Bioconversion of Ferulic Acid to Volatile Phenols by Yeasts
1059
region of the internal transcribed spacer 2 (hereafter, the
ITS sequence). Further, we discuss the conversion
pathway of FA by new isolates.
Pichia guilliermondii species complex proposed by
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Vaughan-Martini et al., they were divided into three
species, (i) P. guilliermondii (its anamorph: Candida
guilliermondii), (ii) Candida carpophila, and (iii) Pichia
caribbica (its anamorph: Candid fermentati). Since the
sex of the seven isolates could not be observed, the
species names of the anamorph of the teleomorphic
species are used in this study. The 26S rDNA sequences
of the five strains, B-2, B-3, I-5, I-6, and K-3, were
identical with that of the type strain of C. fermentati
(AY187283), and their ITS sequences were also iden-
tical with that of the type strain of C. fermentati
YPG agar medium (1% yeast extract, 1% polypep-
tone, 2% glucose, and 2% agar) and the same medium
with the addition of 15% NaCl (hereafter, YPG + 15%
NaCl medium) were used for isolation of yeasts from
soy sauce mashes and miso pastes. Table 1 shows a list
of yeast strains used in this study. HPLC chromatograms
of the bioconversion of FA to 4-VG and 4-EG are
summarized in Fig. 1.
The results of convertibility of FA to 4-VG and to 4-
EG and of 4-VG to 4-EG are summarized in Table 1.
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(AB032175). Therefore, they were identified as C. fer-
mentati (anamorph of P. caribbica). On the other hand,
the 26S rDNA sequences of I-4 and K-2 were identical
with that of the type strain of P. guilliermondii
(U45709), and their ITS sequences were also identical
with that of the type strain of P. guilliermondii
T
Twelve strains of C. versatilis (IFO 10056 , JCM 5974,
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2
M2, 15M2, 25M2, 30M2, 33Z1, J5M2, Miso 19, Miso
2, Miso 83, and OH3G1) showed convertibility of FA
to 4-EG, including that of 4-VG to 4-EG (designated
type 1 FA conversion), in agreement with previous
results5 for C. versatilis strains. However, two strains
)
8)
(AY939792). Therefore, they were also identified as
(
JCM 5958 and KS05) of C. versatilis did not show
C. guilliermondii (anamorph of P. guilliermondii).
Division of the isolates into two species, C. fermentati
and C. guilliermondii, based on the results of the 26S
rDNA and the ITS sequence analyses, is closely
correlated to typing among convertibility of FA to 4-
VG and those of the both compounds to 4-EG, as shown
in Table 1. Thus, since the seven strains can be clearly
divided into the two species by the convertibility of FA
as well as by the results of 26S rDNA and ITS sequence
analyses, the bioconvertibility of FA can be a useful tool
for differentiation between C. fermentati and C. guil-
liermondii, which are indistinguishable on the basis of
convertibility of FA to 4-EG, but did show convertibility
of 4-VG to 4-EG (designated type 2 FA conversion). On
the other hand, three strains of C. etchellsii (9Z1, 25Z1,
and 33Z2) showed convertibility of FA to 4-VG, but did
not show convertibility of 4-VG to 4-EG (designated
type 3 FA conversion). This is the first finding of a
C. etchellsii strain with convertibility of FA to 4-VG.
T
The other four strains of C. etchellsii (IFO 1592 , Miso
0
201, Miso 0208, and Miso 0209) showed neither
convertibility of FA to 4-VG or to 4-EG, nor convert-
ibility of 4-VG to 4-EG (designated type 4 FA
5
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8)
conversion) in agreement with previous results for
C. etchellsii strains. Five strains newly isolated from
miso pastes and miso-koji, B-2, B-3, I-5, I-6, and K-3,
showed convertibility of FA to 4-EG (corresponding to
type 1 of FA conversion). However, two strains isolated
from miso-koji, I-4 and K-2, showed convertibility of
FA to 4-VG, but not of 4-VG to 4-EG (corresponding to
type 3 of FA conversion).
The FA decarboxylase and 4-VG reductase activities
of the strains tested (type 1 to type 4), which were
examined by the method described in footnote ( 4) in
Table 1, and the bioconvertibilities are summarized in
conventional phenotypical criteria.
Conversion of FA by microorganisms under NaCl-
free conditions has frequently been reported. For
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example, it is well known that some microorganisms,
1
0,11)
including wine and beer yeasts and yeasts isolated
1
from frozen concentrated orange juice, can convert
2)
1
0,11)
FA to 4-VG. Chatonnet et al.
found that only three
species, Brettanomyces intermedius, Dekkra interme-
dius, and Brettanomyces lambicus produce both cinna-
mate decarboxylase and vinylphenol reductase under
NaCl-free conditions such as wine making. However,
under high NaCl conditions such as the soy sauce and
miso fermentation processes, convertibility of FA by
microorganisms besides C. versatilis and C. etchellsii
and the degradation pathway have rarely been studied.
In this study, we first obtained four novel isolates with
different functions for conversion of FA and 4-VG in the
presence of 15% NaCl: (i) 4-VG producing strains of
C. etchellsii, (ii) 4-EG producing strains of C. versatilis
that did not convert FA but did convert 4-VG, (iii) 4-VG
producing strains of C. guilliermondii, and (iv) 4-EG
producing strains of C. fermentati. From success in
ꢀ
T
Table 1. C. versatilis IFO 10056 , 8M2, and C. fermen-
tati I-5 (type 1) had both FA decarboxylase and 4-VG
reductase activities, while C. versatilis JCM 5958 and
KS05 (type 2) had only 4-VG reductase activities.
C. etchellsii 9Z1 and C. guillermondii K-2 (type 3)
had only FA decarboxylase activites, while C. etchellsii
T
IFO 1592 and Miso 0208 (type 4) had neither FA
decarboxylase nor 4-VG reductase activities. This result
agrees with those for FA convertibility of the tested
strains.
The results of molecular identification of the isolates
carried out by analyses of the 26S rDNA sequence and
the ITS sequence are summarized in Table 1. The
seven isolates were identified as Picha guilliermondii.
Furthermore, according to the new classification of
5
,6)
obtaining novel isolates and from previous studies,
four types of conversion mode of FA were identified:
type 1, 4-EG production by conversion of FA and 4-VG,
in most C. versatilis strains and C. fermentati; type 2, 4-
EG production by conversion of 4-VG, in atypical
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