Revised 19 December 0888
Accepted 00 January 1999

ABSTRACT] The glycosidically bound volatiles were isolated from dried plant material by percolation with
ethyl acetate and by extraction with water during hydrodistillation of essential oil[ Fifteen volatile aglycones
were identi_ed by gas chromatography!mass spectrometry "GC!MS# on two columns with di}erent polarity of
the stationary phases[ The main aglycones were] thymoquinone\ benzyl alcohol\ thymol\ 1!phenylethanol\
carvacrol and 0!octen!2!ol[ The content of aglycones was 08 and 10 mg kg−0 with respect to the method of
isolation[ The chemical composition of aglycones was compared with the chemical composition of essential oil\
which consisted mainly of thymol and carvacrol[
After the hydrolysis of glycosides\ D!"¦#!glucose and unknown disaccharide were identi_ed[ The hydrolysis
of disaccharide\ D!"¦#!glucose and D!"¦#!galactose were detected by TLC and GC!MS as trimethylsilylethers[
Copyright Þ 1999 John Wiley + Sons\ Ltd[

KEY WORDS] Ori`anum vul`are ssp[ hirtum^ Lamiaceae^ glycosidically bound volatiles^ sugar moiety^ essential
oil\ gas chromatography!mass spectrometry

Introduction antioxidant activity of di}erent Ori`anum species have

Ori`anum vul`are L[ ssp[ hirtum "Link# Ietswaart is a
famous aromatic and medicinal herb[ Due to its high
content of thymol and carvacrol\ the essential oil of
oregano is an interesting product in pharmacology and
food industry[ The herb\ its extracts and its essential oil
serve as folk remedies[ The plant itself is widely used in
the Mediterranean and in the Latin American cuisines
"chilli and pizza#[
Volatile agylcones were detected in many aromatic
and nonaromatic plants[ Among volatile aglycones
from plants of Lamiaceae family aliphatic alcohols\ ter!
penes\ coumarin and phenylpropane compounds were
identi_ed[0 The sugar moiety of these glycosides can be
monosaccharidic and disaccharidic[ b!D!glucopyranose
is the most common monosaccharide found in many
plants of the Lamiaceae1Ð7 family\ and in numerous
other plants[8Ð07 b!D!galatopyranose is present in Thy!
mus vul`aris08 and b!L!arabinopyranose in Gymnocladus
chinensis[19 The disaccharidic sugar moiety of these gly!
cosides can be divided into several groups] rhamnosyl!
glucoside^10\11 arabinosyl!glucoside^10\12 apiosyl!glu!
coside11\13 and galactosyl!glucoside sugar moiety[14
The essential oils\ the geographic variations and the
 Correspondence to] J[ Mastelic\ Department of Organic Chemistry\ Faculty
of Chemical Technology\ N[ Tesle 09:V\ 10999 split\ Croatia[
been intensively investigated[15Ð20 Glycosidically bound
volatiles were partially investigated in Ori`anum vul`are
by Merkx and B[ Svendsen[21 They identi_ed eugenol
and an isomer of methoxy!vinylphenol as the main agly!
cones[
The aim of this study was to determine the com!
position of aglycones and sugar moiety of gly!
cosidically!bound volatile compounds from Ori`anum
vul`are L[ ssp[ hirtum "Link# Ietswaart[ The percolation
with ethyl acetate at room temperature and the extrac!
tion with water during hydrodistillation of essential oil
were compared as methods for the isolation of glyco!
sides[ The composition of volatile aglycones and of
essential oil was also compared[
Experimental
Plant Material
The plant material was collected in the Mediterranean
region of Croatia\ near Split\ after ~owering in Sep!
tember 0887[ The dried plant material "leaves and apical
stalks# was used for investigation[ The voucher speci!
men is deposited at Department of Organic Chemistry\
Faculty of Chemical Technology\ University of Split[

Copyright Þ 1999 John Wiley + Sons\ Ltd[


Isolation of Glycosides
The glycosides were isolated by two methods]
Method A
One hundred grams of plant material was extracted
by exhaustive percolation with ethyl acetate at room
temperature[ Octyl!b!D!glucoside\ 499 mg\ was added as
internal standard22 in ethyl acetate[ After percolation\
the extract was concentrated to dryness in a rotating
evaporator under reduced pressure up to 39>C[ The
residue was dissolved in ethanol and puri_ed by selective
precipitation of the ballast compounds using water[ The
precipitation of acidic balast compounds was performed
in ethanol with conc[ ammonia[23 Finally\ the puri!
_cation was performed by ~ash chromatography on a
silica gel column applying ethyl acetate ] ethanol ]
ammonia 5 ] 2 ] 0 v:v:v[ TLC analyses showed the
absence of free carbohydrates in the glycosidic fraction[
The obtained glycosidic fraction was concentrated to
dryness\ dissolved in a citrate bu}er "pH 4[4^ 4 ml# and
the remaining free terpenes and hydrophobic com!
pounds were removed with penthane!dicloromethane
as described in a previous paper[22
Method B
One hundred grams of plant material was submitted\
for 2 h\ to a simultaneous extraction and hydro!
distillation in Clevenger type apparatus[ Octyl!b!D!glu!
coside\ 499 mg\ was added to water intended for
hydrodistillation as internal standard[ The essential oil
was separated\ successfully dried over Na1SO3 and
stored[ The aqueous extract was separated and the
residual plant material was extracted once more with
299 ml of boiling water[ The pooled aqueous extracts
were concentrated to 29 ml in a rotating evaporator
under reduced pressure\ up to 49>C[ Balast components
were removed by precipitation with ethanol[ Further
puri_cation\ ~ash chromatography\ dissolving in bu}er
and removing the hydrophobic compounds were per!
formed as described in Method A[
Enzymatic Hydrolysis of Glycosides and Separation
of Aglycones
b!Glucosidase from almonds "{Fluka|\ 19 mg# was
added to the glycosidic solutions "from Method A and
Method B# along with 2 ml pentane for the trapping of
liberated aglycones[ The hydrolysis was carried out for
61 h\ at 29>C with the mixture being shaken occasion!
ally[ After the hydrolysis\ the pentane layer was separ!
ated[ The remaining aglycones were extracted from the
aqueous layer with pentane "09×1 ml#[ The combined
pentane extracts were dried over an anhydrous sodium
sulphate\ concentrated to _nal volume of 9[4 ml\ and 0
ml was used for GC!MS analysis[
Copyright Þ 1999 John Wiley + Sons\ Ltd[
ESSENTIAL OIL OF ORIGANUM VULGARE 080
Isolation of Carbohydrates
After removing the aglycones\ the aqueous extracts
"Method A and Method B# were concentrated to 9[4 ml
in a rotating evaporator under reduced pressure\ and
heated in a boiling water bath for 09 min[ The salts and
the denatured enzyme were removed as precipitate by
addition of 09 ml of ethanol[ The ethanol extract was
concentrated to 9[4 ml and this solution was used for
thin layer chromatography "TLC# of carbohydrates[
Isolation and Acidic Hydrolysis of a Disaccharide
Glucose and disaccharide\ which were detected by TLC
analyses of ethanolic extract\ were separated by ~ash
chromatography on silica gel column "09 g silica gel 59\
i[d[ 1[4 cm#[ Elution was performed with 199 ml mixture
of ethyl acetate ] ethanol 5 ] 1 v:v and 199 ml ethanol[
The fraction of disaccharide was hydrolyzed with HC0
"pH 0# for 29 min at 099>C[ After the hydrolysis\ neu!
tralization and drying\ the residue was dissolved in 9[4
ml methanol[ The precipitate of NaCl was removed and
the methanolic solution was subjected to TLC and GC!
MS analyses[
Acidic Hydrolysis of Glycosides
The acidic hydrolysis of isolated glycosides was per!
formed with HC0 "pH 0# for 0 hr at 099>C[ The obtained
aglycones were not isolated and investigated[ The iso!
lation of liberated saccharides was performed as given
in above paragraph "neutralization\ drying and dis!
solving of residue#[
Identification of Carbohydrates by TLC
TLC was employed to separate and identify the carbo!
hydrates from the hydrolysates[ The carbohydrates
were chromatographed on silica gel with di}erent
mobile phases[24Ð26 The authentic samples of carbo!
hydrates as 9[1) methanolic solutions were used for
references[ Detection] aniline!diphenylamine!phos!
phoric acid as reagent[ The individual spots were ident!
i_ed by comparison of their RF!values with those of
authentic samples[
Preparation of Trimethylsilylethers
The solution of carbohydrates was concentrated to dry!
ness and dissolved in 9[4 ml pyridine[ The mixture of
hexamethyldisylazane and trimethylchlorsilane "2 ml
1 ]0 v:v# was added to the solution[ After a 2 h reaction
at room temperature\ the solvent and the remaining
reagents were removed by evaporation under reduced
pressure[ The residue was dissolved in 9[4 ml pentane
and concentrated once more to dryness[ Finally\ the
Flavour Fra`r[ J[ 1999^ 04] 089Ð083
081 J[ MASTELIC
AND I[ JERKOVIC
ł \ M[ MILOS ł

residue was dissolved in 9[4 ml pentane ] ether 0 ]0 v:v Table 1. Constituents and percentage composition of
and this solution was used for GC!MS analyses of essential oil from Origanum vulgare L. ssp. hirtum (Link)
carbohydrates[ Ietswaart isolated by hydrodistillation
Peak Compound I0 Peak area Methods of
No[ "HP!19M# ) identi_cation
Gas Chromatography-Mass Spectrometry (GC-MS)
Hydrocarbons
The volatile aglycones and the essential oil were 0[ a!Thujene 0923 9[4 I0\ I1\ MS
1[ b!Pinene 0986 9[2 I0\ I1\ MS
analyzed by gas chromatography!mass spectrometry 2[ Sabinene 0001 9[1 I0\ I1\ MS
"Hewlett!Packard\ model 4789\ with a mass selective 3[ d!2!Carene 0020 9[2 I0\ I1\ MS
detector\ model 4860A#[ Two columns with di}erent 4[ b!Phelandrene 0101 9[0 I0\ I1\ MS
5[ g!Terpinene 0120 1[9 I0\ I1\ MS
polarity of stationary phases were used[ GC operating 6[ Terpinolene 0159 9[2 I0\ Ð\ MS
conditions were]22\23 column HP!19M "Carbowax 19M#\ 7[ p!Cymene 0162 00[7 I0\ Ð\ MS
49 m×9[1 mm i[d[\ _lm thickness 9[1 mm\ column tem! 8[ Caryophyllene 0467 9[6 I0\ I1\ MS
09[ a!Humulene 0526 9[0 I0\ I1\ MS
perature programmed from 69>C isothermal for 3 min\ 01[ a!Murolene 0582 9[2 I0\ I1\ MS
then increased to 079>C at a rate of 3>C min−0^ column 02[ b!Farnesene 0588 9[2 I0\ I1\ MS
HP!090 "Methylsilicone#\ 14 m×9[1 mm i[d[\ _lm thick! 03[ b!Bisabolene 0690 9[6 I0\ I1\ MS
04[ d!Cadinene 0618 9[1 I0\ I1\ MS
ness 9[1 mm\ column temperature programmed from
69>C isothermal for 1 min\ then increased to 199>C at Oxygene containing compounds
a rate of 2>C mm−0^ carrier gas helium\ ~ow rate 0 ml 05[ 0!Octen!2!ol 0304 1[9 I0\ I1\ MS
06[ Bornyl acetate 0431 9[0 I0\ I1\ MS
min−0\ injector temperature 149>C\ volume injected 0 07[ Terpinen!3!ol 0448 0[9 I0\ I1\ MS
m\ split ratio 0 ] 49[ MS conditions] ionization voltage 08[ Methylthymyl ether 0454 9[4 I0\ Ð\ MS
69 eV\ ion source temperature 179>C\ mass range 29Ð 19[ 1!Isopropyl!0!methoxy!
3!methyl benzene 0465 9[3 I0\ I1\ MS
299 mass units[ The analysis of carbohydrates as tri! 10[ Borneol 0544 0[0 I0\ I1\ MS
methylsilylethers was performed on column HP!090 11[ p!Cymen!7!ol 0676 9[0 I0\ I1\ MS
with the same GC!MS conditions[ In this case mass 12[ Thymol 1012 38[2 I0\ I1\ MS
13[ Carvacrol 1038 13[5 I0\ I1\ MS
range was 29Ð599 mass units[
Total 85[2)

Identification and Quantitative Determination
Individual peaks were identi_ed by comparison of their
retention indices with those of authentic samples\ as
well as by comparison of their mass spectra with those
stored in data base "Wiley library#[ The percentage com!
position of the samples was computed from the GC
peak areas without using correction factors[ The con!
tents of aglycones were calculated from the GC!peak
areas related to the GC!peak area of 0!octanol "lib!
erated from octyl!b!D!glucoside#[ Preliminary GC!MS
analysis showed the absence of 0!octanol as potential
aglycone[
Results and Discussion
Free Volatile Compounds
The yield of essential oil was 1[8)[ The content and
chemical composition of the essential oil is given in
Table 0[ Sixteen compounds were identi_ed rep!
resenting 85[2) of the total oil[ The main components
were thymol "38[2)#\ carvacrol "13[5)# and p!cymene
"00[7)#[ The essential oil also contained smaller quan!
tities of g!terpinene "1[9)#\ 0!octen!2!ol "1[9)#\ bor!
neol "0[0)# and terpinen!3!ol "0[9)#[ Similar results
were reported for the Green origin of Ori`anum vul`are
ssp[ hirtum[20
I0  retention indices on HP!19M^ I1  retention indices on HP!090^
MS  mass spectra^ Ð  not detected[
Glycosidically Bound Volatiles
The content of glycosidically bound volatile compounds
in dried plant material was 08 mg kg−0 "Method A#
and 10 mg kg−0 "Method B#[ Fifteen aglycones were
identi_ed[ Among the aglycones were identi_ed ali!
phatic alcohols\ terpene compounds and derivatives of
phenylpropanes[ The results are shown in Table 1[ The
main aglycones for both methods were] thymoquinone
"36[9 and 39[1)#\ benzyl alcohol "1[9 and 7[8)#\ thy!
mol "5[7 and 6[4)#\ 1!phenylethanol "9[7 and 4[5)#\ 0!
octen!2!ol "4[3 and 0[2)# and carvacrol "4[4 and 0[8)#[
Minor di}erences among the content and the com!
position of aglycones depend on the isolation methods[
By comparison of the chemical composition of the
essential oil "Table 0# and aglycones "Table 1# _ve com!
pounds were established to be identical] 0!octen!2!ol\
terpinen!3!ol\ p!cymen!7!ol\ thymol and carvacrol[ Our
results show only moderate correlation in the chemical
composition of free and glycosidically bound volatiles[
The aglycones\ such as aliphatic alcohols\ 1!phenyl!
ethanol\ benzyl alcohol\ eugenol\ linalool\ geraniol\
neral\ a!terpineol and terpinen!3!ol can\ more or less\
be considered as ubiquitous in aglycone fractions of
Lamiaceae0 family[ From our results it can be seen that

Copyright Þ 1999 John Wiley + Sons\ Ltd[ Flavour Fra`r[ J[ 1999^ 04] 089Ð083
 ESSENTIAL OIL OF ORIGANUM VULGARE 082

Table 2. Identified constituents and percentage com- 080"39#\ 036"13#\ 017"7#\ 092"5#\ 62"78#[ b!anomer of
position of volatile aglycones from Origanum vulgare L. glucopyranose] M¦ 324"1#\ 294"1#\ 180"1#\ 106"06#\
ssp. hirtum (Link) Ietswaart isolated by two different 193"099#\ 080"27#\ 078"3#\ 036"10#\ 017"5#\ 092"5#\
methodsa 62"66#\ a!anomer of galactopyranose] M¦ 324"0#\
Peak Compound Peak area ")# Methods of 294"2#\ 154"1#\ 106"16#\ 194"10#\ 193"099#\ 080"37#\
No[ identi_cation 036"11#\ 017"00#\ 092"5#\ 62"67#\ b!anomer of gal!
A B
actopyranose] M¦ 324"2#\ 250"1#\ 208"3#\ 107"00#\
0[ 2!Hexen!0!ol tr 2[3 I0\ I1\ MS 106"49#\ 194"11#\ 193"099#\ 081"07#\ 080"82#\ 036"23#[ In
1[ 0!Octen!2!ol 4[3 0[2 I0\ I1\ MS
2[ Benzaldehyde 9[7 9[8 I0\ I1\ MS
general\ galactose is rarely found as the glycone moiety
3[ Terpinen!3!ol 9[5 Ð I0\ I1\ MS of glycosidically bound volatiles[ This monosaccharide
4[ Thymoquinone 36[9 39[1 I0\ I1\ MS unity was identi_ed as glycone part in glycosides of
5[ Methyl salicylate Ð 9[5 I0\ I1\ MS
Thymus vul`aris "Lamiaceae#\08 as well as the disac!
6[ Benzyl alcohol 1[9 7[8 I0\ I1\ MS
7[ 1!Phenyl ethanol 9[7 4[5 I0\ I1\ MS charide part in glycosides of b!D!glucopyranosyl!"0!1#!
8[ p!Cymen!7!ol 9[7 0[1 I0\ I1\ MS b!D!galactopyranose in Coleus forskohlii "Lamiaceae#[14
09[ Eugenol 9[3 Ð I0\ I1\ MS
00[ Thymol 5[7 6[4 I0\ I1\ MS Acknowled`ements * This work was supported by Croatian National
01[ Carvacrol 4[4 0[8 I0\ I1\ MS Grant\ Project 900!992[
02[ 0!H!Indole 1[1 9[7 I0\ I1\ MS
03[ 1!"p!Metoxyphenyl# ethanol Ð 0[2 Ð\ Ð\ MS
04[ Butyl phtalate monoester 2[9 tr Ð\ Ð\ MS

Total 64[2) 63[9) References

a
Methods used] A!Percolation with ethyl acetate and enzymatic hydrolysis^
B!Extraction with water during hydrodistillation and enzymatic hydrolysis[
I0  retention indices on HP!19M^ I1  retention indices on HP!090^
MS  mass spectra^ Ð  Not detected^ tr  trace ³ 9[0)[
most of them were not identi_ed among the oregano
aglycone compounds[
Carbohydrates
After removing the aglycones from aqueous solutions
the released carbohydrates were identi_ed by TLC on a
silica gel with solvents of di}erent polarity[ After the
enzymatic hydrolysis of the glycosides D!"¦#!glucose as
the main glycone and one unknown disaccharide was
found\ RF!values of disaccharide in di}erent solvents
were similar to those of "¦#!lactose[ The most e}ective
mobile phase was n!buthanol ] acetic acid ] ether ] water
8 ] 5 ] 2 ] 0 v:v:v with the following RF values] D"¦#!
glucose 9[33\ D"¦#!galactose 9[28\ "¦#!lactose 9[19[ The
acidic hydrolysis of isolated disaccharide produced\
after enzymatic hydrolysis\ D!"¦#!glucose and D!"¦#!
galactose "in 0 ] 0 ratio#[ The same monosaccharides
were identi_ed after a direct acidic hydrolysis of gly!
cosides "without previous enzymatic hydrolysis#[ The
acidic hydrolisate contained D!"¦#!glucose "77)# and
D!"¦#!galactose "01)#[ The agylcones obtained by
acidic hydrolysis of glycosides contained many artefacts
"due to reactions of elimination and:or rearrangement#
and were therefore not further investigated[
Finally\ D!"¦#!glucose and D!"¦#!galactose were
identi_ed by GC!MS as trimethylsilylethers on HP!090
column by coinjection with authentic compounds[ The
anomers of glucose and galactose gave the following
ion fragments\ m:z ")# a!anomer of glucopyranose]
M¦ 324"1#\ 282"1#\ 294"2#\ 180"1#\ 106"06#\ 193"099#\
0[ Stahl!Biskup E\ Intert F\ Holthuijzen J\ Stengele M\ Schulz G[
Flavour Fra`r[ J[ 0882^ 7] 50[
1[ Sakata I\ Mitsui T[ A`ric[ Biol[ Chem[ 0864^ 28] 0218[
2[ Sakata I\ Koshimizu K[ A`ric[ Biol[ Chem[ 0867^ 31] 0848[
3[ Martinkus C\ Croteau R[ Plant Physiol[ 0870^ 57] 88[
4[ Lang E\ Horster H[ Planta Med[ 0866^ 20] 001[
5[ Skopp K\ Horster H[ Planta Med[ 0865^ 18] 197[
6[ Sasaki H\ Tagushi H\ Endo T\ Yoshioka I\ Itaka Y[ Chem[ Pharm[
Bull[ 0870^ 18] 0525[
7[ Cubo M\ Sasaki H\ Endo T\ Tagushi H\ Yoshioka I[ Chem[
Pharm[ Bull[ 0875^ 230] 2986[
8[ Francis MJO\ Allcock C[ Phytochem[ 0858^ 7] 0228[
09[ Willis RDH\ Scriven FM[ Phytochem[ 0868^ 07] 674[
00[ Tackeda Y\ Fukomoto K\ Tachibana M\ Shingu T\ Tetsuro F\
Ichihara T[ Phytochem[ 0889^ 18] 0480[
01[ Takeo T[ Phytochem[ 0870^ 19] 1034[
02[ Banthorpe DV\ Mann J[ Phytochem[ 0860^ 00] 1478[
03[ Myakado M\ Ohno N\ Kirai H\ Yoshioka H[ Phytochem[ 0863^
02] 1770[
04[ Tschesche R\ Ciper F\ Breimaier E[ Chem[ Ber[ 0866^ 009] 2000[
05[ Huiying L\ Shoushen L\ McCabe T\ Clardy J[ Planta Med[ 0873^
49] 490[
06[ Krauss R\ Spiteler G[ Phytochem[ 0880^ 29] 0192[
07[ Inoshiri S\ Saiki M\ Kohda H\ Otsuka H\ Yamasaki K[ Phyto!
chem[ 0877^ 16] 1758[
08[ Mulkens A[ Pharm[ Acta Helv[ 0876^ 51] 118[
19[ Konoshima T\ Sawada T[ Chem[ Pharm[ Bull[ 0873^ 21] 1506[
10[ Williams PJ\ Strauss CR\ Wilson B\ Massy!Westropp R[ Phyto!
chem[ 0871^ 10] 1902[
11[ Nagao T\ Okabe H\ Yamagauchi T[ Pharm[ Bull[ 0877^ 25] 460[
12[ Huiying L\ Shoushen L\ McCabe T\ Clardy J[ Planta Med[ 0873^
49] 490[
13[ Bredenkamp MW\ Drewes SE\ Wenther GL[ Phytochem[ 0878^
17] 152[
14[ Ahmed B\ Vishwakarma RA[ Phytochem[ 0877^ 16] 2298[
15[ Kokkini S\ Karousou A\ Dardioti A\ Krigas N\ Lanaras T[ Phy!
tochem[ 0886^ 33] 772[
16[ Russo M\ Galletti GC\ Bocchini P\ Carnacini A[ J[ A`ric[ Food
Chem[ 0887^ 35] 2630[
17[ Lagouri V\ Blekas G\ Tsimidou M\ Kokkini S\ Boskou D[ Z[
Lebensm Unters Forsch[ 0882^ 086] 19[
18[ Kokkini S\ Vokou D[ Biochem[ Syst[ Ecol[ 0882^ 10] 286[
29[ Kokkini S\ Karousou R\ Vokou D[ Biochem[ Syst[ Ecol[ 0883^
11] 406[
20[ Vokou D\ Kokkini S\ Bessiere JM[ Biochem[ Syst[ Ecol[ 0882^ 10]
176[
21[ Merkx YM\ Baerheim Svendsen A[ Planta Med[ 0878^ 44] 77[

Copyright Þ 1999 John Wiley + Sons\ Ltd[ Flavour Fra`r[ J[ 1999^ 04] 089Ð083
083 J[ MASTELIC AND I[ JERKOVIC
ł \ M[ MILOS ł

22[ Mastelic J\ Milos² M\ Kus²trak D\ Radonic A[ Croat[ Chem[ Acta[
0887^ 60] 036[
23[ Mastelic J\ Kus²trak D[ Acta Pharm[ 0886^ 36] 022[
24[ Bieganowska ML\ Petruzynik A[ Chem[ Anal[ "Warsaw# 0886^
31] 235[
25[ Soczewinski E\ Wojciah M\ Pachowicz K[ Chem[ Anal[ "Warsaw#
0887^ 32] 712[
26[ Franken!Luykx JMM\ Klopper WJ[ Brauwissenschaft 0856^ 19]
062[

Copyright Þ 1999 John Wiley + Sons\ Ltd[ Flavour Fra`r[ J[ 1999^ 04] 089Ð083