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The long-chain fatty acid composition as a tool for differentiating

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 International Journal of Food Microbiology 38 (1997) 143–155

Fatty acid profiling: a feasible typing system to trace yeast

contamination in wine bottling plants

M. Malfeito-Ferreira, M. Tareco, V. Loureiro*

´
Laboratorio ˆ
de Microbiologia, Departamento de Botanica ´
e Engenharia Biologica , Instituto Superior de Agronomia, Tapada da Ajuda,
1399 Lisboa Codex, Portugal

Received 23 March 1997; received in revised form 6 August 1997; accepted 19 September 1997

Abstract

The long-chain fatty acid composition of yeast strains was determined for several species associated with the wine
industry. The Saccharomyces cerevisiae, Zygosaccharomyces bailii, Saccharomycodes ludwigii, Schizosaccharomyces
pombe, Brettanomyces /Dekkera spp., Pichia anomala, Pichia membranaefaciens and Lodderomyces elongisporus species
presented distinct fatty acid profiles after multivariate statistical analysis. The Zygosaccharomyces rouxii species showed
profiles similar to Zygosaccharomyces bailii. The use of fatty acid profiling in wine bottling plants and wines makes it
possible to trace the origin of the strains responsible for spoiling the final product. In one case the origin was found at the
outlet of the finishing filter and identified as Zygosaccharomyces bailii. In the other case the source of contamination was
discovered in the heads of the filling machine and assigned to the Pichia membranaefaciens species. The results point out the
discriminating power and the industrial applicability of the technique described in this work to analyse yeast long-chain fatty
acid compositions.  1997 Elsevier Science B.V.

Keywords: Spoilage yeasts; Wines; Fatty acid composition; Zygosaccharomyces bailii

1. Introduction Therefore, it would be of considerable value to have

One of the major concerns for wine technologists
are stability problems caused by yeasts in wines.
Most wineries rely on plate counts for their routine
microbiological control. The yeasts isolated from
wine plants are quite diversified but only few species
spoil wine (Loureiro and Malfeito-Ferreira, 1993).
*Corresponding author. Tel.: 1 351 1 3638161; fax: 1 351 1
3635031.
a method which could distinguish between ‘innocent’
contamination yeasts and spoilage yeasts. This would
provide a more precise tool for the interpretation of
the results in terms of risk to bottled wine stability.
Several attempts have been made to develop
methods for yeast differentiation, besides conven-
tional identification procedures, such as selective
media, serology, isoenzymes, nucleic acid typing
analysis and the analysis of fatty acid and protein
compositions as reviewed by Fleet (1992), and Van

0168-1605 / 97 / $17.00  1997 Elsevier Science B.V. All rights reserved.

PII S0168-1605( 97 )00096-2
144 M. Malfeito-Ferreira et al. / International Journal of Food Microbiology 38 (1997) 143 – 155
der Vossen and Hofstra (1996). Most of these
methodologies were developed without a detailed
concern for industrial applicability and seem not
efficient enough to differentiate the yeasts associated
with the wine industry.
The use of fatty acid profiling as a biomarker has
been used since the work of Abel et al. (1963) to
type bacteria of clinical interest. With yeasts most of
the research has been done by the group at the
University of Bloemfontein (Kock et al., 1985; Smit
et al., 1988; Augustyn and Kock, 1989; Viljoen et al.,
1988, 1993). One specifically deals with yeast
species associated with the wine industry (Tredoux
et al., 1987).
The work described in this paper represents an
extension of the research initiated by Malfeito-Fer-
reira et al. (1989) into the use of fatty acid profiles as
a biomarker. The technique developed differs mainly
from that of Kock et al. (1985) in the use of a solid
medium for yeast growth which makes it much more
attractive for industrial purposes and it has already
been tried on yeasts associated with breweries
(Moreira-da-Silva et al., 1994). The results presented
in this work include the fatty acid profiling of several
yeast strains isolated from wine bottling plants and
the comparison of the profiles obtained with those of
strains identified by the conventional methodologies
described in the manuals of Barnett et al. (1983) and
Kreger-van Rij (1984).
2. Material and methods
2.1. Yeast strain origin, isolation and identification
The yeast strains analysed were obtained from
several culture collections or isolated from wineries
and bottled wines (Table 1). Wine samples were
analysed by filtering 10 to 100 ml through cellulose
acetate membranes of 0.45 mm pore size and in-
cubating onto GYP agar (glucose (Merck, Darm-
stadt, Germany) 20 g l 21 , yeast extract (Difco
Laboratories, Detroit, USA) 5 g l 21 , peptone (Difco
10 g l 21 and agar 20 g l 21 , pH 6.0). Equipment
contamination was assessed by using cotton swabs
immersed in Ringer solution (Oxoid, Unipath Ltd.,
Basingstoke, England) or by analysing the rinsing
water used in equipment sanitation followed by
membrane filtration and incubation as previously
described. Wines presenting visible sediments were
analysed by pipetting the minimum amount of liquid
necessary to withdraw the sediments from the bottom
of the bottles and inoculating them onto GYP plates.
Air contamination was assessed by placing GYP
petri dishes (9 cm diameter) at several locations in
the bottling line and leaving them open for 10 min.
Corks were analysed by strong agitation for 30 s in
100 ml Ringer solution followed by membrane
filtration and incubation as previously described.
Membrane filtration was also used for analysing
bottle contamination after rinsing with 100 ml of
Ringer solution. All incubations were carried out at
25ºC over a period of three to seven days. Strains
selected according to different colony morphology
were purified by restreaking onto the same medium
and maintained on GYP slants at 4ºC. Fresh cultures
incubated for 24–48 h on GYP slants were used for
the subsequent analysis. Strain identification fol-
lowed the conventional methodologies described by
Barnett et al. (1983) and Kreger-van Rij (1984).
2.2. Long-chain fatty acid analysis
Production of biomass and long-chain fatty acid
analysis have been described elsewhere (Moreira-da-
Silva et al., 1994). Briefly, purified yeast strains were
suspended in Ringer solution and a drop of this
suspension was spread onto GYP agar to form a
large colony, about 2 cm diameter, after 48 h of
incubation at 258C. Biomass extraction and fatty acid
derivatization were attained by alkaline saponifica-
tion (5% w / v NaOH, Merck, in 50% v / v methanol,
Merck, Darmstadt, Germany) and methylation (14%
w / v BF3 in methanol, Merck, Darmstadt, Germany).
Methyl esters were separated by GLC (Perkin Elmer,
model 8410, Norwalk, USA) using a wide-bore DB-
wax column (JW Scientific, Folsom, USA) and
identified by the comparison of relative retention
times of known standards (Sigma Chemical Co., St.
Louis, USA).
Statistical data treatment was performed by Princi-
pal Component Analysis (PCA) using the SPAD
software (Systeme´ Portable d’Analyse des Donnes ´
´
Numeriques, Centre International de Statistique et
d’Informatique Appliquees, ´ Saint-Mande, ´ France,
1993). This analysis groups the yeast strains into
several clusters according to the similarity in their
Table 1 M. Malfeito-Ferreira et al. / International Journal of Food Microbiology 38 (1997) 143 – 155 145
List of studied yeast strains
Species Laboratory number a Origin
Strains from culture collections
S. cerevisiae ISA 1000 IGC 4072, active dry wine yeast
ISA 1011 IGC 4614, active dry wine yeast
ISA 1197 IGC 4455, CBS 1171, type strain
S. cerevisiae var bayanus ISA 1198 IGC 4456, CBS 380, type strain
S. cerevisiae var pastorianus ISA 1207 DBVPG 1243, wine
Zygosaccharomyces bailii (S. acidifaciens)b ISA 1006 CBS 749, type strain
Zygosaccharomyces bailii ISA 1108 IGC 4227, NRRL S9 144
ISA 1149 UCD 801, IGC 5167, type strain
ISA 1206 Lemon concentrate
ISA 1212 DVBPG 6379, CBS 2902
ISA 1213 DVBPG 6453, CBS 3014
ISA 1214 DVBPG 6454, CBS 3016
Zygosaccharomyces rouxii ISA 1188 CECT 1231, orange skin
ISA 1194 CECT 1229, sugar cane
ISA 1322 Soya beans
Pichia membranaefaciens ISA 1005 CBS 107, IGC 3796, type strain
ISA 1195 IGC 3316, CBS 2098, wine
ISA 1196 IGC 3321, CBS 2100, wine
Dekkera intermedia ISA 1146 UCD 605
Brettanomyces bruxellensis ISA 1147 UCD 615
Pichia anomala ISA 1004 CBS 605, IGC 3294
ISA 1185 CECT 1107
Saccharomycodes ludwigii ISA 1093 White wine
ISA 1186 CECT 1382
ISA 1218 DBVPG 2704
S. pombe ISA 1190 CECT 1375
ISA 1191 CECT 1376
ISA 1192 CECT 1377
ISA 1193 CECT 1381
Strains isolated from wineries
S. cerevisiae var bayanus ISA 1028, ISA 1029 Sediments in bottled wines
Zygosaccharomyces bailii ISA 1022, ISA 1023, ISA Sediments in bottled wines
1024, ISA 1025, ISA 1027,
ISA 1031
ISA 1307 Equipment for production of sparkling wine
ISA 1430, ISA 1431, ISA Bottled wine plant A
1432, ISA 1433
Zygosaccharomyces rouxii ISA 1220 Concentrated grape juice
Pichia membranaefaciens ISA 1030 Sediments in bottled wine
ISA 1239, ISA 1241 Contaminants in bottled wine
ISA 1399, ISA 1400, ISA Wine filler plant B
1401, ISA 1403
ISA 1404 Contamination in bottled wint plant B
ISA 1423, ISA 1424 Wine filler plant A
ISA 1429 Bottled wine plant A
Brettanomyces bruxellensis ISA 1327, ISA 1328 Bottled sparkling wine
Pichia anomala ISA 1420 Wine filler plant A
Lodderomyces elongisporus ISA 1421, ISA 1422 Wine filler plant A
Saccharomycodes ludwigii ISA 1083, ISA 1088, ISA Contaminants in sweet bottled wine
1089
Unidentified strains 3 to 43 26 strains isolated from plant A
a
Abbreviations: ISA, Instituto Superior de Agronomia; IGC, Gulbenkian Institute of Science; CBS, Centraalbureau voor Schimmelcultures;
˜
CECT, Coleccion Espanola de Cultivos Tipo; DBVPG, Dipartamento de Biologia Vegetale della Universita´ di Perugia; UCD, University of
California, Davis.
b
Synonim of the species described by Barnett et al. (1983) and Kreger-van Rij (1984).
 146

Table 2
Long-chain fatty acid profiles of analysed yeast strains (NID, unidentified fatty acids)
ISA Fatty acids

strains C 14:0 C 14:1 C 15:0 C 15:1 C 16:0 C 16:1 NID 1 C 17:0 NID 2 NID 3 C 18:0 C 18:1 C 18:2 NID 4 C 18:3

Brettanomyces spp. /Dekkera spp.

1146 3,061,0 0,260,2 0,260,0 0,160,1 20,061,0 47,061,8 3,960,6 0,0 0,0 0,0 1,460,4 15,161,2 9,361,2 0,0 0,0
1147 1,560,3 0,360,1 0,460,1 0,160,1 21,461,0 33,561,2 3,660,4 0,0 0,660,1 0,0 2,260,5 23,461,4 12,961,2 0,0 0,0
1327 1,260,1 0,160,0 0,960,2 0,760,2 20,561,4 29,462,5 3,760,3 0,160,0 1,360,4 1,760,5 5,760,1 19,463,9 9,562,0 4,961,2 0,860,3
1328 1,260,4 0,160,1 1,260,4 0,760,3 21,761,4 28,262,1 3,560,1 0,060,0 0,960,2 2,260,8 5,660,9 18,861,8 9,060,9 5,861,8 0,960,4

Lodderomyces elongisporus
1421 0,360,0 0,060,0 0,260,0 0,060,0 13,461,0 2,760,2 0,0 0,760,2 0,960,1 0,260,1 7,060,4 48,060,5 21,661,1 0,160,0 4,860,7
1422 0,260,0 0,060,0 0,160,0 0,060,1 12,360,2 1,660,1 0,0 0,760,0 0,960,0 0,260,0 9,760,3 51,660,6 18,860,8 0,0 3,960,2

Pichia anomala
1004 0,360,0 0,060,0 0,260,0 0,060,0 18,960,6 3,060,2 0,160,0 0,260,0 0,460,1 0,0 3,660,3 38,061,7 26,661,2 0,0 8,661,2
1185 0,660,2 0,060,0 0,460,1 0,060,0 23,460,3 7,960,7 0,560,1 0,160,0 0,560,2 0,0 1,960,1 30,963,1 25,060,6 0,0 8,861,0
1420 0,360,0 0,160,0 0,260,0 0,060,0 20,760,2 3,260,2 0,260,0 0,260,0 0,260,1 0,160,3 4,760,2 39,960,4 23,760,9 0,0 6,660,5

Pichia membranaefaciens
1005 0,660,0 0,360,1 0,560,0 0,060,0 17,560,4 26,761,9 0,460,0 0,560,0 0,660,1 0,0 3,660,3 33,961,6 11,660,4 0,0 3,860,1
1030 0,160,0 0,0 1,060,1 0,560,0 11,760,8 13,261,2 0,460,1 0,660,0 2,660,4 1,060,0 5,460,1 29,561,1 19,560,3 6,360,4 8,360,2
1195 0,460,1 0,260,1 0,760,2 0,160,0 18,360,9 11,961,5 0,360,0 1,560,4 1,960,0 0,960,3 5,160,9 27,861,9 17,962,2 2,060,4 10,160,1
1196 0,160,0 0,060,0 0,260,1 0,060,0 11,260,8 10,560,3 0,160,1 0,560,1 1,560,5 0,0 2,660,5 46,761,1 17,260,1 0,0 9,460,6
1239 1,160,8 0,560,2 1,460,4 0,960,3 10,861,0 10,761,4 0,760,1 1,060,3 2,860,7 1,260,9 3,760,8 36,360,3 19,060,8 3,162,1 7,160,7
1241 1,060,3 0,660,2 2,861,2 2,761,7 12,060,2 19,461,2 1,260,5 1,160,2 5,761,1 2,161,4 4,060,5 28,366,3 12,061,5 3,961,0 3,860,3
1399 0,360,0 0,060,0 0,560,1 0,060,0 13,760,2 16,760,7 0,360,0 0,960,1 4,260,8 0,0 2,160,2 37,860,3 18,060,9 0,0 5,660,3
1400 0,360,0 0,160,0 0,360,0 0,060,0 14,661,1 19,260,1 0,360,0 0,560,0 2,460,3 0,0 2,160,2 39,960,5 16,260,2 0,0 4,060,4
1401 0,360,0 0,160,0 0,760,1 0,060,0 15,960,9 17,860,5 0,260,0 1,060,0 5,160,0 0,0 1,160,4 36,860,7 16,760,6 0,0 4,560,4
1403 0,460,1 0,160,1 0,360,1 0,160,1 13,760,6 17,360,3 0,560,1 0,560,1 1,560,4 0,0 2,360,5 35,261,3 19,760,5 0,0 8,560,7
1404 0,560,1 0,060,0 0,360,1 0,060,0 15,561,4 19,963,6 0,560,4 0,160,1 0,860,5 0,0 1,660,6 36,561,3 17,560,8 0,0 6,760,4
1423 0,260,0 0,160,0 0,260,0 0,160,0 10,360,4 10,060,5 0,0 0,660,1 3,760,4 0,360,1 1,260,4 52,861,8 14,660,4 0,060,0 6,060,2
1424 0,260,1 0,260,1 0,260,1 0,160,1 10,761,4 19,563,2 0,360,0 0,360,3 2,261,4 0,160,2 2,260,7 49,968,9 16,661,2 0,060,0 6,260,7
1429 0,360,2 0,160,1 0,460,1 0,160,1 10,460,8 19,561,9 0,560,1 0,660,1 3,460,2 0,460,0 1,660,1 39,461,8 16,761,1 0,060,0 6,860,7

S. cerevisiae
1000 0,460,2 0,160,0 0,360,2 0,360,1 8,660,7 39,261,6 0,360,2 0,160,1 0,860,5 2,061,7 4,260,6 38,261,9 0,560,4 3,261,1 0,260,0
M. Malfeito-Ferreira et al. / International Journal of Food Microbiology 38 (1997) 143 – 155

1011 0,760,1 0,360,0 0,160,0 0,260,0 13,460,6 39,560,9 0,060,0 0,260,0 0,360,1 0,060,0 3,560,2 41,560,3 0,360,2 0,0 0,260,0
1028 1,460,1 0,460,0 0,360,1 0,160,0 17,361,8 47,160,9 0,060,0 0,160,0 0,460,1 0,060,0 2,660,1 30,062,3 0,360,1 0,0 0,0
1029 1,560,4 0,960,9 0,360,1 0,160,1 18,060,7 39,462,4 0,0 0,260,1 0,160,1 0,0 3,160,5 35,463,2 0,860,4 0,0 0,460,3
1197 0,960,2 0,460,1 0,160,0 0,260,0 11,060,5 34,863,0 0,060,0 0,160,0 0,360,0 0,160,0 6,660,8 45,362,6 0,260,2 0,0 0,160,1
1198 0,360,5 0,260,1 0,160,0 0,160,0 18,760,5 43,863,1 0,0 0,260,0 0,360,0 0,260,1 2,860,5 33,262,3 0,160,1 0,0 0,060,1
1207 1,160,1 0,660,0 0,260,0 0,160,0 12,160,9 47,061,9 0,0 0,360,1 0,460,1 0,060,0 3,260,4 34,562,1 0,360,1 0,360,1 0,060,0
S. ludwigii
1083 0,960,1 0,560,1 0,360,0 0,360,1 9,260,3 59,262,6 0,0 0,460,2 0,460,1 0,360,1 1,360,3 24,761,8 0,060,0 2,060,1 0,660,1
1088 0,860,1 0,560,1 0,460,3 0,560,3 8,460,7 58,164,8 0,0 0,560,3 0,560,2 0,360,1 1,660,4 25,763,0 0,460,0 2,460,1 0,660,3
1089 0,860,2 0,660,2 0,360,1 0,360,1 8,260,6 58,661,8 0,060,0 0,260,0 0,260,1 0,760,1 1,960,2 25,061,3 0,360,1 2,460,4 0,660,3
1093 0,760,1 0,860,4 0,560,1 0,560,2 8,560,3 55,663,6 0,760,4 0,060,1 0,960,4 0,560,3 1,860,6 26,861,8 0,260,0 2,060,3 0,760,4
1186 0,660,1 0,560,1 0,260,1 0,260,1 8,460,6 55,161,0 0,0 0,660,3 0,860,3 0,560,2 1,660,1 29,362,8 0,360,1 1,360,6 0,760,8
1218 1,360,1 0,660,1 0,060,0 0,260,3 11,161,2 59,161,4 0,060,0 0,160,0 0,160,0 0,460,5 1,760,4 25,061,0 0,360,1 0,060,1 0,160,0

S. pombe
1190 0,960,2 0,260,3 0,660,4 0,760,4 11,760,8 2,360,3 0,160,2 0,860,1 0,960,2 0,560,1 4,060,2 74,362,5 0,560,1 2,060,3 0,560,3
1191 0,860,1 0,160,0 0,760,2 0,560,1 10,560,9 0,860,1 0,0 1,060,6 0,660,4 0,460,4 5,360,1 74,160,2 0,660,1 3,860,6 0,760,2
1192 0,860,1 0,060,0 0,360,4 0,560,1 11,660,5 0,860,0 0,0 1,060,1 0,760,1 0,560,0 5,760,5 73,460,9 0,360,1 3,360,1 0,760,0
1193 0,660,0 0,160,0 0,160,0 0,160,0 11,960,1 3,160,1 0,160,1 0,460,2 0,460,3 0,0 5,460,3 70,960,2 0,660,4 6,560,0 0,0

Zygosaccharomyces bailii
1006 0,260,0 0,260,0 0,660,3 0,560,2 6,960,5 9,960,6 0,360,1 0,160,2 0,760,2 1,160,2 6,760,5 44,162,4 25,960,6 2,761,0 0,360,1
1022 0,160,0 0,060,0 0,560,0 0,460,0 8,160,0 6,361,0 0,260,0 0,060,0 0,460,0 0,460,1 5,960,0 37,960,7 37,160,7 2,560,3 0,560,0
1023 0,360,0 0,160,0 0,660,2 0,560,2 9,760,7 6,860,9 0,0 0,660,2 0,760,3 0,660,3 7,760,7 36,762,8 31,762,1 3,762,0 0,860,3
1024 0,260,1 0,160,0 0,560,1 0,460,2 9,560,7 6,061,3 0,0 0,760,5 0,360,3 0,560,3 6,160,4 39,263,3 32,760,7 3,160,7 0,760,2
1025 0,260,1 0,060,0 0,260,1 0,360,1 8,960,6 9,461,2 0,460,1 0,460,1 0,960,1 0,560,1 4,960,5 38,760,1 32,962,1 1,760,7 0,660,2
1027 0,260,1 0,060,0 0,260,1 0,360,2 9,660,9 6,661,1 0,0 0,760,3 0,460,0 0,760,5 6,360,8 40,162,5 33,160,8 2,361,1 0,560,2
1031 0,260,1 0,060,0 0,260,2 0,260,2 10,761,6 7,861,6 0,160,1 0,260,1 0,560,1 0,360,1 5,961,0 44,261,7 27,165,0 2,461,1 0,360,4
1108 0,360,1 0,160,0 0,060,0 0,160,0 8,460,8 10,861,3 0,160,0 0,160,0 0,160,0 0,060,0 4,460,2 41,360,8 34,261,2 0,060,0 0,160,1
1149 2,261,0 0,160,1 0,060,0 0,160,0 14,161,3 11,660,4 0,260,0 0,160,1 0,260,1 0,060,0 2,660,4 43,360,8 25,460,2 0,060,0 0,060,0
1206 0,260,0 0,060,0 0,160,0 0,260,0 10,360,4 12,261,2 0,460,2 0,460,0 0,760,0 0,360,4 5,660,3 36,061,3 33,261,6 1,760,0 0,460,0
1212 0,260,1 0,060,0 0,460,2 0,260,2 9,160,3 10,161,3 0,0 1,060,4 0,560,0 0,460,1 5,360,5 41,461,9 28,961,2 2,160,8 1,561,0
1213 0,360,1 0,160,0 0,460,0 0,460,1 12,060,7 20,861,1 2,060,4 0,0 0,560,2 0,760,4 5,860,2 22,660,9 30,760,5 2,760,2 1,160,2
1214 0,160,0 0,060,0 0,160,0 0,160,0 8,960,1 13,460,4 0,560,3 0,460,2 0,860,5 0,960,7 4,060,2 34,062,1 34,360,7 1,260,7 0,860,7
1307 0,260,4 0,260,3 0,060,0 0,160,1 9,060,7 10,760,6 0,260,1 0,160,1 0,160,0 0,0 4,660,3 38,361,5 36,360,5 0,060,0 0,260,1
1430 0,260,1 0,160,0 0,160,0 0,360,1 9,761,3 7,561,2 0,160,0 0,160,0 0,360,1 0,260,2 4,560,3 38,961,3 37,961,8 0,060,0 0,160,0
1431 0,260,0 0,160,0 0,060,0 0,160,0 9,060,1 11,560,6 0,160,1 0,060,0 0,160,0 0,160,0 3,260,3 41,961,2 33,861,0 0,0 0,060,0
1432 0,260,0 0,060,0 0,060,0 0,160,0 9,660,3 9,561,0 0,160,1 0,060,0 0,160,0 0,160,1 4,160,5 36,561,4 39,560,7 0,160,1 0,260,2
1433 0,160,0 0,160,0 0,060,0 0,160,0 8,860,4 8,560,5 0,0 0,660,2 0,160,0 0,0 4,360,3 39,460,4 38,260,5 0,0 0,0

Zygosaccharomyces rouxii
1188 0,760,1 0,360,1 0,160,0 0,060,0 10,861,1 39,161,1 0,0 0,160,1 0,360,1 0,0 4,360,2 44,160,7 0,360,1 0,0 0,0
1194 0,560,1 0,160,1 0,160,0 0,160,1 10,060,5 11,361,2 0,460,1 0,0 0,0 0,0 2,260,6 42,761,0 32,760,4 0,0 0,0
1220 0,660,3 0,160,0 0,060,0 0,060,0 13,961,0 9,661,0 0,260,0 0,160,0 0,160,0 0,360,4 2,760,5 43,860,2 28,362,0 0,060,0 0,360,1
1322 0,460,1 0,060,0 0,760,0 0,260,1 17,361,0 10,160,3 1,060,3 0,860,1 1,960,2 0,460,1 5,060,4 35,960,7 18,860,5 1,860,6 5,860,5
M. Malfeito-Ferreira et al. / International Journal of Food Microbiology 38 (1997) 143 – 155

Unidentified strains
3 0,560,2 0,060,0 0,160,0 0,060,0 18,761,3 0,460,2 0,0 0,460,1 0,260,0 0,160,0 5,160,6 49,361,9 17,961,1 0,0 7,2160,9
4 0,360,0 0,060,0 0,360,1 0,360,2 20,061,0 2,360,4 0,260,1 0,360,1 0,560,3 0,160,1 5,560,5 38,060,8 25,160,6 0,0 7,460,7
5 0,460,1 0,060,0 0,260,0 0,060,0 19,961,7 0,460,1 0,060,1 0,560,1 0,160,0 0,060,0 9,860,8 33,261,8 29,560,9 0,0 5,960,2
6 0,460,0 0,160,0 0,260,0 0,160,0 22,160,3 3,260,1 0,260,1 0,260,1 0,460,1 0,160,1 4,160,5 40,061,0 22,060,6 0,0 7,060,7
7 0,360,0 0,160,0 0,560,1 0,160,1 12,160,2 10,860,5 0,0 1,460,1 4,160,2 0,460,1 2,560,3 42,161,3 18,960,5 0,160,0 7,460,2
9 0,260,0 0,060,0 0,160,0 0,060,0 16,760,3 3,560,2 0,360,1 0,460,1 0,760,2 0,160,1 4,760,9 53,761,3 13,660,6 0,060,0 5,860,3
147
 148

Table 2. Continued
ISA Fatty acids

strains C 14:0 C 14:1 C 15:0 C 15:1 C 16:0 C 16:1 NID 1 C 17:0 NID 2 NID 3 C 18:0 C 18:1 C 18:2 NID 4 C 18:3

13 0,360,0 0,160,0 0,260,0 0,060,0 13,160,6 2,860,2 0,0 0,760,1 1,060,2 0,260,0 7,460,7 48,660,9 21,461,1 0,160,0 4,260,4
15 0,360,0 0,060,0 0,160,0 0,060,0 12,260,4 2,360,2 0,060,0 0,760,1 0,960,1 0,260,1 8,160,5 46,560,3 23,160,7 0,160,0 5,560,4
16 0,560,4 0,160,0 0,160,0 0,160,0 11,360,8 1,6360,1 0,0 0,960,0 0,060,1 0,160,0 8,261,4 50,761,0 20,561,9 0,160,0 4,960,7
18 0,260,0 0,160,0 1,060,7 0,160,0 11,560,1 11,660,7 0,0 1,560,1 4,760,2 0,360,0 1,660,2 38,661,8 19,460,7 0,160,0 9,560,3
19 0,260,0 0,160,0 0,360,1 0,160,0 11,760,4 8,160,8 0,0 1,260,1 3,060,6 0,460,1 2,660,3 41,261,4 18,460,3 0,160,0 12,860,1
20 0,960,2 0,060,0 0,160,1 0,060,0 20,960,6 1,160,6 0,0 0,260,1 0,360,2 0,060,0 2,960,3 49,961,7 14,760,8 0,060,0 8,960,5
23 0,760,2 0,160,0 0,360,1 0,160,0 16,160,9 25,461,9 0,0 0,560,1 1,160,1 0,0 3,460,7 52,462,2 0,160,0 0,060,0 0,060,0
24 0,860,2 0,060,0 0,160,0 0,060,0 21,060,5 0,760,4 0,0 0,360,1 0,560,1 0,060,0 5,160,3 48,060,8 16,960,8 0,060,0 6,760,8
26 0,360,1 0,160,0 0,460,1 0,060,0 19,461,5 3,260,4 0,0 0,660,0 0,760,0 0,160,1 7,360,4 51,460,8 12,860,9 0,160,0 3,860,7
27 0,360,1 0,060,0 0,160,0 0,060,0 12,660,7 2,660,1 0,0 0,760,1 0,560,5 0,160,0 8,660,7 47,162,6 22,561,7 0,160,0 4,760,6
28 0,360,0 0,160,0 0,460,1 0,060,0 12,660,7 13,561,2 0,0 0,960,1 3,760,9 0,260,1 1,960,3 43,960,9 17,060,4 0,060,0 5,860,6
29 0,260,0 0,160,1 0,260,0 0,160,0 9,960,5 14,961,2 0,160,6 0,360,1 1,860,5 0,260,1 1,860,3 47,561,7 16,160,3 0,060,0 7,060,5
30 0,560,0 0,260,1 0,160,1 0,160,1 10,460,1 46,463,1 0,0 0,160,1 0,460,1 0,060,0 2,460,4 38,662,3 0,660,3 0,0 0,360,2
31 0,360,0 0,260,1 0,260,1 0,260,2 9,460,7 18,860,4 0,360,0 0,460,1 2,861,0 0,260,1 1,660,1 44,061,1 15,361,2 0,060,0 6,461,3
32 0,360,0 0,060,0 0,560,0 0,060,0 13,860,6 11,660,3 0,260,0 0,960,0 3,160,3 0,360,0 2,060,1 38,160,6 17,260,2 0,160,0 12,060,1
33 0,160,1 0,160,0 0,260,0 0,060,0 11,060,1 13,060,3 0,260,0 0,560,1 3,360,4 0,260,1 1,560,1 46,460,1 17,460,4 0,060,0 6,160,2
36 0,360,1 0,160,1 0,060,0 0,260,1 9,861,0 11,361,3 0,0 0,160,0 0,160,0 0,160,1 3,360,2 40,261,1 34,561,5 0,060,0 0,060,0
39 0,360,1 0,160,0 0,060,0 0,160,0 8,760,3 9,160,9 0,160,1 0,060,1 0,160,0 0,160,1 3,660,5 43,960,5 33,860,4 0,0 0,160,0
41 0,260,1 0,160,1 0,060,0 0,160,0 10,962,4 7,261,0 0,160,0 0,660,7 0,360,3 0,060,0 4,860,2 36,461,0 39,163,2 0,060,0 0,360,5
43 0,260,0 0,160,0 0,060,0 0,260,0 9,860,5 8,560,6 1,461,0 0,160,1 0,160,1 0,060,0 4,260,6 37,260,7 38,062,0 0,0 0,160,0
M. Malfeito-Ferreira et al. / International Journal of Food Microbiology 38 (1997) 143 – 155
 M. Malfeito-Ferreira et al. / International Journal of Food Microbiology 38 (1997) 143 – 155
fatty acid compositions. The statistical stability of
the partition is weighed by its inertia relation. A
particular partition is considered stable if an increase
in the number of clusters does not significatively
extend this parameter, the maximum theoretical
value of which is 1 (Lebart et al., 1984).
3. Results
The fatty acid compositions of the analysed yeast
strains are presented in Table 2. The major fatty
acids have 16 or 18 carbon atoms with a variable
degree of unsaturation. Primarily the identified yeast
strains were divided into three groups according to
the composition of the acids with 18 carbon atoms:
(i) group I, including species without C18:2 and
C18:3 acids, such as Saccharomyces cerevisiae,
Schizosaccharomyces pombe and Saccharomycodes
ludwigii; (ii) group II, including species without
C18:3 acid, such as Zygosaccharomyces bailii and
Brettanomyces /Dekkera spp. ; (iii) group III, includ-
ing species with C18:3 acid, such as Pichia mem-
branaefaciens, Pichia anomala and Lodderomyces
elongisporus. Then each group was subjected to a
principal component analysis (PCA) the graphical
results of which are shown in Fig. 1 (group I), Fig. 2
(group II) and Fig. 3 (group III). The PCA was
performed using the most significant fatty acids (C
16:0, C 16:1, C 18:0, C 18:1, C 18:2 and C 18:3) the
variability coefficient of which was less than 10%.
For each group the number of initial clusters was
established as equal to the number of species tested,
then the number of clusters was increased to assess
the statistical stability of each partition.
The three species from group I were clearly
separated into three clusters with an inertia relation
of 0.73. If an increase in the number of clusters is
performed by the PCA the Saccharomyces cerevisiae
var bayanus strains (ISA 1028, ISA 1029 and ISA
1198) are separated from the other Saccharomyces
cerevisiae strains and gathered in an additional
cluster. This partition into four clusters had an inertia
relation of 0.89. The division into five clusters
increased the inertia relation to 0.92, the new cluster
being formed only by the Saccharomyces cerevisiae
type strain (ISA 1197). A possible partition into six
clusters did not significantly increase the inertia
relation (0.93) and the new cluster obtained by
149
splitting Saccharomycodes ludwigii strains does not
have any taxonomical meaning (results not shown).
Thus the partition into four clusters appeared to be
the most suitable for separating the yeasts in this
group.
The two species in group II were firstly separated
into two clusters, one for Brettanomyces /Dekkera
and the other for Zygosaccharomyces bailii strains,
with an inertia relation of 0.66. An increase in the
number of clusters to three splits the
Zygosaccharomyces bailii cluster, with an inertia
relation of 0.77. The division of this species did not
show any correlation with taxonomical or tech-
nological characteristics such as strain origin or
resistance to preservatives (unpublished results).
Further increases in the number of clusters although
improving the inertia relation do not present any
advantages in species differentiation (results not
shown).
The three species of group III were separated into
three distinct clusters with an inertia relation of 0.47.
The increase to four (0.64 of inertia relation) and five
clusters (0.78 of inertia relation) was achieved by
splitting the Pichia membranaefaciens cluster. The
significant increase in the value of the inertia relation
may be regarded as an indicator of the heterogeneity
of this species. The division of this species did not
indicate any apparent technological meaning.
A survey of yeast contaminants was performed in
a wine bottling plant in order to trace the source of
the strain responsible for the formation of sediments
in bottled wine (strains from plant A, Table 1). The
microbiological analysis showed that yeast contami-
nants were present all over the bottling line. The
fatty acid profiles of these isolates were determined
(Table 2). The visual comparison and PCA (Fig. 2)
of fatty acid profiles indicated that the source of
contamination was situated at the outlet of the
finishing filter before the filler. One strain isolated
from this source (number 41) was similar to those
isolated from the newly bottled wine (ISA 1430) and
from sediments from wine bottled two months earlier
(ISA 1432 and ISA 1433) and from wine stored for
four months (ISA 1431). They shared a common
fatty acid profile which seemed to be of
Zygosaccharomyces bailii. Later identification by
conventional methodology confirmed this hypohesis.
Another strain (ISA 1429) with a different fatty acid
profile was isolated from the newly bottled wine but
150 M. Malfeito-Ferreira et al. / International Journal of Food Microbiology 38 (1997) 143 – 155

Fig. 1. Principal Component Analysis (PCA). Yeast strains without polyunsaturated C 18 fatty acids. The strains are grouped in three (solid
line), four (dashed line) or five clusters (dotted line). Cluster I, Saccharomycodes ludwigii; cluster II, Schizosaccharomyces pombe and
cluster III, Saccharomyces cerevisiae strains. (Yeasts are numbered according to , TBLR . 1 , / TBLR . .)
 M. Malfeito-Ferreira et al. / International Journal of Food Microbiology 38 (1997) 143 – 155 151

Fig. 2. Principal Component Analysis (PCA). Yeast strains without C 18:3 fatty acid. The strains are grouped in two (solid line) and three
clusters (dashed line). The 0035, 0037, 0040 and 0042 strains are the same as the ISA 1430, ISA 1431, ISA 1432 and ISA 1433 strains,
respectively. The 1146 and 1328 strains are covered by 1147 and 1327 strains. Cluster I, Brettanomyces /Dekkera spp. and cluster II,
Zygosaccharomyces bailii strains. (Yeasts are numbered according to , TBLR . 1 , / TBLR . .)
152 M. Malfeito-Ferreira et al. / International Journal of Food Microbiology 38 (1997) 143 – 155

Fig. 3. Principal Component Analysis (PCA). Yeast strains with C 18:3 fatty acid. The strains are grouped in three (solid line), four (dashed
line) or five clusters (dotted line). The 0010, 0012, 0014, 0017 and 0022 strains are the same as the ISA 1420, ISA 1421, ISA 1422, ISA
1423 and ISA 1424 strains, respectively. Cluster I, Lodderomyces elongisporus; cluster II, Pichia anomala and cluster III, Pichia
membranaefaciens strains. (Yeasts are numbered according to , TBLR . 1 , / TBLR . .)
 M. Malfeito-Ferreira et al. / International Journal of Food Microbiology 38 (1997) 143 – 155
it was not found in the sediments of the wine after
several months of storage indicating that it could not
survive in such environmental conditions. The prob-
able origin of this strain was in the filler (ISA 1424)
and at the filter inlet (strain 31) and the fatty acid
composition of both was similar to Pichia mem-
branaefaciens. This possibility of identification as
Pichia membranaefaciens was also confirmed by
conventional methodology. All other isolates from
the bottling plant were not found in bottled wine
indicating, in case they contaminate wine, their
inability to grow under such conditions for they lose
their viability immediately after coming into contact
with wine. Some of these isolates were also conven-
tionally identified. One strain isolated from the filler
(ISA 1420) had a fatty acid profile similar to Pichia
anomala and was identified as such. Two other
strains isolated from the filler showed an unusual
spore shape and were identified as Lodderomyces
elongisporus (ISA 1421 and ISA 1422). Their fatty
acid profile was quite distinct from all other isolates.
A survey of another bottling line (strains in plant
B, Table 1) revealed that final product contamination
was due to strains of Pichia membranaefaciens (ISA
1404) which were also recovered from the filling
machine (ISA 1399, ISA 1400, ISA 1401 and ISA
1403).
Other strains occasionally isolated from spoiled
wines or from winery equipment were conventional-
ly identified to check if they corresponded to the
species to be expected after fatty acid profiling. In
fact the identity of the ISA 1307 strain was con-
firmed as Zyosaccharomyces bailii while ISA 1239
and ISA 1241 strains were Pichia membranaefa-
ciens. ISA 1327 and ISA 1328 strains isolated from
spoiled sparkling wine showed profiles which were
distinct from all the other strains and were identified
later as Dekkera bruxellensis.
4. Discussion
Strains from the conventionally identified Sac-
charomyces cerevisiae, Zygosaccharomyces bailii,
Saccharomycodes ludwigii, Schizosaccharomyces
pombe, Brettanomyces /Dekkera spp., Pichia
anomala, Pichia membranaefaciens and Lod-
deromyces elongisporus species presented distinct
fatty acid profiles after multivariate statistical analy-
153
sis. Several species are heterogenous as regards their
fatty acid composition. This was the case of Pichia
membranaefaciens and Zygosaccharomyces rouxii.
In spite of this heterogeneity the former species
could be distinguished from the others because the
clusters given by PCA did not present strains from
other species. Noronha-da-Costa et al. (1996) sug-
gested that the heterogeneity of Pichia mem-
branaefaciens may be related with the inconsistency
of the identification given by conventional method-
ologies when compared with either fatty acid profiles
or DNA homology with the respective type strain.
On the contrary, Zygosaccharomyces rouxii strains
were included in the clusters of Saccharomyces
cerevisiae, Zygosaccharomyces bailii and Pichia
membranaefaciens. This observation may indicate
that conventional procedures lead to misidentification
of Zygosaccharomyces rouxii strains. In fact ISA
1188, ISA 1194 and ISA 1322 strains which were
clustered together with Saccharomyces cerevisiae
(see Fig. 1), Zygosaccharomyces bailii (see Fig. 2)
and Pichia membranaefaciens (see Fig. 3), respec-
tively, did not hybridize with the respective type
strain (unpublished results). Thus, from the four
strains of Zygosaccharomyces rouxii analysed only
ISA 1220 was confirmed as such and clustered
together with Zygosaccharomyces bailii. However,
the number of strains analysed should be increased to
confirm these results.
The differentiating ability of the technique proved
adequate for the characterization of yeasts associated
with the wine industry for it was possible to dis-
tinguish strains with different abilities to spoil wine
and to trace back contaminants of final products. In
practical terms the results can be obtained within two
days after yeast isolation and purification. It may
take longer if strain purification is required or if slow
growing species such as Brettanomyces /Dekkera are
to be analysed.
Although the medium and growth conditions were
different from other studies, the fatty acid com-
positions of Zygosaccharomyces bailii, Pichia mem-
branaefaciens, Pichia anomala, Lodderomyces elon-
gisporus, Schizosaccharomyces pombe and Sac-
charomycodes ludwigii strains analysed were similar,
in relative terms, to those of the same species
reported in the literature (Rattray, 1988; Tredoux et
al., 1987; Viljoen et al., 1988; Jeffery et al., 1995).
This similarity might be somewhat unexpected be-
154 M. Malfeito-Ferreira et al. / International Journal of Food Microbiology 38 (1997) 143 – 155
cause of the well known dependence of fatty acid
compositions on growth conditions. However, other
authors have reported that under certain conditions
fatty acid profiles do not vary or that the degree of
variation is species or strain dependent (Johnson et
al., 1972; Suutari et al., 1990). Unpublished results
from our laboratory concur with these observations.
Probably the alteration of cellular fatty acid com-
position is only significant when there are consider-
able variations in environmental conditions. How-
ever, for an accurate comparison of fatty acid
profiles, biomass must be grown under the same
environmental conditions. For this the use of a solid
medium for yeast growth, as it is currently referred
for bacterial analysis (e.g. Bousfield et al., 1983;
Decallonne et al., 1991; Svetashev et al., 1995),
proved to be a suitable mean of decreasing analysis
time and simultaneously of simplifying extraction
procedures by avoiding the washing and centrifuga-
tion of the cells which may also contribute to the
appearance of artifacts (Ratledge and Evans, 1989).
Guerzoni et al. (1993) showed that fatty acid
composition could be correlated with strain origin or
resistance to thermal stress. However, the division of
Saccharomyces cerevisiae, Zygosaccharomyces bailii
and Pichia membranaefaciens observed could not be
related to strain origin or tolerance to sulphur dioxide
or sorbic acid (unpublished results).
The use of fatty acid profiling may separate
species according to their spoilage potential. This
possibility has already been suggested by Botha and
Kock (1993) to monitor fungal contamination in
bioprotein production. In the wine industry the most
potential spoilage yeasts would be indicated by
significant amounts of C18:1 and C18:2 and by the
absence of C 18:3, because it corresponds to
Zygosaccharomyces spp. In fact unpublished results
from our laboratory revealed that several samples of
spoiled bottled wine with levels as high as 60 ppm of
free sulphur dioxide and pH 3.3, contained sediments
due solely to growth of Zygosaccharomyces bailii.
The absence of polyunsaturated C 18 acids would be
an indicator of the presence of Saccharomyces
cerevisiae or of other contamination species such as
Schizosaccharomyces pombe or Saccharomycodes
ludwigii. The presence of C18:2 and C18:3 reflects
the presence of yeasts like Pichia membranaefaciens
and Pichia anomala associated with poor hygiene or
insufficient use of preservatives and could be re-
garded as indicating lack of proper sanitation or of
operating efficiency.
Acknowledgements
We are indebted to Professor I. Spencer Martins
for allowing the conventional identification of yeast
strains to be carried out at the Gulbenkian Institute of
Science, Oeiras, Portugal. This work was partially
funded by the EU project AIR-2-CT93-830.
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