Biotransformation of Linalool by Botrytis cinerea

G. BOCK, I. BENDA, and P. SCHREIER

ABSTRACT Grape Juice

Biotransformation of linalool was studied using grape must and three
strains of Botrytis cinerea (.5901/2; 5909/l; 5899/4). Capillary gas
chromatography and combined capillary gas chromatography-mass + Terpene
spectrometry revealed predominant conversion (>90%) of linalool to
1
(E)-2,6-dimethyl-2,7-octadiene-1,6-diol. In minor concentrations
I Terpene alcohol I
(<lo%), the corresponding (Z)-isomer, 2-vinyl-2-methyl-tetrahydro-
furan-S-one, the four (E)-and (Z)-linalool oxides in their furanoid and 0
I I
pyranoid forms, the (E)- and (Z)-acetates of pyranoid linalool oxides Filtration of mycetium
as well as 3,9-epoxy-p-menth- 1-ene were identified.

INTRODUCTION
Solvent extraction
ALCOHOLIC BEVERAGES such as wine and beer are clas-
sical examples of biotechnological production of foods. During
Concentration
their processing, various enzyme activities of yeasts catalyze
the biotransformation of plant constituents leading to a com- I
plex mixture of flavor compounds, which determine, in a char- Extract
acteristic quantitative distribution, the quality of the final product
(Schreier, 1984). In winemaking, in certain cases the influence NaHC03 5 010
of a fungus, i.e. Botrytis cinerea, also has to be considered
(Dittrich, 1977; Krogh and Carlton, 1982). In an early, unripe I 1
state of maturation, the infection of grapes by B. cinerea is Aqueous phase Organic phere
I
feared, as the grapes become moldy. With fully ripe grapes,
however, the growth of B. cinerea is promoted, as grapes Acidification
infected by the “noble rot” deliver the famous sweet wines,
Dlethylether extraction LC preseparation
such as Sauternes of France, Tokay Aszu of Hungary, or
I
Trockenbeerenauslese wines of Germany.
CH2N2
The high metabolic activity of B. cinerea is well-known.
Thus, the formation of glycerol and gluconic acid as well as Mothylester I II III
citric acid in grape musts infected by B. cinerea has been
observed (Bertrand et al., 1976; Dittrich, 1977). As to the
volatiles of grapes and wines, some years ago, several key
L
HRGC . HRGC-MS -I
components were identified by our group (Schreier et al., 1976;
Schreier, 1984) consisting of terpenes and derived terpenoids. Fig. l-Scheme of sample preparation steps. l-grape juice vol-
As some of these compounds, including terpene alcohols such atiles; P-botrytized musts (3 strains, 590112; 590911; 589914); 3-
as, e.g., linalool, have been found to be affected by B. cinerea, addition of linalool to the botrytized musts; I-addition of linalool
in the course of our work on microbial transformation of vol- to the must. B.C. = B. cinerea.
atiles we dealt with the metabolization of terpene alcohols by
this fungus. It was the aim of this study to identify the struc-
Incubation conditions
tures of fungal metabolization products, which had not been
characterized in the previous work (Boidron, 1978; Boidron The sugar and acid content of the grape must (cultivar, Milller-
and Torn%, 1978; Shimizu et al., 1982). Thurgau) used were adjusted to 200 g/L and 8.5 g/L (= pH 3.5),
respectively. After sterilization (30 min at 110°C) the grape must (700
mL) was filled into 1 L-Erlenmayer flasks using 0.2 pm membrane
MATERIALS & METHODS filter. Each flask was inoculated with a pure B. cinereu strain and
incubated at 25°C for 2 wk. The mycelium was removed and the
Botrytis cinerea strains solutions analyzed by capillary gas chromatography (HRGC) and
combined capillary gas chromatography-mass spectrometry (HRGC-
The B. cinerea strains 5901/2, 5901/l, and S899/4, used in this MS). In like manner, blank tests without B. cinerea incubation, and
study, were obtained from the collection of the Bayerische Landesan- experiments after addition of 50 mg/L linalool to the must with and
stalt fur Weinbau und Gartenbau, Wiirzburg. From the original cul- without B. cinerea incubation were carried out (cf. Fig. 1). Addition-
tures, a part was transferred to malt agar slants and incubated at 25°C ally, in two control experiments, 2,6-dimethyl-3,7-octadiene-2,6-dial
for 7 days. and 2,6-dimethyl-1,7-octadiene-3,6-diol were added (10 mg/L) to the
botrytized must. All steps were performed under strictly sterile con-
ditions.

Author Benda is with the Bayerische Landesanstalt fur Weinbau

und Gartenbau, Residenzplatz 3, D-8700 Wiirzburg, West Ger-
many. Authors Bock and Schreier are with the Lehrstuhl fur
Lebensmittelchemie, Universitat Wiinborg, Am Hubland, D-8700
Wiirzburg, West Germany.
Isolation of volatiles and preseparation
After addition of internal standards (ethyl (E)-2-butenoate, 0.40 mg/
L; 4-methyl-1-pentanol, 0.49 mg/L) to the untreated and botrytized
musts solvent extraction was carried out using a pentane-dichloro-

Volume 51, No. 3, 198fZ-JOlJRNAL OF FOOD SCIENCE-659

BIOTRANSFORMATION OF LINALOOL. . .

Table I-Mass spectrometric data of linalool biotransformation products

M S data
Comoound m/z f%la
(E)-2,6-dimethyl-2,7-octadiene-l,6-diol (19) 43(100) 71(41) 67(38) 41(28)
55(26) 68(15) 82(6) 81(4)
(Zl-2,6-dimethyl-2,7-octadiene-1,6-diol (20) 431100) 71(U) 67151) 41(36)
55(30) 68(20) 82(12) El(E)
(Z)-linalool oxide, furanoid (21) 43125) 55(20) 59(100) 67(20)
68125) 93125) 94(35) ill(5)
(El-linalool oxide, furanoid (22) cf. (Z)-isomer
(Z)-linalool oxide, pyranoid (23) 41(26) 43(50) 59(80) 87(53)
68(100) 79(20) 78(19) 94(63)
(E)-linalool oxide, pyranoid (24) cf. (Z)-isomer
(Zblinalool oxide acetate, pyranoid (25) 41(10) 43(100) 5519) 59(11)
67(E) 68(17) 9318) 94(29)
(E)-linalool oxide acetate, pyranoid (26) cf. (Z)-isomer
3,9-epoxy-p-menth-1-ene (27) 137(100) 69(50) 41(37) lOS(30)
79(31) 91(15) 55(14) 93(12)
2-vinyl-2-methyl-tetrahydrofuran-5-one (28) 27(76) 41(35) 43(95) 55(78)
56(34) 67(52) 71(40) 111(100)
a The eight most intense peaks are represented.

3c

I /OH I I /OH I ‘; 2c

1 15 16 17
1c

9 10 11 12 13 14 a bed
Fig. 2-Structures of terpenoids chemically formed from lina- Fig. 3-Formation of P-methyl-l-propanol, 3-methyl-I-butanol
loo1 (11 under acidic conditions (experiment 1, Fig. 1). (2) 2,4(8)- and P-phenylethanol by three strains of B. cinerea (a = grape
p-m&thadiene; (3) p-myrcene;. (4j a-phellandrene; (5) a-terpi- must; b = 590112; c = 590911; d = 589914).
nene; (6) limonene; (7) P-phellandrene; (8) (Z)-ocimene; (9) y-
terpinene; (10) (El-ocimene; (11) p-cymene; (12) terpinolene;
(13) (E,Z)-alloocimene; (14) (E,E)-alloocimene; (15) a-terpineol; detector temperature was kept at 220°C. Volumes of 0.3 PL were
(16) 3,Fdimethyl-I-octene-3,7-diol; (17) 1,8-cineole; (18) 2,2,6- injected.
trimeth yl-2-vin yl-tetrah ydrop yran. Results of qualitative analyses were verified by comparison of HRGC
retention and mass spectral data with those of authentic reference
substances. Quantitative determinations were carried out by standard
methane mixture (2:l) as described by Drawert and Rapp (1968). controlled calculations using a Hewlett-Packard 3388 A laboratory
Acids were removed from the extracts by separation with 5% NaHC03 data system considering extraction yields and HRGC response factors.
solution, and neutral volatiles were carefully concentrated to 1 mL
using a Vigreux column (45°C). The concentrates were preseparated Capillary gas chromatography-mass spectrometry
into fractions of different polarity by adsorption chromatography on
silica gel with a pentane-diethyl ether gradient (60 mL/hr) (Idstein and Instrument: Finnigan M A T 44 quadrupole mass spectrometer cou-
Schreier, 1985). Fraction I: 200 mL pentane; fraction II: 200 mL pled by an open-split interface with a Varian Aerograph 1440 equipped
pentane + diethyl ether (9 + 1 v/v); fraction III: 200 mL diethyl ether. with a water-cooled on-column injector. A J & W DB-Wax (30 m x
The eluates were dried over anhydrous sodium sulfate and carefully 0.32 m m i.d.; df = 0.25 pm) fused silica capillary column connected
concentrated using a Vigreux column (45°C) to 0.1 mL for HRGC to a 2m uncoated piece of fused silica capillary column as the “re-
and HRGC-MS analysis. tention gap” (Grob and Miiller, 1982) was used. The conditions were
as follows: temperature, isothermal for 5 min at 60°C and then from
60” to 240°C at 5Ymin; carrier gas flow rate, 2.5 mL/min He; tem-
Capillary gas chromatography (HRGC) perature of ion source and all connection parts, 200°C; electron en-
ergy, 70 eV; cathodic current, 0.8 mV; injection volumes, 0.3 JLL.
Instrument: Carlo Erba Fractovap 4160 fitted with a flame ioniza-
tion detector (FID) and an air-cooled on-column injector. Column: J Reference compounds
& W DB-Wax (30 m X 0.32 m m i.d.; df = 0.25 pm) fused silica
capillary, connected with a 2m uncoated fused silica precolumn (“re- The preparation of (E)- and (Z)-2,6-diiethyl-2,7-octadiene- 1,6-diol
tention gap”) (Grob and Miiller, 1982). On-column injection was was performed by SeOz oxidation of linalool according to Behr et al.
used. The temperature program was isothermal for 2 min at 5O”C, (1978). The M S data are outlined in Table 1. Syntheses of 2,6-di-
then 50” to 240°C at 5”CYmin. The flow rates for the carrier gas were methyl-3,7-octadiene-2,6-diol [m/z (o/o): 43(100)-82(84)-71(70)-67(48)-
2 mL/min He, for the make-up gas 30 mUmin N2 as well as for the 41(23)-55( 16)-85(5)-81(3)] and 2,6-dimethyl-1,7-octadiene-3,6-dial
detector gases 30 mL/min Hz and 300 mL/min air, respectively. The [m/z (o/o): 43(100)-67(78)-71(62)-55(41)-41(40)-82(36)-68(26)-69(15)]

660-JOURNAL OF FOOD SCIENCE-Volume 51, No. 3, 1986

 \9 r
3
OH OH

”... H
27
0
J

29
OH
Q \

30
I
OH

.:= .:=
HO 0 no3 oh
-’ 0 o&k
+
21 22 28
1

OH I

23 24

5
OH

I
I +
vo... --
I
-f” ,.:= ‘d ._.. I - P CH,OH
9 0 0 0 1
>@I% x5%
25 26 1
Fig. 4-Structures of terpenoids formed from linalool (1) by 6. 19 20
cinerea. (19),(20) (EJ- and (ZJ-2,bdimeth yl-2,7-octadiene- 1,Sdiol;
(2 1),(22J (ZJ- and (EJ-linalool oxides, furanoid; (231,124) (ZJ- and
(EJ-linalool oxides, pyranoid; (25),(26) (ZJ- and (El-linalool oxide
acetates, pyranoid; (27) 3,9-epoxy-p-menth-I-ene; (28) P-vinyl-
2-methyl-tetrahydrofuran-&one.
I

were carried out by photooxygenation of linalool followed by mild

reduction (Kjesen and Liaaen-Jensen, 1973).

RESULTS & DISCUSSION

IN THE LINALOOL control experiment (line 4 in Fig. 1)
linalool (1) underwent a variety of well-known chemical re-
actions [hydrolysis, deprotonation, hydration, cyclization (Mot-in
and Richard, 1985)] leading to a series of hydrocarbons (2)-
(14) as well as a-terpineol (15), 3,7-dimethyl-1-octene-3,7-
diol(16), 1,8-cineole (17) and 2,6,6-trimethyl-2-vinyl-tetrahy-
dropyran (18) shown in Fig. 2.
When B. cinerea was added to the grape must (line 2 in
Fig. 1) higher alcohols originating from the amino acid me-
tabolism, such as 2-methyl-1-propanol, 3-methyl-1-butanol and
2-phenylethanol, were found as metabolic products from B.
cinerea (Fig. 3). There were distinct quantitative differences
depending on the strain used. With additional control experi-
ments it could be demonstrated that these alcohols were formed
exclusively by B . cinerea and not by an eventual contamination
by yeasts. This fact has to be stressed as contradictory results
have been published (Bertrand et al., 1976; Dittrich, 1977).
After addition of linalool to the botrytized must (line 3 in
Fig. I), a series of transformation products was identified by
HRGC and HRGC-MS (Fig. 4). The conversion products com-
prised (E)- (19) and (Z)-2,6-dimethyl-2,7-octadiene-1,6-diol
(20), the furanoid (Z)-(21) and (E)-linalool oxides (22), the
pyranoid (Z)- (23) and (E)-linalool oxides (24), the isomer
acetates of the latter ones (25)) (26)) 3,9-epoxy-p-menth- 1 -ene
(27) and 2-vinyl-2-methyl-tetrahydrofuran-S-one (28). The MS
data of these fungal conversion products of linalool are pre-
sented in Table 1. Quantitative HRGC showed that linalool
was predominately (>90%) metabolized to (E)-2,6-dimethyl-
2,7-octadiene-1,6-diol (19) by B. cinerea; the compounds (20)-
(28) were only found as by-products in minor concentrations.
As to the possible biogenetic pathways of metabolic prod-
ucts formed from linalool by B. cinerea, the terpene diols 2,6-
dimethyl-3,7-octadiene-2,6-diol(29) and 2,6-dimethyl-1,7-oc-
27
g. 6-Scheme of biogenetic formation of (19) and (20) by di-
r&t o-hydroxylation of linalool (I) and exclusion of dials (29)
and (301 as precursors of (19) and (20). Formation of (27) by
allylic rearrangement and cyclization from (19) (Kitagawa et al.,
1983).
tadiene-3,6-diol (30) (Fig. 5), both detected among the natural
grape must constituents (Fig. 1, l), might function as inter-
mediates in the formation of 2,6-dimethyl-2,7-octadiene-1,6-
diol (19). However, in control experiments, in which the diols
(29) and (30) were added to the botrytized must instead of
linalool, no formation of (19) or (20) could be observed. Con-
sequently, for the production of (E)-(19) a direct enzymic w-
hydroxylation of linalool can be considered, as previously pro-
posed for analogous reactions of bacterial metabolization of
linalool (Fig. 5) (Devi et al.; 1977; 1978; Madyastha, 1984).
Contrary to the above-mentioned odorless diols (29) and (30),
2,6-dimethyl-2,7-octadiene-1,6-diol is an odoriferous com-
pound, useful in perfume and flavor industry (Jap. Pat., 1983).
As to the by-products of linalool transformation by B. ci-
nerea the formation of 3,9-epoxy-p-menth-1-ene (27), the
character impact compound of fresh dill herb (Schreier et al.,
1981), can be understood by allylic rearrangement of (E)-2,6-
dimethyl-2,7-octadiene-1,6-diol (19) to the corresponding hy-
droxygeraniol (-nerol), which is known to undergo cyclization
to the epoxy derivative (27) under acidic conditions (Kitagawa
et al., 1983).
For the formation of isomer hydroxy ethers (21)-(24), the

Volume 51, No. 3, 19864OlJRNAL OF FOOD SCIENCE-661

I BlOTRANSFORMATlON OF LINALOOL.. .
3
Q
OH OH
I - I-
I acetates (25),(26) from linalool (1).
x
1 31
23
25
so-called linalool oxides, the diastereoisomers of 6,7-epoxy-
linalool(3 1) have been proposed as biogenetic precursors (Fig.
6) (Ohloff et al., 1985). Recently, in our studies of the pre-
cursors of papaya fruit volatiles, the two 6,7-epoxy-linalool
isomers have been detected as natural constituents of this fruit
(Winterhalter et al., 1986). Due to the acidic nature of the
medium (pH 3.5) in the present study, the detection of the
labile epoxy derivatives (31) could not be expected. From the
hydroxy ethers (23) and (24) the formation of acetates is easy
to understand.
Finally, the lactone (28) is generally regarded as a formal
oxidation product of furanoid linalool oxides (21) and (22),
but the biogenetic formation pathway of this compound has
not been elucidated as yet.
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MS received 8130185;revised 12/17/85; accepted 12/19/85.
The authors thank Dr. Imenz Nei&adt for providing different B. cinereastrains.