FLAVOUR AND FRAGRANCE JOURNAL

Flavour Fra`r[ J[ 1999^ 04] 219Ð211 "1999#

Volatile flavour compounds from oyster mushroom

(Pleurotus florida) in submerged culture
G. Venkateshwarlu,1$, M. V. Chandravadana,1 Meera Pandey,1 R. P. Tewari1 and Y. Selvaraj1
1
Indian Institute of Horticultural Research, Hessaraghatta Lake Post, Bangalore 560 089, India

Received 10 February 1999

Revised 14 April 1999
Accepted 14 April 1999

ABSTRACT] The ~avour extract from the mycelial pellets of oyster mushroom "Pleurotus ~orida# grown in
submerged culture was isolated by simultaneous distillation and solvent extraction under reduced pressure[
Nineteen volatile ~avour compounds were identi_ed after analysing the extract by capillary gas chromatography[
Among the most abundant compounds were anisaldehyde\ 2!methyl!0!butanol\ 2!methyl!0!propanol\ ben!
zaldehyde and 0!octen!2!one[ The ~avour pro_le from P[ ~orida in submerged culture was found to be sig!
ni_cantly di}erent from that of mushrooms grown on natural substrate[ The e}ect of addition of precursors like
phenylalanine\ linoleic acid and linolenic acid on production of volatile ~avour compounds by oyster mushroom
in submerged culture is discussed[ Copyright Þ 1999 John Wiley + Sons\ Ltd[

KEY WORDS] Pleurotus ~orida^ submerged culture^ precursors^ volatile aroma compounds

Introduction Experimental
Mushrooms have attracted attention as potential sour! Materials
ces to produce natural ~avours through biotech!
The mushroom Pleurotus ~orida was grown on paddy
nological utilization of their mycelium by submerged
straw6 at the Indian Institute of Horticultural Research\
culture[0\1 The technique of growing mushroom
Hessaraghatta\ Bangalore[ On the peak day of ~ush\
mycelium in liquid medium is being attempted for a
mushrooms of uniform size and maturity were har!
rapid and controlled production of ~avour components[
vested for the study[
The array of ~avour compounds produced by several
The mycelium of P[ ~orida was pre!cultured in 149
species of basidiomycetes cultured in liquid medium has
ml conical ~asks containing 099 ml synthetic medium\
been reported[0\2Ð5 However\ the published literature did
containing glucose 29 g:l\ asparagine 3[4 g:l and yeast
not encompass articles providing direct correlation of
extract 2 g:l[ It was allowed to grow for 14 days on a
aroma compounds produced by a species in synthetic
shaker kept at 049 rpm and 29>C[ This culture was
liquid medium as against those formed when grown on
then inoculated into 099 ml sterile medium and cultured
traditional solid substrates[ Such knowledge enhances
under the same conditions as pre!culture[
understanding of the metabolic capabilities of basidio!
mycetes for ~avour production under altered culture
conditions[ With this background\ the present study Isolation of Volatile Flavour Compounds
elucidates the pro_le of volatile ~avour compounds gen!
After a culture period of 29 days\ the mycelial pellets
erated by Pleurotus ~orida in submerged culture and its
grown from _ve ~asks were pooled and macerated using
comparison with the mushrooms grown on a natural
a pestle and mortar[ The homogenate was left for 04
solid substrate[
min to maximize the enzymatic production of ~avour
compounds[7 The homogenized material\ along with
culture _ltrate\ was subjected to simultaneous steam
 Correspondence to] Y[ Selvaraj\ Indian Institute of Horticultural Research\
Hessaraghatta Lake Post\ Bangalore 459 978\ India[ E!mail]
distillation and solvent extraction under reduced pres!
rootÝiihr[kar[nic[in[ sure in Likens and Nickerson!type apparatus^ using
Contribution No[ 092:88 of IIHR\ Bangalore 459 978\ India[
$
Present address] Division of Biochemistry\ Central Institute of Fisheries Edu!
diethyl ether as extraction solvent\ as described by Ven!
cation\ Versova\ Andheri "W#\ Mumbai!399 950\ India[ kateshwarlu et al[09 The ~avour extra was dried over

Copyright Þ 1999 John Wiley + Sons\ Ltd[

 VOLATILE FLAVOUR COMPOUNDS OF PLEUROTUS FLORIDA 210
sodium sulphate "anhydrous# and concentrated to 9[0
ml at 24>C bath temperature[ A recovery of 79) of
added n!heptanol was realized by this method[ This
procedure was replicated thrice[
Effect of Addition of Precursors
In experiments involving added precursors\ the requisite
quantity of L!phenylalanine was added to the culture
medium before autoclaving and linoleic and linolenic
acids "as sodium salts# were added after autoclaving the
medium but before the inoculation of mycelial cells[
Gas Chromatography
Analysis was performed on a Perkin!Elmer Autosystem
XL gas chromatograph _tted with 29 m×9[14 mm i[d[
PE!WAX "stationary phase\ polyethylene glycol# and
PE!0 "stationary phase\ dimethylpolysiloxane# fused sil!
ica capillary columns "Perkin!Elmer\ USA#[ The oper!
ational conditions were] 59>C for 3 min\ then
temperature raised to 199>C at a rate of 4>C:min and
held at 199>C for 4 min[ Injector and detector "FID#
temperatures were 129>C and 159>C\ respectively^ car!
rier gas\ N1 at a pressure of 9[57 Bar "09 psi#^ split ratio
0 ] 099^ sample size\ 9[4 ml[ The following standards\
procured from Sigma and Aldrich Chemical Company\
USA\ were chromatographed under identical con!
ditions^ 0!octen!2!ol\ benzaldehyde\ n!hexanal\ benzyl
alcohol\ n!octanol\ phenyl ethanol\ benzyl acetate\ ani!
saldehyde\ 2!methyl!0!butanol\ 1!methyl!0!propanol
and hexanol[ The volatile compounds were identi_ed
based on retention times and Kovats indices00 calculated
using n!alkane "C!7 to C!21# standards "Sigma\ USA#[
Assessment of absolute amounts was made by an
internal standard method using PE Nelson Turbochrom
software\ the standard being L!citronellol added to the
sample after distillation[ A sample size of 9[4 ml con!
tained 29 mg L!citronellol[
Results and Discussion
P[ ~orida mycelium\ when grown in synthetic liquid
medium\ produced a sweet anise and almond!like
odour\ in contrast to the characteristic mushroom
odour of fully!grown sporophores of the species[ In the
aroma concentrate of the culture broth after 29 days of
cultivation\ about 24 di}erent compounds were detected
by gas chromatography\ of which 08 were identi_ed[
The important compounds identi_ed were anis!
aldehyde\ 2!methyl!0!butanol\ 1!methyl!0!propanol\
benzaldehyde\ 2!methyl!1!pentanol\ n!hexanol\ 0!
Copyright Þ 1999 John Wiley + Sons\ Ltd[
Table 1. Concentration of volatile compounds in culture
media of Pleurotus florida
Component Concentration "mg:l#
n!Hexanal 09Ð54
1!Methyl!l!propanol 039Ð314
n!butanol 09Ð19
2!Methyl!1!pentanol 29Ð189
2!Methyl!0!butanol 014Ð0949
2!Octanone 04Ð19
0!Octen!2!one 14Ð109
n!Hexanol 09Ð19
1!Octenal 19Ð29
Ethyl anisate 04Ð19
0!Octen!2!ol 09Ð099
Benzaladehyde 19Ð119
n!Octanol 79Ð099
Ethyl benzoate 4Ð19
1\2!Butanediol 49Ð69
Benzyl acetate 09Ð209
Benzyl alcohol 09Ð029
Phenyl ethanol 04Ð29
Anisaldehyde 029Ð0349
Ranges includes the minimum and maximum concentrations of three rep!
lications studied[
 Identi_ed tentatively[
octen!2!one and benzyl acetate "Table 0#[ The con!
centration levels of anisaldehyde\ 2!methyl!0!butanol
and 1!methyl!0!propanol were in the ranges 029Ð0349\
014Ð0949 and 039Ð314 mg:l\ respectively[ Anisaldehyde
and 1!methyl!0!propanol were reported as major con!
stituents of Pleurotus sapidus species in liquid medium\0
while 2!hydroxy!1!butanone and 1\2!butanediol were
the major aroma compounds of Pleurotus sajorkaju[5
This is the _rst report on the aroma compounds gen!
erated by P[ ~orida in liquid medium[
Striking di}erences were observed in the composition
of aroma concentrate of liquid culture when compared
to that of mushroom sporophores[ The major aroma
compound of naturally grown P[ ~orida sporophores is
0!octen!2!ol "C!7 alcohol\ responsible for typical mush!
room odour#\ contributing to 54Ð69) of aroma con!
centrate[09 Surprisingly\ this was a minor constituent in
liquid medium "09Ð099 mg:l#[ The other C!7
compounds\ such as 0!octanol\ 2!octanone\ 2!octanol
and 0!octen!2!one\ which contribute signi_cantly to the
typical odour of mushroom sporophores\ were not pro!
duced in considerable amounts in liquid cultures[ In
mushrooms\ the C!7 volatile compounds are derived
enzymatically from unsaturated fatty acids by lipox!
ygenase\01 but in the present study\ when linoleic and
linolenic acids were added as precursors to the liquid
medium up to 499 ppm\ it did not result in enhanced
formation of C!7 ~avour compounds[
It has been reported that usually lipoxygenase cat!
alysed degradation of fatty acids occurs at a late phase
of cultivation\ due to increased contact of enzyme and
substrate as a result of enhanced cell lysis[ However\ it
was observed in this study that even when the cul!
Flavour Fra`r[ J[ 1999^ 04] 219Ð211
211 G[ VENKATESHWARLU ET AL[

tivation period was extended up to 49 days\ the synthesis Table 2. Effect of phenylalanine (500 ppm) addition on
of C!7 alcohols and ketones did not improve[ The myce! production of flavour compounds in 30 days-old liquid
lial pellets after 29 days of cultivation\ when subjected culture of Pleurotus florida
to shearing stress in the presence of linoleic acid\ also Compound Maximum concentration "mg:l#
failed to enhance the formation of 0!octen!2!ol[ This
Control Treatment
suggests that the conversion of linoleic acid to C!7 alco!
hols and ketones is impaired in liquid medium\ unlike Benzaldehyde 119 0459
Benzyl alcohol 029 434
in fresh mushrooms\ where lipoxygenase activity is pre! Benzyl acetate 209 054
dominantly responsible for production of the C!7 ~av! Phenyl ethanol 29 028
our compounds[ Anisaldehyde 0349 0523
Ethyl benzoate 19 61
Abraham and Berger0 investigated 19 strains of
basidiomycetes for generating aroma components by
submerged culture[ However\ except in the case of Sar!
codonta setosa\ 0!octen!2!ol was generated in con! ther\ along with enzyme co!factors\ to improve speci_!
centrations below 09Ð099 mg:l levels\ in spite of the fact cally the yields of C!7 ~avour compounds in liquid
that some species examined\ such as Pleurotus\ were cultures[
typical C!7 alcohol!generating basidiomycetes in
nature[02 This corroborates our _ndings in P[ ~orida[ Acknowled`ements * The authors express their thanks to the Direc!
tor\ I[I[H[R[\ for his interest in this work[ We thank Mr C[ S[ Bujji
In contrast\ many aromatic compounds\ such as ani! Babu\ Technical o.cer\ for assistance in carrying out this work[
saldehyde and benzaldehyde\ were found in liquid med!
ium at higher levels than are present in mushroom
sporophores[ The ability of the synthetic nutrient med! References
ium for the production of aromatic compounds was
further tested by supplementing L!phenylalanine to the 0[ Abraham BG\ Berger RG[ J[ A`ric[ Food Chem[ 0883^ 31] 1233[
1[ Lomascolo A\ Stentelaire C\ Asther M\ Lesage Meessen L[ Trends
medium[ Benzaldehyde concentration increased appre! Biotechnol[ 0888^ 06] 171[
ciably to 0459 mg:l\ as against the control\ 199 mg:l\ 2[ Grove JF[ Phytochemistry 0870^ 19] 1910[
contributing 29Ð39) in the aroma concentrate "Table 3[ Drawert F\ Berger RG\ Neuhauser K[ Eur[ J[ Appl[ Microbiol[
Biotechnol[ 0872^ 07] 013[
1#[ This suggested that the strain of P[ ~orida has a 4[ Kawabe T\ Morita H[ J[ A`ric[ Food Chem[ 0883^ 31] 1445[
pathway to metabolize L!phenylalanine to ben! 5[ Krings U\ Abraham BG\ Berger RG[ Perfum[ Flavor[ 0884^ 19]
zaldehyde more pronouncedly in liquid medium[ 68[
6[ Bano Z\ Srivastava HC[ Food Sci[ 0851^ 01] 252[
The ability of mycelium to produce aromatic com! 7[ Hong JS\ Lee JY\ Kim YH\ Kim MK\ Jung GR\ Lee KR[ Koreah[
pounds and its positive response to the addition of J[ Mycol[ 0875^ 03] 20[
precursors like phenylalanine\ revealed that it is possible 8[ Picardi SM\ Issenberg P[ J[ A`ric[ Food Chem[ 0862^ 10] 848[
09[ Venkateshwarlu G\ Chandravadana MV\ Tewari RP[ Flavour
to obtain large amounts of benzaldehyde and ani! Fra`[ J[ 0888^ 03] 080[
saldehyde by controlled cultivation of P[ ~orida in sub! 00[ Jennings W\ Shibamoto T[ Qualitative analysis of Flavour and
merged culture[ However\ since 0!octen!2!ol and 0! Fra`rance Volatiles by Glass Capillary Gas Chromato`raphy\ Aca!
demic Press] London^ 0879[
octen!2!one constitute most important components of 01[ Tressl R\ Bahri D\ Engel KH[ J[ A`ric[ Food Chem[ 0871^ 29] 78[
mushroom volatiles\ it is appropriate to investigate fur! 02[ Hader Y\ Dosoretz CG[ Trends Food Sci[ Technol[ 0880^ 1] 103[

Copyright Þ 1999 John Wiley + Sons\ Ltd[ Flavour Fra`r[ J[ 1999^ 04] 219Ð211