I;(/ CltnmX~ric.Vol.13No. 3.pp.343-347. 19x5
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0278-69 I S/85 t3.00
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Copyright0 1985Pergamon
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MUTAGEN CONTENT OF WINES CONTAMINATED
COMMON YEASTS
WITH
R. E. SURDENand A. KRIZUS
Department of Botany and Genetics
and
D. JOSEPHY
Department of Chemistry,University of Guelph, Guelph. Ontario NlG ZWI, Canada
(Receiued
Abstract-Wines
Contaminant
yeast
exhibiting
species
a microbial
were cultured
4 June
1984)
haze were collected
in grape musts under
and their
controlled
microbial
conditions
contents
identified.
and extracts
of the
resultant wines were assayed for mutagen content using four strains of Salmonella in the Ames
Salmonella/microsomc/faecalase
system.The
wine extracts
exhibited
some
mutagen
content.
It would appear that mutagen
content
is more a function
preparation
than of microbial
metabolism
during fermentation.
INTRODIJCITON
Many naturally occurring products (MacGregor &
Jurd. 1978) and fruit juices (Mazaki, Ishii & Uyeta.
1982)
contain
mutagenic
constituents.
Wines
and
other fermented fruit juices (Lin & Tai, 1980; Stolz,
Stavric, Krewski et al. 1982; Tamura, Gold, FerroLuzzi & Ames, 1980) and their distillates (Lee &
Fong. 1979; Loquet, Toussaint & LeTalaer, 1981;
Nagao, Takahashi, Wakabayashi & Sugimura, 1981)
have also been shown to contain mutagens.
Correlations between certain alcoholic beverages and
specific cancer types such as nasopharyngeal and
oesophageal cancers have been demonstrated
(Loquet et al. 1981; Warwick & Harington, 1973). It
has also been demonstrated that certain moulds and
contaminants growing on grapes (Scott, Fuleki &
Harwig, 1977) and other fruits (Scott & Stoltz, 1980)
produce substances with mutagenic activity.
About 20% of the world’s annual production of
3.0 x IO’ litres of wine is made at home with little
regard for asepsis. Much of the wine contains
microbial contamination or chemical faults such as
those resulting from oxidation or the presence of
mercaptoethanol. Most homemade wine is consumed
regardless of its organoleptic faults. It is therefore of
interest to inquire into the identity of the contaminants and the mutagenic nature of their metabolites.
This paper reports on the incidence of mutagens in
wines contaminated with yeasts other than
Saccharornyces
cereuisiae.
Commonly found yeasts
contaminating homemade wines were isolated and
identified. Type cultures of the commonly found
contaminants were grown on white wine musts that
contained little or no natural mutagens. Extracts of
the contaminated wines were tested in the Ames
Salmonella/microsomaI mutagen assay.
EXPERIMENTAL
Isolariorl
ad
ider~tijcation
of yeasts.
The contaminated wines were brought into our lab by local
home wine makers. The homemade wines were made
from fresh grapes imported from California, fresh
343
toxicity
to Salmonella
but no
of the grape species and must
Ontario grapes, juice from California, Germany or
Spain, or concentrate from Spain or California. Only
grape wines with a marked cloudiness or obvious
odour or taste defect were tested. Samples of 0.01,0.2,
1.0 and 100 ml of wine were filtered through a 0.45
llrn filter and cultured on WL medium (Difco).
Single clones or isolated colonies were picked off
the filter paper and streaked prior to analysis on an
API 2OC clinical yeast identification strip. Profile
numbers were obtained and compared with those of
known wine-contaminating yeasts, and supplementary assimilation and fermentation tests were performed as required (Subden, Cornell & Noble, 1980).
bfrres ord sample
preparation.
To study the mutagenic metabolites of the contaminants and to exclude
the effects of wine mutagens it was necessary to
obtain a wine with little or no mutagen content.
Becausewhite wines in general h:.ve a lower mutagen
content than red wines (Subden, Haffie & Rancourt,
1983; Subden, Krizus & Rancourt, 1984; Tamura et
al. 1980) the white grape variety “Palomino” was
selected. Wines made from Palomino and Foch
grapes were used to test activation and toxicity.
Wine made from Concord grapes was used to test the
efficacy of the extraction and activation procedures.
The Palomino juice from California was obtained
commercially. The Concord and Foch grapes were
grown in the Niagara peninsula using pathogencontrol spray programmes recommended by the
Ontario Ministry of Agriculture and Food. The
harvested grapes were washed, and given an initial
sulphuring with 100 mg K&O&g
grapes. Musts
were chaptalized to a sugar level of 210 g/litre and
inoculated with yeast strain VI80 (obtained from R.
Chudyk, Ontario Ministry of Agriculture and Food)
to give an initial concentration of lo8 cells/ml. The
red Concord wine was fermented on the skins until it
contained approximately 80 g residual sugar/litre
must (5’ Brix), and was then pressed. Finished wines
were given a final sulphuring, to give 30 mg free
SO,/litre, and were then bottled.
The XAD-2 apolar resin (Sigma Chemical Co., St
Louis, MO, USA) was washed five times with IO vols
344
R. E SUBDEN
acetone followed by ov wash with methanol and one
with distilled water. The resin was then poured into a
battery of a dozen 0.7 cm x 10 cm Econo colums
(Bio-Rad Laboratories, Mississauga, Ontario) to a
volume of 1.5 cm3 in each. Columns were then
washed with 50 ml water. Excesswater was flushed
out with COZ. Wine (50 ml) was applied to each
column and eluted at 2 ml/min; then each column
was washed with 1.5 ml distilled water and flushed
with CO,. Adsorbed compounds were eluted with
5ml acetone and dried at 65°C. The residue was
redissolved in 0.2 ml spectroscopy-grade dimethylsulphoxide/50ml wine and all fractions were pooled
and used immediately or stored at 4°C.
Mutagenicity
assay.
Salmonella
typhimurium
strains TA97, TA98, TAlOO and TAlO2 were obtained from Professor B. N. Ames (University of
Cahfomia, Berkeley). Genotypes were confirmed
according to the method of Ames, McCann &
N-Methyl-N’-nitro-N-nitroYamasaki
(1975)
soguanidine and nitrofluorene were used as positive
controls. S-9 hepatic enzyme in KC1 was purchased
from Inter-Medico (Willowdale, Ontario) and its
activity was confirmed by assaying the S-9 activation
of 2-aminoanthracene (de Serres & Shelby, 1979).
Faecalase preparation and activation assays were
those described by Subden et al. (1983). Spot tests
(Ames et al. 1975) were performed on extracts of
white wines each fermented with a yeast strain (other
than S. cereuisiae) isolated from homemade wines.
Complete mutagen assays were performed on extracts of white wines fermented by “type” strains
corresponding to the species found in homemade
wines.
Toxicity assay. One ml of extract was obtained
from 250 ml of wine. To determine the toxicity of the
extract, aliquots of 2. 5, 10 and 20 ~1 of extract were
added to 0.1 ml of overnight cultures of TA98 with
various activation systemsand incubated at 37°C for
30 min, after which they were diluted with physiological saline (0.9 % NaCl) to approximately
8 x 10’ cells/ml and 0.1 ml plated on minimal
medium
supplemented
with
0.1 ml Oxoid
broth/plate, 1.6 mg histidine/plate and 25 mg
biotin/plate. Extracts of Foch and Palomino wines
were used in toxicity tests as they have been shown to
have low mutagen contents (Subden et al. 1984).
et al.
The ability of the XAD-2 column to extract
organic mutagens from dilute solutions has been
demonstrated (Heartlein, de Marini, Katz et al. 1981;
Honer, Ashwood-Smith & Warby, 1980; Yamasaki &
Ames, 1977). Using Concord wine, which is known to
contain mutagens (Subden et al. 1984) the XAD-2
columns consistently removed mutagens over the
extract concentration ranges used, as shown in Fig. 1.
Figure 2 shows the low reversion frequency of the
white wine made from Palomino grape juice fermented with Saccharomyces
cereuisiae
(UCD522
“Montrachet” industrial yeast) used in this study. In
contrast, the data for another white wine (from a
concentrate of blended Spanish grape juices and
fermented with UCD522), which showed significant
mutagenic activity, are presented in Fig. 3.
Table I. Yeas1 species tested
Candida
nlhicons
NRC
Candida
valida
ATCC
Candida
uiui
ATCC
AND
ATCC10231
ATCC22687
ATCC20217
ATCC14442
UCD-605
UCD-664
ATCCI0606
ATCC2252
ATCC28484
NRC204008
UCD
UCD
NRC
NRC
NRC
NRC
UCD
UCD
NRC
NRC
UCD-519
UCD-522
NCYC-132
NRC202050
NRC = National Research Council. Ottawa
= American Type Culture Collection,
Rockville. Maryland.
USA
UCD = University
of California.
Davis. USA
= National Collection
of Yeast Cultures, Nutfield, Surrey,
England
ATCC
NCYC
Table
2. Spontaneous
reversion
frequencies
of Sa/ntorw//a rppltistrains with or without activation by S-9 and/or faecalase
Added enzyme
preparation
DISCUSSION
The yeast species we identified as frequent contaminants of homemade wines are listed in Table 1.
The yeast specieslisted are not unexpected as all have
been found in wines previously (Barnett, Payne &
Yarrow, 1979). The API 2OC is an excellent inital
screening device for identifications of the genus but
requires subsequent assimilation and fermentation
studies for identification of the species.
The spontaneous reversion frequencies of TA97,
TA98 and TAlOO (Table 2) are well within the limits
stated previously (Ames et al. 1975; Levin, Hollstein,
Christman et al. 1982a; Levin, Yamasaki & Ames,
1982b). The lower than expected reversion frequency
of TA102 made these results less reliable than those
from the other strains.
Source
number
NRC
mwiwn
RESULTS
Obtained
from
Species
None
s-9
Faecalase
S-9 + Faecalase
Values are means f
Strain
TA97
TA98
TAIM)
TAlO2
TA91
TA98
TAIOO
TAIOZ
TA97
TA98
TAIOO
TAIOZ
TA97
TA98
TAIOO
TAlO2
SD for 24 plates from
Reversion
frequency
I64
27
135
193
179
38
I31
234
219
42
171
267
I87
52
I51
224
*
+
+
f
_+
f
+
_+
+
f
f+_
f
*
+
f
I2 experiments.
63
I4
38
65
82
20
38
63
81
I7
38
79
70
I9
46
63
Mutagenicity
400
r
of yeast-contaminated
wines
(a)
60
60
40
20
0
Extract vol. (~1)
Extract vol. (~1)
Fig. I. Reversion rrequencies of Salmonella ryphirnurium strains (a) TA97, (b) TA98, (c) TAIOO and (d)
TAlO2 exposed to several doses of XAD-2 resin bound extract of wine vinified from Concord grapes using
Sacchnromyces cereuisiae. Frequencies have been corrected for spontaneous, nutritional and other effects.
The arrow on the ordinate indicates the reversion frequency of statistical significance. 0 = extract, 0 =
extract plus S-9, A = extract plus faecalase, n = extract plus S-9 and faecalase.
(cl
Extract vol. +I)
Extract vol. (~1)
Fig. 2. Reversion frequencies of Salmonella typhimurium strains (a) TA97, (b) TA98, (c) TAIOO and (d)
TA102 exposed to several doses of XAD-2 resin-bound extract of wine vinified from Palomino grape must
using Saccharomyces cereuisiae (strain Montrachet). Frequencies have been corrected for spontaneous,
nutritional and other effects. The arrow on the ordinate indicates the reversion frequency of statistical
significance. o = extract, l = extract plus S-9, A = extract plus faecalase, n = extract plus S-9 and
faecalase
345
R. E SUBDEN et al.
346
I
I
I
I
0
2
5
10
Extract
vol.
The data in Table 3 reveal that there is statistically
significant toxicity of the Palomino and Foch
extracts at doses of 20 ~1 and IO ~1,respectively. The
data in Table 3 also indicate that while S-9 is
somewhat toxic on its own, it did appear to inactivate
the extract toxin. Faecalase was not toxic on its own
and had little effect on the toxicity of the extract.
In a previous study (Subden er al. 1984) it was
noted that wine extracts exhibiting a positive
mutagenic response gave the highest reversion frequency at IOpl extract with S-9 and faecalase activation. This finding was confirmed by the data in Figs
l-3. It was therefore deemed necessaryto present only
the reversion frequencies for 10 ~1 extract with S-9
and faecalase activation (Table 4); readers who wish
to see the complete data for reversion frequencies for
the other extract doses and activation systems should
contact the first author. None of the base wines
fermented with yeast strains isolated from homemade wines exhibited a positive mutagenic response
using the Ames spot test.
The data, part of which is presented in Table 4,
indicate that none of the wines contaminated with
“type” strains left a significant level of mutagenic
(~1)
Fig. 3. Reversion frequencies of Salmondla
typltimurium
strain TA98 exposed to several doses of XAD-2 resin-bound
extract of wine vinified from a white grape concentrate
cereuisiae
(UCD522
strain
using
Saccharomyces
Montrachet). Frequencies have been corrected for spontaneous, nutritional and other effects. The arrow on the
ordinate indicates the level of statistical significance.
0 = extract, 0 = extract plus S-9, A = extract plus faecalase,
H = extract plus S-9 and faecalase.
Table
3. Toxicity
or extracts
with or without
or Palomino and Foch wines towards SuL~nrllo
ryphia~wian~
activation
by S-9 and/or faecalase enzyme preparations
Survival
Extract
vol. (PI).
Activation
0
53 +
100 +
72 *
Foeh wine
loo *
51 +
86 f
54 +
None
s-9
Faecalase
S-9 + laecalase
Values are means
(7; or control)
2
5
Palomino wine extract
loo ?O 125 +
None
s-9
Faecalase
S-9 + raecalase
+ 95 % confidence
limits
I2
65
23 103
31
50
extract
0
133
I4
64
26 II8
38
58
(determined
32
+ I2
k 47
* 27
110
75
71
59
+
f
f
+
II7 k 26
66+21
106 f 76
60 + I8
38
I4
42
22
using Student’s
f
+
f
k
IO
32
23
34
32
84
67
55
63
f
+
f
f
20
21
23
21
38
38
65
23
55
t distribution).
No. of revertants/plate
Carldida alhicons
Candida valida
Candida uini
Debarpmyces
palyr~~orphas
Dekkera iarennedia
Hansstiaspora
uvarton
Kluyuerotnpu
atanianus
Pichia firiaosa
Pichiafermenrwu
Pi&a kudriauxuii
Saccharofnyces heficrrs
(cerevisiae)
Saccharontyces cereuisiae
Schisosaccharoatyres
pornhe
Z~~ggosaccllaro,tl~~es rourii
Values are means
TA97
ND
f
f
*
f
+
k
+
ND
I64 f
170
176
II2
94
I84
I58
208
TA98
30
2
19
106
I7
22
I3
37
I50 k 24
136 It 44
203 f 22
ND
56 +
40 +
53 f
57+26
60 +
53 f
33 +
62 +
50 +
33 k
43
28
46
61
TAIOO
22
26
I2
8
I3
4
20
I8
II
f I8
f 4
+ I5
f 23
TAlO2
126 f
I58 f
II5 f
15Ok24
I45 +
II6 f
166 f
198 +
II2 f
I50 +
30
34
27
90
I9
I9
ND
I83 +
ND
256+92
175 f
158 f
190 +
214 f
ND
263 f
II9
I84
I58
130
35
22
38
26
I83 f 18
212 f I2
205 f 38
ND
ND = Not determined
+ SD from four plates from two separate
+
f
f
+
I8
25
37
f 15
+ 9
f 22
+ 43
5’2 + 33 6 f 65
74526
74k20
55 f 45 23 f 24
59 f
5 58 k I9
Table 4. Reversion lrequencies or Sabnonelko tgphbmtriron strains TA97, TA98.
TAIOO and TAIOZ, with activation
by IO pl S-9 and laecalase. exposed to IO-111
extracts of Palomino must inoculated with various yeast species
Yeast species
TA98
experiments.
I41
I4
31
30
I4
72
Mutagenicity
of yeast-contaminated
metabolite in the wine. As previously noted, there are
few negative reports with which to put the positive
findings into perspective (Sugimura,
1978). These
data are preliminary
in as much as they include only
the common coniaminants
of homemade wine from
Southern Ontario and include only one strain from
each species. However, they indicate that these wines
are less of a health hazard than several other
common fluids tested under similar conditions (Lin,
Wang & Yeh, 1978; Nagao, Takahashi, Yamanaka &
Sugimura,
1979; Pamakcu, Ertiirk,
YalGiner et al.
1978; Uyeta, Taue & Mazaki, 1981).
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