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Fatty acid profiling: a feasible typing system to trace yeast contamination in wine bottling plants
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* ´ ˆ ´ Laboratorio 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 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.
Therefore, it would be of considerable value to have 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 conventional 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
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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-Ferreira 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 incubating onto GYP agar (glucose (Merck, Darmstadt, 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 followed 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-daSilva 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 saponification (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 DBwax 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 Principal Component Analysis (PCA) using the SPAD ´ ´ software (Systeme Portable d’Analyse des Donnes ´ Numeriques, Centre International de Statistique et ´ ´ France, d’Informatique Appliquees, Saint-Mande, 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 List of studied yeast strains Species Strains from culture collections S. cerevisiae
S. cerevisiae var bayanus S. cerevisiae var pastorianus Zygosaccharomyces bailii (S. acidifaciens)b Zygosaccharomyces bailii
Zygosaccharomyces rouxii
Pichia membranaefaciens
Dekkera intermedia Brettanomyces bruxellensis Pichia anomala Saccharomycodes ludwigii
S. pombe
Strains isolated from wineries S. cerevisiae var bayanus Zygosaccharomyces bailii
Zygosaccharomyces rouxii Pichia membranaefaciens
Brettanomyces bruxellensis Pichia anomala Lodderomyces elongisporus Saccharomycodes ludwigii Unidentified strains a
Laboratory number a
Origin
ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA
IGC 4072, active dry wine yeast IGC 4614, active dry wine yeast IGC 4455, CBS 1171, type strain IGC 4456, CBS 380, type strain DBVPG 1243, wine CBS 749, type strain IGC 4227, NRRL S9 144 UCD 801, IGC 5167, type strain Lemon concentrate DVBPG 6379, CBS 2902 DVBPG 6453, CBS 3014 DVBPG 6454, CBS 3016 CECT 1231, orange skin CECT 1229, sugar cane Soya beans CBS 107, IGC 3796, type strain IGC 3316, CBS 2098, wine IGC 3321, CBS 2100, wine UCD 605 UCD 615 CBS 605, IGC 3294 CECT 1107 White wine CECT 1382 DBVPG 2704 CECT 1375 CECT 1376 CECT 1377 CECT 1381
1000 1011 1197 1198 1207 1006 1108 1149 1206 1212 1213 1214 1188 1194 1322 1005 1195 1196 1146 1147 1004 1185 1093 1186 1218 1190 1191 1192 1193
ISA 1028, ISA 1029 ISA 1022, ISA 1023, ISA 1024, ISA 1025, ISA 1027, ISA 1031 ISA 1307 ISA 1430, ISA 1431, ISA 1432, ISA 1433 ISA 1220 ISA 1030 ISA 1239, ISA 1241 ISA 1399, ISA 1400, ISA 1401, ISA 1403 ISA 1404 ISA 1423, ISA 1424 ISA 1429 ISA 1327, ISA 1328 ISA 1420 ISA 1421, ISA 1422 ISA 1083, ISA 1088, ISA 1089 3 to 43
145
Sediments in bottled wines Sediments in bottled wines
Equipment for production of sparkling wine Bottled wine plant A Concentrated grape juice Sediments in bottled wine Contaminants in bottled wine Wine filler plant B Contamination in bottled wint plant B Wine filler plant A Bottled wine plant A Bottled sparkling wine Wine filler plant A Wine filler plant A Contaminants in sweet bottled wine 26 strains isolated from plant 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).
0,260,0 0,460,1 0,260,0
0,560,0 1,060,1 0,760,2 0,260,1 1,460,4 2,861,2 0,560,1 0,360,0 0,760,1 0,360,1 0,360,1 0,260,0 0,260,1 0,460,1
0,360,2 0,160,0 0,360,1 0,360,1 0,160,0 0,160,0 0,260,0
0,060,0 0,060,0 0,160,0
Pichia anomala 1004 0,360,0 1185 0,660,2 1420 0,360,0
Pichia membranaefaciens 1005 0,660,0 0,360,1 1030 0,160,0 0,0 1195 0,460,1 0,260,1 1196 0,160,0 0,060,0 1239 1,160,8 0,560,2 1241 1,060,3 0,660,2 1399 0,360,0 0,060,0 1400 0,360,0 0,160,0 1401 0,360,0 0,160,0 1403 0,460,1 0,160,1 1404 0,560,1 0,060,0 1423 0,260,0 0,160,0 1424 0,260,1 0,260,1 1429 0,360,2 0,160,1
S. cerevisiae 1000 0,460,2 1011 0,760,1 1028 1,460,1 1029 1,560,4 1197 0,960,2 1198 0,360,5 1207 1,160,1
0,160,0 0,360,0 0,460,0 0,960,9 0,460,1 0,260,1 0,660,0
0,260,0 0,160,0
C 15:0
Lodderomyces elongisporus 1421 0,360,0 0,060,0 1422 0,260,0 0,060,0
C 14:1
0,260,0 0,460,1 0,960,2 1,260,4
C 14:0
strains
Brettanomyces spp. /Dekkera spp. 1146 3,061,0 0,260,2 1147 1,560,3 0,360,1 1327 1,260,1 0,160,0 1328 1,260,4 0,160,1
Fatty acids
ISA
0,360,1 0,260,0 0,160,0 0,160,1 0,260,0 0,160,0 0,160,0
0,060,0 0,560,0 0,160,0 0,060,0 0,960,3 2,761,7 0,060,0 0,060,0 0,060,0 0,160,1 0,060,0 0,160,0 0,160,1 0,160,1
0,060,0 0,060,0 0,060,0
0,060,0 0,060,1
0,160,1 0,160,1 0,760,2 0,760,3
C 15:1
8,660,7 13,460,6 17,361,8 18,060,7 11,060,5 18,760,5 12,160,9
17,560,4 11,760,8 18,360,9 11,260,8 10,861,0 12,060,2 13,760,2 14,661,1 15,960,9 13,760,6 15,561,4 10,360,4 10,761,4 10,460,8
18,960,6 23,460,3 20,760,2
13,461,0 12,360,2
20,061,0 21,461,0 20,561,4 21,761,4
C 16:0
39,261,6 39,560,9 47,160,9 39,462,4 34,863,0 43,863,1 47,061,9
26,761,9 13,261,2 11,961,5 10,560,3 10,761,4 19,461,2 16,760,7 19,260,1 17,860,5 17,360,3 19,963,6 10,060,5 19,563,2 19,561,9
3,060,2 7,960,7 3,260,2
2,760,2 1,660,1
47,061,8 33,561,2 29,462,5 28,262,1
C 16:1
0,360,2 0,060,0 0,060,0 0,0 0,060,0 0,0 0,0
0,460,0 0,460,1 0,360,0 0,160,1 0,760,1 1,260,5 0,360,0 0,360,0 0,260,0 0,560,1 0,560,4 0,0 0,360,0 0,560,1
0,160,0 0,560,1 0,260,0
0,0 0,0
3,960,6 3,660,4 3,760,3 3,560,1
NID 1
Table 2 Long-chain fatty acid profiles of analysed yeast strains (NID, unidentified fatty acids)
0,160,1 0,260,0 0,160,0 0,260,1 0,160,0 0,260,0 0,360,1
0,560,0 0,660,0 1,560,4 0,560,1 1,060,3 1,160,2 0,960,1 0,560,0 1,060,0 0,560,1 0,160,1 0,660,1 0,360,3 0,660,1
0,260,0 0,160,0 0,260,0
0,760,2 0,760,0
0,0 0,0 0,160,0 0,060,0
C 17:0
0,860,5 0,360,1 0,460,1 0,160,1 0,360,0 0,360,0 0,460,1
0,660,1 2,660,4 1,960,0 1,560,5 2,860,7 5,761,1 4,260,8 2,460,3 5,160,0 1,560,4 0,860,5 3,760,4 2,261,4 3,460,2
0,460,1 0,560,2 0,260,1
0,960,1 0,960,0
0,0 0,660,1 1,360,4 0,960,2
NID 2
2,061,7 0,060,0 0,060,0 0,0 0,160,0 0,260,1 0,060,0
0,0 1,060,0 0,960,3 0,0 1,260,9 2,161,4 0,0 0,0 0,0 0,0 0,0 0,360,1 0,160,2 0,460,0
0,0 0,0 0,160,3
0,260,1 0,260,0
0,0 0,0 1,760,5 2,260,8
NID 3
4,260,6 3,560,2 2,660,1 3,160,5 6,660,8 2,860,5 3,260,4
3,660,3 5,460,1 5,160,9 2,660,5 3,760,8 4,060,5 2,160,2 2,160,2 1,160,4 2,360,5 1,660,6 1,260,4 2,260,7 1,660,1
3,660,3 1,960,1 4,760,2
7,060,4 9,760,3
1,460,4 2,260,5 5,760,1 5,660,9
C 18:0
38,261,9 41,560,3 30,062,3 35,463,2 45,362,6 33,262,3 34,562,1
33,961,6 29,561,1 27,861,9 46,761,1 36,360,3 28,366,3 37,860,3 39,960,5 36,860,7 35,261,3 36,561,3 52,861,8 49,968,9 39,461,8
38,061,7 30,963,1 39,960,4
48,060,5 51,660,6
15,161,2 23,461,4 19,463,9 18,861,8
C 18:1
0,560,4 0,360,2 0,360,1 0,860,4 0,260,2 0,160,1 0,360,1
11,660,4 19,560,3 17,962,2 17,260,1 19,060,8 12,061,5 18,060,9 16,260,2 16,760,6 19,760,5 17,560,8 14,660,4 16,661,2 16,761,1
26,661,2 25,060,6 23,760,9
21,661,1 18,860,8
9,361,2 12,961,2 9,562,0 9,060,9
C 18:2
3,261,1 0,0 0,0 0,0 0,0 0,0 0,360,1
0,0 6,360,4 2,060,4 0,0 3,162,1 3,961,0 0,0 0,0 0,0 0,0 0,0 0,060,0 0,060,0 0,060,0
0,0 0,0 0,0
0,160,0 0,0
0,0 0,0 4,961,2 5,861,8
NID 4
0,260,0 0,260,0 0,0 0,460,3 0,160,1 0,060,1 0,060,0
3,860,1 8,360,2 10,160,1 9,460,6 7,160,7 3,860,3 5,660,3 4,060,4 4,560,4 8,560,7 6,760,4 6,060,2 6,260,7 6,860,7
8,661,2 8,861,0 6,660,5
4,860,7 3,960,2
0,0 0,0 0,860,3 0,960,4
C 18:3
146 M. Malfeito-Ferreira et al. / International Journal of Food Microbiology 38 (1997) 143 – 155
0,660,3 0,560,0 0,660,2 0,560,1 0,260,1 0,260,1 0,260,2 0,060,0 0,060,0 0,160,0 0,460,2 0,460,0 0,160,0 0,060,0 0,160,0 0,060,0 0,060,0 0,060,0
0,160,0 0,160,0 0,060,0 0,760,0
0,160,0 0,360,1 0,260,0 0,260,0 0,560,1 0,160,0
Zygosaccharomyces bailii 1006 0,260,0 0,260,0 1022 0,160,0 0,060,0 1023 0,360,0 0,160,0 1024 0,260,1 0,160,0 1025 0,260,1 0,060,0 1027 0,260,1 0,060,0 1031 0,260,1 0,060,0 1108 0,360,1 0,160,0 1149 2,261,0 0,160,1 1206 0,260,0 0,060,0 1212 0,260,1 0,060,0 1213 0,360,1 0,160,0 1214 0,160,0 0,060,0 1307 0,260,4 0,260,3 1430 0,260,1 0,160,0 1431 0,260,0 0,160,0 1432 0,260,0 0,060,0 1433 0,160,0 0,160,0
Zygosaccharomyces rouxii 1188 0,760,1 0,360,1 1194 0,560,1 0,160,1 1220 0,660,3 0,160,0 1322 0,460,1 0,060,0
Unidentified strains 3 0,560,2 4 0,360,0 5 0,460,1 6 0,460,0 7 0,360,0 9 0,260,0
0,060,0 0,060,0 0,060,0 0,160,0 0,160,0 0,060,0
0,660,4 0,760,2 0,360,4 0,160,0
0,260,3 0,160,0 0,060,0 0,160,0
0,960,2 0,860,1 0,860,1 0,660,0
S. pombe 1190 1191 1192 1193
0,360,0 0,460,3 0,360,1 0,560,1 0,260,1 0,060,0
0,560,1 0,560,1 0,660,2 0,860,4 0,560,1 0,660,1
S. ludwigii 1083 0,960,1 1088 0,860,1 1089 0,860,2 1093 0,760,1 1186 0,660,1 1218 1,360,1
0,060,0 0,360,2 0,060,0 0,160,0 0,160,1 0,060,0
0,060,0 0,160,1 0,060,0 0,260,1
0,560,2 0,460,0 0,560,2 0,460,2 0,360,1 0,360,2 0,260,2 0,160,0 0,160,0 0,260,0 0,260,2 0,460,1 0,160,0 0,160,1 0,360,1 0,160,0 0,160,0 0,160,0
0,760,4 0,560,1 0,560,1 0,160,0
0,360,1 0,560,3 0,360,1 0,560,2 0,260,1 0,260,3
18,761,3 20,061,0 19,961,7 22,160,3 12,160,2 16,760,3
10,861,1 10,060,5 13,961,0 17,361,0
6,960,5 8,160,0 9,760,7 9,560,7 8,960,6 9,660,9 10,761,6 8,460,8 14,161,3 10,360,4 9,160,3 12,060,7 8,960,1 9,060,7 9,761,3 9,060,1 9,660,3 8,860,4
11,760,8 10,560,9 11,660,5 11,960,1
9,260,3 8,460,7 8,260,6 8,560,3 8,460,6 11,161,2
0,460,2 2,360,4 0,460,1 3,260,1 10,860,5 3,560,2
39,161,1 11,361,2 9,661,0 10,160,3
9,960,6 6,361,0 6,860,9 6,061,3 9,461,2 6,661,1 7,861,6 10,861,3 11,660,4 12,261,2 10,161,3 20,861,1 13,460,4 10,760,6 7,561,2 11,560,6 9,561,0 8,560,5
2,360,3 0,860,1 0,860,0 3,160,1
59,262,6 58,164,8 58,661,8 55,663,6 55,161,0 59,161,4
0,0 0,260,1 0,060,1 0,260,1 0,0 0,360,1
0,0 0,460,1 0,260,0 1,060,3
0,360,1 0,260,0 0,0 0,0 0,460,1 0,0 0,160,1 0,160,0 0,260,0 0,460,2 0,0 2,060,4 0,560,3 0,260,1 0,160,0 0,160,1 0,160,1 0,0
0,160,2 0,0 0,0 0,160,1
0,0 0,0 0,060,0 0,760,4 0,0 0,060,0
0,460,1 0,360,1 0,560,1 0,260,1 1,460,1 0,460,1
0,160,1 0,0 0,160,0 0,860,1
0,160,2 0,060,0 0,660,2 0,760,5 0,460,1 0,760,3 0,260,1 0,160,0 0,160,1 0,460,0 1,060,4 0,0 0,460,2 0,160,1 0,160,0 0,060,0 0,060,0 0,660,2
0,860,1 1,060,6 1,060,1 0,460,2
0,460,2 0,560,3 0,260,0 0,060,1 0,660,3 0,160,0
0,260,0 0,560,3 0,160,0 0,460,1 4,160,2 0,760,2
0,360,1 0,0 0,160,0 1,960,2
0,760,2 0,460,0 0,760,3 0,360,3 0,960,1 0,460,0 0,560,1 0,160,0 0,260,1 0,760,0 0,560,0 0,560,2 0,860,5 0,160,0 0,360,1 0,160,0 0,160,0 0,160,0
0,960,2 0,660,4 0,760,1 0,460,3
0,460,1 0,560,2 0,260,1 0,960,4 0,860,3 0,160,0
0,160,0 0,160,1 0,060,0 0,160,1 0,460,1 0,160,1
0,0 0,0 0,360,4 0,460,1
1,160,2 0,460,1 0,660,3 0,560,3 0,560,1 0,760,5 0,360,1 0,060,0 0,060,0 0,360,4 0,460,1 0,760,4 0,960,7 0,0 0,260,2 0,160,0 0,160,1 0,0
0,560,1 0,460,4 0,560,0 0,0
0,360,1 0,360,1 0,760,1 0,560,3 0,560,2 0,460,5
5,160,6 5,560,5 9,860,8 4,160,5 2,560,3 4,760,9
4,360,2 2,260,6 2,760,5 5,060,4
6,760,5 5,960,0 7,760,7 6,160,4 4,960,5 6,360,8 5,961,0 4,460,2 2,660,4 5,660,3 5,360,5 5,860,2 4,060,2 4,660,3 4,560,3 3,260,3 4,160,5 4,360,3
4,060,2 5,360,1 5,760,5 5,460,3
1,360,3 1,660,4 1,960,2 1,860,6 1,660,1 1,760,4
49,361,9 38,060,8 33,261,8 40,061,0 42,161,3 53,761,3
44,160,7 42,761,0 43,860,2 35,960,7
44,162,4 37,960,7 36,762,8 39,263,3 38,760,1 40,162,5 44,261,7 41,360,8 43,360,8 36,061,3 41,461,9 22,660,9 34,062,1 38,361,5 38,961,3 41,961,2 36,561,4 39,460,4
74,362,5 74,160,2 73,460,9 70,960,2
24,761,8 25,763,0 25,061,3 26,861,8 29,362,8 25,061,0
17,961,1 25,160,6 29,560,9 22,060,6 18,960,5 13,660,6
0,360,1 32,760,4 28,362,0 18,860,5
25,960,6 37,160,7 31,762,1 32,760,7 32,962,1 33,160,8 27,165,0 34,261,2 25,460,2 33,261,6 28,961,2 30,760,5 34,360,7 36,360,5 37,961,8 33,861,0 39,560,7 38,260,5
0,560,1 0,660,1 0,360,1 0,660,4
0,060,0 0,460,0 0,360,1 0,260,0 0,360,1 0,360,1
0,0 0,0 0,0 0,0 0,160,0 0,060,0
0,0 0,0 0,060,0 1,860,6
2,761,0 2,560,3 3,762,0 3,160,7 1,760,7 2,361,1 2,461,1 0,060,0 0,060,0 1,760,0 2,160,8 2,760,2 1,260,7 0,060,0 0,060,0 0,0 0,160,1 0,0
2,060,3 3,860,6 3,360,1 6,560,0
2,060,1 2,460,1 2,460,4 2,060,3 1,360,6 0,060,1
7,2160,9 7,460,7 5,960,2 7,060,7 7,460,2 5,860,3
0,0 0,0 0,360,1 5,860,5
0,360,1 0,560,0 0,860,3 0,760,2 0,660,2 0,560,2 0,360,4 0,160,1 0,060,0 0,460,0 1,561,0 1,160,2 0,860,7 0,260,1 0,160,0 0,060,0 0,260,2 0,0
0,560,3 0,760,2 0,760,0 0,0
0,660,1 0,660,3 0,660,3 0,760,4 0,760,8 0,160,0
M. Malfeito-Ferreira et al. / International Journal of Food Microbiology 38 (1997) 143 – 155 147
Fatty acids
C 14:0
0,360,0 0,360,0 0,560,4 0,260,0 0,260,0 0,960,2 0,760,2 0,860,2 0,360,1 0,360,1 0,360,0 0,260,0 0,560,0 0,360,0 0,360,0 0,160,1 0,360,1 0,360,1 0,260,1 0,260,0
ISA
strains
13 15 16 18 19 20 23 24 26 27 28 29 30 31 32 33 36 39 41 43
0,160,0 0,060,0 0,160,0 0,160,0 0,160,0 0,060,0 0,160,0 0,060,0 0,160,0 0,060,0 0,160,0 0,160,1 0,260,1 0,260,1 0,060,0 0,160,0 0,160,1 0,160,0 0,160,1 0,160,0
C 14:1
Table 2. Continued
C 15:0 0,260,0 0,160,0 0,160,0 1,060,7 0,360,1 0,160,1 0,360,1 0,160,0 0,460,1 0,160,0 0,460,1 0,260,0 0,160,1 0,260,1 0,560,0 0,260,0 0,060,0 0,060,0 0,060,0 0,060,0
C 15:1 0,060,0 0,060,0 0,160,0 0,160,0 0,160,0 0,060,0 0,160,0 0,060,0 0,060,0 0,060,0 0,060,0 0,160,0 0,160,1 0,260,2 0,060,0 0,060,0 0,260,1 0,160,0 0,160,0 0,260,0
C 16:0 13,160,6 12,260,4 11,360,8 11,560,1 11,760,4 20,960,6 16,160,9 21,060,5 19,461,5 12,660,7 12,660,7 9,960,5 10,460,1 9,460,7 13,860,6 11,060,1 9,861,0 8,760,3 10,962,4 9,860,5
2,860,2 2,360,2 1,6360,1 11,660,7 8,160,8 1,160,6 25,461,9 0,760,4 3,260,4 2,660,1 13,561,2 14,961,2 46,463,1 18,860,4 11,660,3 13,060,3 11,361,3 9,160,9 7,261,0 8,560,6
C 16:1
NID 1 0,0 0,060,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,160,6 0,0 0,360,0 0,260,0 0,260,0 0,0 0,160,1 0,160,0 1,461,0
C 17:0 0,760,1 0,760,1 0,960,0 1,560,1 1,260,1 0,260,1 0,560,1 0,360,1 0,660,0 0,760,1 0,960,1 0,360,1 0,160,1 0,460,1 0,960,0 0,560,1 0,160,0 0,060,1 0,660,7 0,160,1
NID 2 1,060,2 0,960,1 0,060,1 4,760,2 3,060,6 0,360,2 1,160,1 0,560,1 0,760,0 0,560,5 3,760,9 1,860,5 0,460,1 2,861,0 3,160,3 3,360,4 0,160,0 0,160,0 0,360,3 0,160,1
NID 3 0,260,0 0,260,1 0,160,0 0,360,0 0,460,1 0,060,0 0,0 0,060,0 0,160,1 0,160,0 0,260,1 0,260,1 0,060,0 0,260,1 0,360,0 0,260,1 0,160,1 0,160,1 0,060,0 0,060,0
C 18:0 7,460,7 8,160,5 8,261,4 1,660,2 2,660,3 2,960,3 3,460,7 5,160,3 7,360,4 8,660,7 1,960,3 1,860,3 2,460,4 1,660,1 2,060,1 1,560,1 3,360,2 3,660,5 4,860,2 4,260,6
C 18:1 48,660,9 46,560,3 50,761,0 38,661,8 41,261,4 49,961,7 52,462,2 48,060,8 51,460,8 47,162,6 43,960,9 47,561,7 38,662,3 44,061,1 38,160,6 46,460,1 40,261,1 43,960,5 36,461,0 37,260,7
C 18:2 21,461,1 23,160,7 20,561,9 19,460,7 18,460,3 14,760,8 0,160,0 16,960,8 12,860,9 22,561,7 17,060,4 16,160,3 0,660,3 15,361,2 17,260,2 17,460,4 34,561,5 33,860,4 39,163,2 38,062,0
NID 4 0,160,0 0,160,0 0,160,0 0,160,0 0,160,0 0,060,0 0,060,0 0,060,0 0,160,0 0,160,0 0,060,0 0,060,0 0,0 0,060,0 0,160,0 0,060,0 0,060,0 0,0 0,060,0 0,0
4,260,4 5,560,4 4,960,7 9,560,3 12,860,1 8,960,5 0,060,0 6,760,8 3,860,7 4,760,6 5,860,6 7,060,5 0,360,2 6,461,3 12,060,1 6,160,2 0,060,0 0,160,0 0,360,5 0,160,0
C 18:3
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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, including species with C18:3 acid, such as Pichia membranaefaciens, 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 technological 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 contaminants 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
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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 . .)
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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 . .)
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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 . .)
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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 probable 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 membranaefaciens. 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 conventionally 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 conventionally 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 confirmed as Zyosaccharomyces bailii while ISA 1239 and ISA 1241 strains were Pichia membranaefaciens. 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 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 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) suggested that the heterogeneity of Pichia membranaefaciens may be related with the inconsistency of the identification given by conventional methodologies 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), respectively, 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 distinguish 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 compositions of Zygosaccharomyces bailii, Pichia membranaefaciens, Pichia anomala, Lodderomyces elongisporus, Schizosaccharomyces pombe and Saccharomycodes 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-
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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 composition is only significant when there are considerable variations in environmental conditions. However, 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 centrifugation 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.
References Abel, K., Schmertzing, H., Peterson, J., 1963. Classification of microorganisms by analysis of chemical composition. 1. Feasibility of utilizing gas chromatography. J. Bacteriol. 83, 1039–1044. Augustyn, O.P.H., Kock, J.L.F., 1989. Differentiation of yeast species and strains within a species by cellular fatty acid analysis. 1. Application of an adapted technique to differentiate between strains of Saccharomyces cerevisiae. J. Microbiol. Meth. 10, 9–23. Barnett, J.A., Payne, R.W., Yarrow, D., 1983. Yeasts, characteristics and identification. Cambridge University Press, Cambridge. Botha, A., Kock, J.L.F., 1993. Application of fatty acid profiles in the identification of yeasts. Int. J. Food Microbiol. 19, 35–51. Bousfield, I.J., Smith, G.L., Dando, T.R., Hobbs, G., 1983. Numerical analysis of total fatty acid profiles in the identification of coryneform, nocardioform and some other bacteria. J. Gen. Microbiol. 129, 375–394. ´ M., Wauthoz, P., Lioui, M., Lambert, R., Decallonne, J., Delmee, 1991. A rapid procedure for identification of lactic acid bacteria based on the gas chromatographic analysis of the cellular fatty acid. J. Food Prot. 54, 217–224. Fleet, G., 1992. Spoilage yeasts. Crit. Rev. Biotechnol. 12, 1–14. Guerzoni, M.E., Sinigaglia, M., Gardini, F., 1993. Interaction between isolation source, yeast fatty acid composition and stress tolerance in Saccharomyces cerevisiae and its subspecies. J. Appl. Bacteriol. 75, 588–594. Jeffery, J., Kock, J.L.F., Botha, A., Coetzee, D.J., Botes, P.J., 1995. Changes in lipid composition over the growth cycle of Schizosaccharomyces pombe CBS 356T. Syst. Appl. Microbiol. 18, 335–337. Johnson, B., Nelson, S.J., Brown, C.M., 1972. Influence of glucose concentration on the physiology and lipid composition of some yeasts. Antoine van Leeuwenhoek 38, 129–136. Kock, J.L.F., Botes, P.J., Erasmus, S.C., Lategan, P.M., 1985. A rapid method to differentiate between four species of the Endomycetaceae. J. Gen. Microbiol. 131, 3393–3396. Kreger-van Rij, N.J.W., (Ed.), 1984. The Yeasts, a taxonomic study (3rd ed.). Elsevier Science Publisher, B.V., Amsterdam.
M. Malfeito-Ferreira et al. / International Journal of Food Microbiology 38 (1997) 143 – 155 Lebart, L., Morineau, A., Warwick, R., 1984. Multivariate descriptive statistical analysis. John Wiley and Sons, New York. Loureiro, V., Malfeito-Ferreira, M., 1993. Yeasts in food spoilage. In: Macrae, R., Robinson, R.K., Sadler, M.J., (Eds.), Encyclopaedia of Food Science, Technology and Nutrition (Vol. 7). Academic Press, London, pp. 4344–4349. Malfeito-Ferreira, M., St. Aubyn, A., Loureiro, V., 1989. The long-chain fatty acid composition as a tool for differentiating spoilage wine yeasts. Mycotaxon 36, 35–42. Moreira-da-Silva, M., Malfeito-Ferreira, M., St. Aubyn, A., Loureiro, V., 1994. Long-chain fatty acid composition as a criterion for yeast distinction in the brewing industry. J. Inst. Brew. 100, 17–22. Noronha-da-Costa, P., Rodrigues, C., Spencer-Martins, I., Loureiro, V., 1996. Fatty acid patterns of film forming yeasts and new evidence for the heterogeneity of Pichia membranaefaciens. Lett. Appl. Microbiol. 23, 79–84. Ratledge, C., Evans, C.T., 1989. Lipids and their metabolism. In: Rose, A.H., Harrison, J.S., (Eds.), The Yeasts (Vol. 3, Ch. 10). Academic Press, London, pp. 367–444. Rattray, J.B.M., 1988. Yeasts. In: Ratledge, C., Wilkinson, S.G. (Eds.), Microbial Lipids (Vol. 1). Academic Press, London, pp. 555–697. Smit, E.J., Kock, J.L.F., van der Westhuizen, J.P.J., Britz, T.J.,
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1988. Taxonomic Relationships of Cryptococcus and Tremella Based on Fatty Acid Composition and Other Phenotypic Characters. J. Gen. Microbiol. 134, 2849–2855. Suutari, M., Liukkonem, K., Laakso, S., 1990. Temperature adaptation in yeasts: the role of fatty acids. J. Gen. Microbiol. 136, 1469–1474. Svetashev, V., Vysotskii, V., Ipanova, E.P., Mikhailov, V.V., 1995. Cellular fatty acids of Alteromonas species. Syst. Appl. Microbiol. 18, 37–43. Tredoux, H.G., Kock, J.L.F., Lategan, P.M., 1987. The use of cellular long-chain fatty acid composition in the identification of some yeasts associated with the wine industry. Syst. Appl. Microbiol. 9, 299–306. Van der Vossen, J.M., Hofstra, H., 1996. DNA based typing, identification and detection systems for food spoilage microorganisms: development and implementation. Int. J. Food Microbiol. 33, 35–49. Viljoen, B.C., Kock, J.L.F., Britz, T.J., 1988. The significance of long-chain fatty acid composition and other phenotypic characteristics in determining relationships among some Pichia and Candida species. J. Gen. Microbiol. 134, 1893–1899. Viljoen, B.C., Dykes, G.A., Callis, M., von Holy, A., 1993. Yeasts associated with Vienna sausage packaging. Int. J. Food Microbiol. 18, 53–62.
Fatty acid profiling: a feasible typing system to trace yeast contamination in wine bottling plants M. Malfeito-Ferreira, M. Tareco, V. Loureiro* ´ ˆ ´ Laboratorio 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 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.
Therefore, it would be of considerable value to have 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 conventional 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
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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-Ferreira 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 incubating onto GYP agar (glucose (Merck, Darmstadt, 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 followed 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-daSilva 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 saponification (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 DBwax 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 Principal Component Analysis (PCA) using the SPAD ´ ´ software (Systeme Portable d’Analyse des Donnes ´ Numeriques, Centre International de Statistique et ´ ´ France, d’Informatique Appliquees, Saint-Mande, 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 List of studied yeast strains Species Strains from culture collections S. cerevisiae
S. cerevisiae var bayanus S. cerevisiae var pastorianus Zygosaccharomyces bailii (S. acidifaciens)b Zygosaccharomyces bailii
Zygosaccharomyces rouxii
Pichia membranaefaciens
Dekkera intermedia Brettanomyces bruxellensis Pichia anomala Saccharomycodes ludwigii
S. pombe
Strains isolated from wineries S. cerevisiae var bayanus Zygosaccharomyces bailii
Zygosaccharomyces rouxii Pichia membranaefaciens
Brettanomyces bruxellensis Pichia anomala Lodderomyces elongisporus Saccharomycodes ludwigii Unidentified strains a
Laboratory number a
Origin
ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA ISA
IGC 4072, active dry wine yeast IGC 4614, active dry wine yeast IGC 4455, CBS 1171, type strain IGC 4456, CBS 380, type strain DBVPG 1243, wine CBS 749, type strain IGC 4227, NRRL S9 144 UCD 801, IGC 5167, type strain Lemon concentrate DVBPG 6379, CBS 2902 DVBPG 6453, CBS 3014 DVBPG 6454, CBS 3016 CECT 1231, orange skin CECT 1229, sugar cane Soya beans CBS 107, IGC 3796, type strain IGC 3316, CBS 2098, wine IGC 3321, CBS 2100, wine UCD 605 UCD 615 CBS 605, IGC 3294 CECT 1107 White wine CECT 1382 DBVPG 2704 CECT 1375 CECT 1376 CECT 1377 CECT 1381
1000 1011 1197 1198 1207 1006 1108 1149 1206 1212 1213 1214 1188 1194 1322 1005 1195 1196 1146 1147 1004 1185 1093 1186 1218 1190 1191 1192 1193
ISA 1028, ISA 1029 ISA 1022, ISA 1023, ISA 1024, ISA 1025, ISA 1027, ISA 1031 ISA 1307 ISA 1430, ISA 1431, ISA 1432, ISA 1433 ISA 1220 ISA 1030 ISA 1239, ISA 1241 ISA 1399, ISA 1400, ISA 1401, ISA 1403 ISA 1404 ISA 1423, ISA 1424 ISA 1429 ISA 1327, ISA 1328 ISA 1420 ISA 1421, ISA 1422 ISA 1083, ISA 1088, ISA 1089 3 to 43
145
Sediments in bottled wines Sediments in bottled wines
Equipment for production of sparkling wine Bottled wine plant A Concentrated grape juice Sediments in bottled wine Contaminants in bottled wine Wine filler plant B Contamination in bottled wint plant B Wine filler plant A Bottled wine plant A Bottled sparkling wine Wine filler plant A Wine filler plant A Contaminants in sweet bottled wine 26 strains isolated from plant 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).
0,260,0 0,460,1 0,260,0
0,560,0 1,060,1 0,760,2 0,260,1 1,460,4 2,861,2 0,560,1 0,360,0 0,760,1 0,360,1 0,360,1 0,260,0 0,260,1 0,460,1
0,360,2 0,160,0 0,360,1 0,360,1 0,160,0 0,160,0 0,260,0
0,060,0 0,060,0 0,160,0
Pichia anomala 1004 0,360,0 1185 0,660,2 1420 0,360,0
Pichia membranaefaciens 1005 0,660,0 0,360,1 1030 0,160,0 0,0 1195 0,460,1 0,260,1 1196 0,160,0 0,060,0 1239 1,160,8 0,560,2 1241 1,060,3 0,660,2 1399 0,360,0 0,060,0 1400 0,360,0 0,160,0 1401 0,360,0 0,160,0 1403 0,460,1 0,160,1 1404 0,560,1 0,060,0 1423 0,260,0 0,160,0 1424 0,260,1 0,260,1 1429 0,360,2 0,160,1
S. cerevisiae 1000 0,460,2 1011 0,760,1 1028 1,460,1 1029 1,560,4 1197 0,960,2 1198 0,360,5 1207 1,160,1
0,160,0 0,360,0 0,460,0 0,960,9 0,460,1 0,260,1 0,660,0
0,260,0 0,160,0
C 15:0
Lodderomyces elongisporus 1421 0,360,0 0,060,0 1422 0,260,0 0,060,0
C 14:1
0,260,0 0,460,1 0,960,2 1,260,4
C 14:0
strains
Brettanomyces spp. /Dekkera spp. 1146 3,061,0 0,260,2 1147 1,560,3 0,360,1 1327 1,260,1 0,160,0 1328 1,260,4 0,160,1
Fatty acids
ISA
0,360,1 0,260,0 0,160,0 0,160,1 0,260,0 0,160,0 0,160,0
0,060,0 0,560,0 0,160,0 0,060,0 0,960,3 2,761,7 0,060,0 0,060,0 0,060,0 0,160,1 0,060,0 0,160,0 0,160,1 0,160,1
0,060,0 0,060,0 0,060,0
0,060,0 0,060,1
0,160,1 0,160,1 0,760,2 0,760,3
C 15:1
8,660,7 13,460,6 17,361,8 18,060,7 11,060,5 18,760,5 12,160,9
17,560,4 11,760,8 18,360,9 11,260,8 10,861,0 12,060,2 13,760,2 14,661,1 15,960,9 13,760,6 15,561,4 10,360,4 10,761,4 10,460,8
18,960,6 23,460,3 20,760,2
13,461,0 12,360,2
20,061,0 21,461,0 20,561,4 21,761,4
C 16:0
39,261,6 39,560,9 47,160,9 39,462,4 34,863,0 43,863,1 47,061,9
26,761,9 13,261,2 11,961,5 10,560,3 10,761,4 19,461,2 16,760,7 19,260,1 17,860,5 17,360,3 19,963,6 10,060,5 19,563,2 19,561,9
3,060,2 7,960,7 3,260,2
2,760,2 1,660,1
47,061,8 33,561,2 29,462,5 28,262,1
C 16:1
0,360,2 0,060,0 0,060,0 0,0 0,060,0 0,0 0,0
0,460,0 0,460,1 0,360,0 0,160,1 0,760,1 1,260,5 0,360,0 0,360,0 0,260,0 0,560,1 0,560,4 0,0 0,360,0 0,560,1
0,160,0 0,560,1 0,260,0
0,0 0,0
3,960,6 3,660,4 3,760,3 3,560,1
NID 1
Table 2 Long-chain fatty acid profiles of analysed yeast strains (NID, unidentified fatty acids)
0,160,1 0,260,0 0,160,0 0,260,1 0,160,0 0,260,0 0,360,1
0,560,0 0,660,0 1,560,4 0,560,1 1,060,3 1,160,2 0,960,1 0,560,0 1,060,0 0,560,1 0,160,1 0,660,1 0,360,3 0,660,1
0,260,0 0,160,0 0,260,0
0,760,2 0,760,0
0,0 0,0 0,160,0 0,060,0
C 17:0
0,860,5 0,360,1 0,460,1 0,160,1 0,360,0 0,360,0 0,460,1
0,660,1 2,660,4 1,960,0 1,560,5 2,860,7 5,761,1 4,260,8 2,460,3 5,160,0 1,560,4 0,860,5 3,760,4 2,261,4 3,460,2
0,460,1 0,560,2 0,260,1
0,960,1 0,960,0
0,0 0,660,1 1,360,4 0,960,2
NID 2
2,061,7 0,060,0 0,060,0 0,0 0,160,0 0,260,1 0,060,0
0,0 1,060,0 0,960,3 0,0 1,260,9 2,161,4 0,0 0,0 0,0 0,0 0,0 0,360,1 0,160,2 0,460,0
0,0 0,0 0,160,3
0,260,1 0,260,0
0,0 0,0 1,760,5 2,260,8
NID 3
4,260,6 3,560,2 2,660,1 3,160,5 6,660,8 2,860,5 3,260,4
3,660,3 5,460,1 5,160,9 2,660,5 3,760,8 4,060,5 2,160,2 2,160,2 1,160,4 2,360,5 1,660,6 1,260,4 2,260,7 1,660,1
3,660,3 1,960,1 4,760,2
7,060,4 9,760,3
1,460,4 2,260,5 5,760,1 5,660,9
C 18:0
38,261,9 41,560,3 30,062,3 35,463,2 45,362,6 33,262,3 34,562,1
33,961,6 29,561,1 27,861,9 46,761,1 36,360,3 28,366,3 37,860,3 39,960,5 36,860,7 35,261,3 36,561,3 52,861,8 49,968,9 39,461,8
38,061,7 30,963,1 39,960,4
48,060,5 51,660,6
15,161,2 23,461,4 19,463,9 18,861,8
C 18:1
0,560,4 0,360,2 0,360,1 0,860,4 0,260,2 0,160,1 0,360,1
11,660,4 19,560,3 17,962,2 17,260,1 19,060,8 12,061,5 18,060,9 16,260,2 16,760,6 19,760,5 17,560,8 14,660,4 16,661,2 16,761,1
26,661,2 25,060,6 23,760,9
21,661,1 18,860,8
9,361,2 12,961,2 9,562,0 9,060,9
C 18:2
3,261,1 0,0 0,0 0,0 0,0 0,0 0,360,1
0,0 6,360,4 2,060,4 0,0 3,162,1 3,961,0 0,0 0,0 0,0 0,0 0,0 0,060,0 0,060,0 0,060,0
0,0 0,0 0,0
0,160,0 0,0
0,0 0,0 4,961,2 5,861,8
NID 4
0,260,0 0,260,0 0,0 0,460,3 0,160,1 0,060,1 0,060,0
3,860,1 8,360,2 10,160,1 9,460,6 7,160,7 3,860,3 5,660,3 4,060,4 4,560,4 8,560,7 6,760,4 6,060,2 6,260,7 6,860,7
8,661,2 8,861,0 6,660,5
4,860,7 3,960,2
0,0 0,0 0,860,3 0,960,4
C 18:3
146 M. Malfeito-Ferreira et al. / International Journal of Food Microbiology 38 (1997) 143 – 155
0,660,3 0,560,0 0,660,2 0,560,1 0,260,1 0,260,1 0,260,2 0,060,0 0,060,0 0,160,0 0,460,2 0,460,0 0,160,0 0,060,0 0,160,0 0,060,0 0,060,0 0,060,0
0,160,0 0,160,0 0,060,0 0,760,0
0,160,0 0,360,1 0,260,0 0,260,0 0,560,1 0,160,0
Zygosaccharomyces bailii 1006 0,260,0 0,260,0 1022 0,160,0 0,060,0 1023 0,360,0 0,160,0 1024 0,260,1 0,160,0 1025 0,260,1 0,060,0 1027 0,260,1 0,060,0 1031 0,260,1 0,060,0 1108 0,360,1 0,160,0 1149 2,261,0 0,160,1 1206 0,260,0 0,060,0 1212 0,260,1 0,060,0 1213 0,360,1 0,160,0 1214 0,160,0 0,060,0 1307 0,260,4 0,260,3 1430 0,260,1 0,160,0 1431 0,260,0 0,160,0 1432 0,260,0 0,060,0 1433 0,160,0 0,160,0
Zygosaccharomyces rouxii 1188 0,760,1 0,360,1 1194 0,560,1 0,160,1 1220 0,660,3 0,160,0 1322 0,460,1 0,060,0
Unidentified strains 3 0,560,2 4 0,360,0 5 0,460,1 6 0,460,0 7 0,360,0 9 0,260,0
0,060,0 0,060,0 0,060,0 0,160,0 0,160,0 0,060,0
0,660,4 0,760,2 0,360,4 0,160,0
0,260,3 0,160,0 0,060,0 0,160,0
0,960,2 0,860,1 0,860,1 0,660,0
S. pombe 1190 1191 1192 1193
0,360,0 0,460,3 0,360,1 0,560,1 0,260,1 0,060,0
0,560,1 0,560,1 0,660,2 0,860,4 0,560,1 0,660,1
S. ludwigii 1083 0,960,1 1088 0,860,1 1089 0,860,2 1093 0,760,1 1186 0,660,1 1218 1,360,1
0,060,0 0,360,2 0,060,0 0,160,0 0,160,1 0,060,0
0,060,0 0,160,1 0,060,0 0,260,1
0,560,2 0,460,0 0,560,2 0,460,2 0,360,1 0,360,2 0,260,2 0,160,0 0,160,0 0,260,0 0,260,2 0,460,1 0,160,0 0,160,1 0,360,1 0,160,0 0,160,0 0,160,0
0,760,4 0,560,1 0,560,1 0,160,0
0,360,1 0,560,3 0,360,1 0,560,2 0,260,1 0,260,3
18,761,3 20,061,0 19,961,7 22,160,3 12,160,2 16,760,3
10,861,1 10,060,5 13,961,0 17,361,0
6,960,5 8,160,0 9,760,7 9,560,7 8,960,6 9,660,9 10,761,6 8,460,8 14,161,3 10,360,4 9,160,3 12,060,7 8,960,1 9,060,7 9,761,3 9,060,1 9,660,3 8,860,4
11,760,8 10,560,9 11,660,5 11,960,1
9,260,3 8,460,7 8,260,6 8,560,3 8,460,6 11,161,2
0,460,2 2,360,4 0,460,1 3,260,1 10,860,5 3,560,2
39,161,1 11,361,2 9,661,0 10,160,3
9,960,6 6,361,0 6,860,9 6,061,3 9,461,2 6,661,1 7,861,6 10,861,3 11,660,4 12,261,2 10,161,3 20,861,1 13,460,4 10,760,6 7,561,2 11,560,6 9,561,0 8,560,5
2,360,3 0,860,1 0,860,0 3,160,1
59,262,6 58,164,8 58,661,8 55,663,6 55,161,0 59,161,4
0,0 0,260,1 0,060,1 0,260,1 0,0 0,360,1
0,0 0,460,1 0,260,0 1,060,3
0,360,1 0,260,0 0,0 0,0 0,460,1 0,0 0,160,1 0,160,0 0,260,0 0,460,2 0,0 2,060,4 0,560,3 0,260,1 0,160,0 0,160,1 0,160,1 0,0
0,160,2 0,0 0,0 0,160,1
0,0 0,0 0,060,0 0,760,4 0,0 0,060,0
0,460,1 0,360,1 0,560,1 0,260,1 1,460,1 0,460,1
0,160,1 0,0 0,160,0 0,860,1
0,160,2 0,060,0 0,660,2 0,760,5 0,460,1 0,760,3 0,260,1 0,160,0 0,160,1 0,460,0 1,060,4 0,0 0,460,2 0,160,1 0,160,0 0,060,0 0,060,0 0,660,2
0,860,1 1,060,6 1,060,1 0,460,2
0,460,2 0,560,3 0,260,0 0,060,1 0,660,3 0,160,0
0,260,0 0,560,3 0,160,0 0,460,1 4,160,2 0,760,2
0,360,1 0,0 0,160,0 1,960,2
0,760,2 0,460,0 0,760,3 0,360,3 0,960,1 0,460,0 0,560,1 0,160,0 0,260,1 0,760,0 0,560,0 0,560,2 0,860,5 0,160,0 0,360,1 0,160,0 0,160,0 0,160,0
0,960,2 0,660,4 0,760,1 0,460,3
0,460,1 0,560,2 0,260,1 0,960,4 0,860,3 0,160,0
0,160,0 0,160,1 0,060,0 0,160,1 0,460,1 0,160,1
0,0 0,0 0,360,4 0,460,1
1,160,2 0,460,1 0,660,3 0,560,3 0,560,1 0,760,5 0,360,1 0,060,0 0,060,0 0,360,4 0,460,1 0,760,4 0,960,7 0,0 0,260,2 0,160,0 0,160,1 0,0
0,560,1 0,460,4 0,560,0 0,0
0,360,1 0,360,1 0,760,1 0,560,3 0,560,2 0,460,5
5,160,6 5,560,5 9,860,8 4,160,5 2,560,3 4,760,9
4,360,2 2,260,6 2,760,5 5,060,4
6,760,5 5,960,0 7,760,7 6,160,4 4,960,5 6,360,8 5,961,0 4,460,2 2,660,4 5,660,3 5,360,5 5,860,2 4,060,2 4,660,3 4,560,3 3,260,3 4,160,5 4,360,3
4,060,2 5,360,1 5,760,5 5,460,3
1,360,3 1,660,4 1,960,2 1,860,6 1,660,1 1,760,4
49,361,9 38,060,8 33,261,8 40,061,0 42,161,3 53,761,3
44,160,7 42,761,0 43,860,2 35,960,7
44,162,4 37,960,7 36,762,8 39,263,3 38,760,1 40,162,5 44,261,7 41,360,8 43,360,8 36,061,3 41,461,9 22,660,9 34,062,1 38,361,5 38,961,3 41,961,2 36,561,4 39,460,4
74,362,5 74,160,2 73,460,9 70,960,2
24,761,8 25,763,0 25,061,3 26,861,8 29,362,8 25,061,0
17,961,1 25,160,6 29,560,9 22,060,6 18,960,5 13,660,6
0,360,1 32,760,4 28,362,0 18,860,5
25,960,6 37,160,7 31,762,1 32,760,7 32,962,1 33,160,8 27,165,0 34,261,2 25,460,2 33,261,6 28,961,2 30,760,5 34,360,7 36,360,5 37,961,8 33,861,0 39,560,7 38,260,5
0,560,1 0,660,1 0,360,1 0,660,4
0,060,0 0,460,0 0,360,1 0,260,0 0,360,1 0,360,1
0,0 0,0 0,0 0,0 0,160,0 0,060,0
0,0 0,0 0,060,0 1,860,6
2,761,0 2,560,3 3,762,0 3,160,7 1,760,7 2,361,1 2,461,1 0,060,0 0,060,0 1,760,0 2,160,8 2,760,2 1,260,7 0,060,0 0,060,0 0,0 0,160,1 0,0
2,060,3 3,860,6 3,360,1 6,560,0
2,060,1 2,460,1 2,460,4 2,060,3 1,360,6 0,060,1
7,2160,9 7,460,7 5,960,2 7,060,7 7,460,2 5,860,3
0,0 0,0 0,360,1 5,860,5
0,360,1 0,560,0 0,860,3 0,760,2 0,660,2 0,560,2 0,360,4 0,160,1 0,060,0 0,460,0 1,561,0 1,160,2 0,860,7 0,260,1 0,160,0 0,060,0 0,260,2 0,0
0,560,3 0,760,2 0,760,0 0,0
0,660,1 0,660,3 0,660,3 0,760,4 0,760,8 0,160,0
M. Malfeito-Ferreira et al. / International Journal of Food Microbiology 38 (1997) 143 – 155 147
Fatty acids
C 14:0
0,360,0 0,360,0 0,560,4 0,260,0 0,260,0 0,960,2 0,760,2 0,860,2 0,360,1 0,360,1 0,360,0 0,260,0 0,560,0 0,360,0 0,360,0 0,160,1 0,360,1 0,360,1 0,260,1 0,260,0
ISA
strains
13 15 16 18 19 20 23 24 26 27 28 29 30 31 32 33 36 39 41 43
0,160,0 0,060,0 0,160,0 0,160,0 0,160,0 0,060,0 0,160,0 0,060,0 0,160,0 0,060,0 0,160,0 0,160,1 0,260,1 0,260,1 0,060,0 0,160,0 0,160,1 0,160,0 0,160,1 0,160,0
C 14:1
Table 2. Continued
C 15:0 0,260,0 0,160,0 0,160,0 1,060,7 0,360,1 0,160,1 0,360,1 0,160,0 0,460,1 0,160,0 0,460,1 0,260,0 0,160,1 0,260,1 0,560,0 0,260,0 0,060,0 0,060,0 0,060,0 0,060,0
C 15:1 0,060,0 0,060,0 0,160,0 0,160,0 0,160,0 0,060,0 0,160,0 0,060,0 0,060,0 0,060,0 0,060,0 0,160,0 0,160,1 0,260,2 0,060,0 0,060,0 0,260,1 0,160,0 0,160,0 0,260,0
C 16:0 13,160,6 12,260,4 11,360,8 11,560,1 11,760,4 20,960,6 16,160,9 21,060,5 19,461,5 12,660,7 12,660,7 9,960,5 10,460,1 9,460,7 13,860,6 11,060,1 9,861,0 8,760,3 10,962,4 9,860,5
2,860,2 2,360,2 1,6360,1 11,660,7 8,160,8 1,160,6 25,461,9 0,760,4 3,260,4 2,660,1 13,561,2 14,961,2 46,463,1 18,860,4 11,660,3 13,060,3 11,361,3 9,160,9 7,261,0 8,560,6
C 16:1
NID 1 0,0 0,060,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,160,6 0,0 0,360,0 0,260,0 0,260,0 0,0 0,160,1 0,160,0 1,461,0
C 17:0 0,760,1 0,760,1 0,960,0 1,560,1 1,260,1 0,260,1 0,560,1 0,360,1 0,660,0 0,760,1 0,960,1 0,360,1 0,160,1 0,460,1 0,960,0 0,560,1 0,160,0 0,060,1 0,660,7 0,160,1
NID 2 1,060,2 0,960,1 0,060,1 4,760,2 3,060,6 0,360,2 1,160,1 0,560,1 0,760,0 0,560,5 3,760,9 1,860,5 0,460,1 2,861,0 3,160,3 3,360,4 0,160,0 0,160,0 0,360,3 0,160,1
NID 3 0,260,0 0,260,1 0,160,0 0,360,0 0,460,1 0,060,0 0,0 0,060,0 0,160,1 0,160,0 0,260,1 0,260,1 0,060,0 0,260,1 0,360,0 0,260,1 0,160,1 0,160,1 0,060,0 0,060,0
C 18:0 7,460,7 8,160,5 8,261,4 1,660,2 2,660,3 2,960,3 3,460,7 5,160,3 7,360,4 8,660,7 1,960,3 1,860,3 2,460,4 1,660,1 2,060,1 1,560,1 3,360,2 3,660,5 4,860,2 4,260,6
C 18:1 48,660,9 46,560,3 50,761,0 38,661,8 41,261,4 49,961,7 52,462,2 48,060,8 51,460,8 47,162,6 43,960,9 47,561,7 38,662,3 44,061,1 38,160,6 46,460,1 40,261,1 43,960,5 36,461,0 37,260,7
C 18:2 21,461,1 23,160,7 20,561,9 19,460,7 18,460,3 14,760,8 0,160,0 16,960,8 12,860,9 22,561,7 17,060,4 16,160,3 0,660,3 15,361,2 17,260,2 17,460,4 34,561,5 33,860,4 39,163,2 38,062,0
NID 4 0,160,0 0,160,0 0,160,0 0,160,0 0,160,0 0,060,0 0,060,0 0,060,0 0,160,0 0,160,0 0,060,0 0,060,0 0,0 0,060,0 0,160,0 0,060,0 0,060,0 0,0 0,060,0 0,0
4,260,4 5,560,4 4,960,7 9,560,3 12,860,1 8,960,5 0,060,0 6,760,8 3,860,7 4,760,6 5,860,6 7,060,5 0,360,2 6,461,3 12,060,1 6,160,2 0,060,0 0,160,0 0,360,5 0,160,0
C 18:3
148 M. Malfeito-Ferreira et al. / International Journal of Food Microbiology 38 (1997) 143 – 155
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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, including species with C18:3 acid, such as Pichia membranaefaciens, 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
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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 technological 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 contaminants 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
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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 . .)
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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 . .)
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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 . .)
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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 probable 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 membranaefaciens. 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 conventionally 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 conventionally 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 confirmed as Zyosaccharomyces bailii while ISA 1239 and ISA 1241 strains were Pichia membranaefaciens. 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 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 analy-
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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) suggested that the heterogeneity of Pichia membranaefaciens may be related with the inconsistency of the identification given by conventional methodologies 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), respectively, 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 distinguish 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 compositions of Zygosaccharomyces bailii, Pichia membranaefaciens, Pichia anomala, Lodderomyces elongisporus, Schizosaccharomyces pombe and Saccharomycodes 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-
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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 composition is only significant when there are considerable variations in environmental conditions. However, 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 centrifugation 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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