Characterization of oenological strains of
Saccharomyces cerevisiae using cellular fatty acid and mtDNA restriction polymorphism analysis
Ana R. GUTIERREZ 1*, Rosa LÓPEZ 2, Pilar SANTAMARÍA 2, María J. SEVILLA 3
RÉSUMÉ Caractérisation de souches œnologiques de Saccharomyces cerevisiae par l’analyse des acides gras cellulaires et analyse des profils de restriction de l’ADN mitochondrial.
L’analyse des acides gras cellulaires et l’analyse du DNA mitochondrial ont été comparées pour la caractérisation de souches de Saccharomyces cerevisisae.
Les deux méthodes ont donné des résultats similaires dans l’étude des sept souches isolées, qui ont été groupées en trois types. Cependant, les résultats obtenus par l’analyse de restriction du DNA ont été plus clairs et plus faciles à interpréter. De plus, la technique d’identification par l’analyse des acides gras cellulaires n’est pas appropriée pour l’étude d’un grand nombre d’échantillons.
Mots clés : caractérisation clonale, acides gras cellulaires, DNA mitochondrial, Saccharomyces cerevisiae, vin.
SUMMARY
Cellular fatty acid analysis and mitochondrial DNA restriction analysis are com- pared for categorizing isolates of Saccharomyces cerevisiae. The two methods gave similar results when seven isolates were studied, with three groups being differentiated. However, the results obtained with the mitochondrial DNA res- triction analysis were clearer and easier to interpret than the results obtained with the cellular fatty acid analysis. Moreover, this latter technique was not adequate when a high number of samples were studied.
Key-words: clonal characterisation, cellular fatty acids, mitochondrial DNA, Saccharomyces cerevisiae, wine.
1. Departamento de Agricultura y Alimentación, Universidad de La Rioja, Avda. de la Paz 105, 26004 Logroño, España.
2. Centro de Investigación y Desarrollo Agrario de La Rioja, Apdo, 433, 26080 Logroño, España.
3. Departamento de Inmunología, Microbiología y Parasitología, Universidad del País Vasco, 48080 Bilbao, España.
* Correspondence
1 - INTRODUCTION
Wine quality is significantly affected by the particular strain of Saccharo- myces cerevisiae used in the fermentation. Thus, important quality parameters such as volatile acidity, sulphide production, aromatic character and dryness of fermentation can be determined by the strain of Saccharomyces cerevisiae (GIU- DICIand ZAMBONELLI, 1992; GIUDICIet al., 1993). Wine fermentation was traditio- nally carried out by indigenous yeast associated with the grapes and the cellar equipment. In the last twenty years, winemakers have begun employing starter cultures of yeast for specific fermentations and numerous programmes have been developed to select strains which are better adapted to both different regions of the world and to their respective grape varietals.
After the addition of starter yeast, more than one strain of Saccharomyces cerevisiae may be present during wine fermentation, because the initial must is not sterilized (FREZIERand DUBOURDIEU, 1992; QUEROLet al., 1994; BRIONES et al., 1996; CONSTANTIet al., 1997). It is therefore necessary to monitor yeast cell population kinetics in order to measure the success of the inoculation. Conse- quently, the ability to differentiate between strains is becoming an important requirement of modern winemaking (FLEET, 1994).
Because conventional identification schemes do not differentiate between strains, several technologies have been developed to distinguish types within species. These techniques can be classified in two groups. The first group is based on the analysis of metabolic compounds, such as exocellular fractions (BOUIXet al., 1981), cellular proteins (DEGRÈet al., 1990; VAN DER WESTHUIZEN and PRETORIUS, 1992), or cellular fatty acids (TREDOUXet al., 1987; AUGUSTYN, 1989; AUGUSTYNet al., 1992; JOLLYet al., 1993). The second group is based on the analysis of the yeast genome: total DNA analysis (DEGRÈet al., 1989), karyo- typing or chromosomal pattern analysis (VEZINHET et al., 1990; AZIAC et al., 1991; VERSAVAUDet al., 1993, IZQUIERDOet al., 1997) or mitochondrial DNA ana- lysis (DUBOURDIEUet al., 1987; QUEROLet al., 1992b; 1992 c).
The first group of techniques has to be performed under carefully defined culture conditions, as the biosynthesis of the compounds analyzed will vary according to the conditions employed (HIDALGOet al., 1992). The second group of techniques is independent of the culture conditions and offers great advan- tages because the identity of the strains can be studied by their genetic charac- teristics and not by their physiological profile.
In this paper we compare two techniques, cellular fatty acid analysis and the mitochondrial DNA restriction pattern, to differentiate indigenous strains of S.
cerevisiae isolated from wine fermentations in La Rioja (Spain). OOSTHUIZEN et al. (1987) and MALFEITO-FERREIRA et al. (1989) reported that chromatographic profiles of the fatty acids comprising cell lipids are sufficiently discriminating to allow differentiation of Saccharomyces cerevisiae wine strains. On the other hand, mitochondrial DNA restriction analysis is a widely accepted technique for this purpose because it has a high discriminatory power, is quick and is not a technically complex method (QUEROLet al., 1992a; VEZINHETet al., 1994). Many cellars and oenology laboratories do not have the instruments and personnel needed to carry out molecular biology techniques; however, instruments nee-
ded to perform fatty acid analysis are widely available. Validating the fatty acid analysis against the molecular technique may therefore be valuable.
2 - MATERIALS AND METHODS
2.1 Yeast
Twenty indigenous strains of Saccharomyces cerevisiae (table 1) from La Rioja (Spain) were used. These twenty strains came from the end stage of a selection program carried out with 250 strains of S. cerevisiae from wineries located in different places of La Rioja (Spain) (GUTIERREZ, 1994). The strains were grown on Malt-agar slants at 28ºC, for 48 h, and maintained at 4 ºC. They were identified following the classification schemes described in KREJER-VAN RIJ (1984) and BARNETTet al. (1990).
Table 1
Indigenous Saccharomyces cerevisiae from the Rioja region which were analyzed in the present study
Strains Village Isolation
TA.32 Aldea Beginning of fermentation
GA.71 Aldea Wine
RA.54 Aldea End of fermentation
RA.63 Aldea Wine
1.55 Ausejo* Vigorous fermentation 3.22 Ausejo* End of fermentation 3.53 Ausejo* End of fermentation 1.95 Ausejo* Vigorous fermentation 1.94 Ausejo* Vigorous fermentation
BT.5 Ausejo* Wine
V.22 Logroño Wine
V.21 Logroño Wine
RSV.45 San Vicente Vigorous fermentation RSV.51 San Vicente End of fermentation
RSV.73 San Vicente Wine
RSV.71 San Vicente Wine
TH.44 Haro Vigorous fermentation
TH.54 Haro End of fermentation
GH.21 Haro Grape
GH.64 Haro Wine
* Experimental winery of the Centro de Investigaciones Agra- rias de La Rioja.
In some of the analyses, only the seven strains that finished the selection program were used.
2.2 Cellular fatty acid analysis
Each strain was grown in a culture (1.7 g·L–1 yeast nitrogen base, 40 g·L–1 glucose, 5 g·L–1peptone) at 28ºC for 48 h. Samples from each culture contai- ning equivalent amounts of biomass (determinated by absorbance at 610 nm) were extracted according to RIBES et al. (1988) with two minor modifications:
the methyl esters of the fatty acids were extracted three times with hexane ins- tead twice with pentane. The fatty acid methyl esters were analyzed by gas chromatography with nitrogen as the carrier gas in a HEWLETT PACKARD 5890 gas chromatograph with a capillary column FFAP (HP), 25 m long and 0.2 mm (I.D.). A 2 µL fraction of each sample was injected. The oven temperatures were 50 ºC (0.50 min) to 220 ºC (10 ºC·min–1). Injector and detector temperatures were set at 220 ºC. Fatty acid extraction and gas chromatography analysis of one strain was repeated 10 times to verify the repeatibility of the method.
With the relative percentages of fatty acids from each strain, a cluster analy- sis was performed with SYSTAT(1992) statistical package.
2.3 Mitochondrial DNA (mtDNA) analysis
Yeast cells were grown overnight in 5 ml of a yeast extract peptone glucose medium (YEPD; 1% yeast extract, 2% peptone, 2% glucose). DNA extraction and mtDNA profiles were determined by the rapid method developed by QUE- ROL and BARRIO (1990) after applying the restriction endonucleases Hinf 1 and Alu 1. This method permits the analysis of a large number of samples, by avoi- ding the process of isolating mitochondria. Restriction fragments were separa- ted on horizontal 1% agarose gels (Boehringer Mannheim) in 1 × TBE buffer (45 mM Tris-borate, 1 mM EDTA, pH 8) and visualized in a UV transiluminator after ethidium bromide staining.
Table 2
Fatty acid composition of seven strains of Saccharomyces cerevisiaea Strains
Fatty acids
V.22 RSV.45 RSV.73 TH.44 TH.54 3.22 3.53
C6:0 3.31 0.00 0.36 0.00 0.11 0.21 0.31
C8:0 2.49 2.16 3.35 2.38 0.77 1.61 1.96
C10:0 7.40 6.23 7.14 3.98 3.85 4.95 5.55
X 1.07 0.85 0.99 0.65 0.85 0.86 0.58
C12:0 2.68 2.71 3.49 2.53 2.53 2.27 2.72
C14:0 1.89 1.95 2.86 1.84 1.47 1.33 1.21
C14:1 0.52 0.41 0.40 0.42 0.40 0.24 0.21
C15:0 0.26 0.32 0.19 0.86 0.18 0.10 0.12
C16:0 23.89 24.67 24.07 28.76 19.63 23.78 22.84
C16:1 25.36 26.41 24.83 23.87 32.91 24.85 21.48
C18:0 7.87 8.34 8.14 8.50 7.07 7.41 12.24
C18:1 16.47 17.35 16.69 16.41 24.45 22.85 14.09
C18:2 0.25 0.34 0.00 0.80 0.21 0.18 0.20
Y 9.51 8.24 8.50 8.01 5.56 9.37 16.38
aThe values are expressed in relative percentages.
3 - RESULTS AND DISCUSSION
3.1 Cellular fatty acid
The chromatographic analysis of the cellular fatty acids allowed us to iden- tify up to 14 different compounds (figure 1). Two of them were not identified, but as they appeared systematically, we called them X and Y. No fatty acids of 20 and 22 carbon atoms appeared in any of the strains, which is in good agree- ment with the results previously observed for Saccharomyces cerevisisae by ROZESand LONVAUD-FUNEL(1991).
Seven Saccharomyces cerevisisae isolates were analyzed (RSV.45, RSV.73, V.22, TH.44, TH.54, 3.22 and 3.53). Except for strain 3.53, where the un-identi- fied compound Y was more abundant than stearic and oleic acids, the main cel- lular fatty acids in the strains studied were palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0) and oleic (C18:1) acids (table 2), making up to 70% of the total
Figure 1
Gas chromatography of the methyl esters of the cellular fatty acids of Saccharomyces cerevisiae (strain RSV.73)
1: caproic acid (C6: 0); 2: caprilic acid (C8: 0); 3: nonanoic acid (C9: 0) (internal standard); 4: capric acid (C10: 0); X: unidentified compound; 5: lauric acid (C12: 0); 6: miristic acid (C14: 0); 7: miristoleic acid (C14: 1); 8: pentadecanoic acid (C15: 0); 9: palmitic acid (C16: 0); 10: palmitoleic acid (C16: 1);
11: heptadecanoic acid (C17: 0; internal standard); 12: stearic acid (C18: 0); 13: oleic acid (C18: 1); 14:
linoleic acid (C18: 2); Y: unidentified compound.
cellular fatty acids, as has been previously reported by other authors (TREDOUX et al., 1987; RIBESet al., 1988).
The relative percentages of the long-chain fatty acids (C12-C18) were consi- dered for the cluster analysis, following previous methods (TREDOUXet al., 1987;
RIBESet al., 1988; AUGUSTYNet al., 1992). Three categories (A, B and C) resul- ted from this analysis (figure 2). Group A, was made up of two strains, 3.53 and 3.22, with less C14:0 and C14:1 than the remaining five strains. Secondly, group B was made up of strains RSV.73., RSV.45, V.22 and TH.44. The first three of these strains showed no significant differences in the abundance of the fatty acids considered and we can speculate that they originate from a unique clone. Finally, we identified group C containing only one strain, TH.54, which produced the lowest amount of C16:0 and the highest amounts of C16:1 and C18:1.
Figure 2
Grouping of seven strains of Saccharomyces cerevisiae according to their fatty acid composition (C12-C18)
Some authors have suggested that fatty acid analysis is only useful for a small number of clones (AUGUSTYNet al., 1992; JOLLYet al., 1993), or to distin- guish between different genera (ROZES and LONVAUD-FUNEL, 1991). For these reasons, fatty acid analysis of another thirteen strains, that arrived during the last stage of the selection program, was carried out. As suggested by AUGUS- TYNet al., (1992), when the number of clones under study was increased and all of the fatty acids distinguished (C6-C18) were considered, the meaning of the results was even less clear (figure 3). Not even the three strains previously considered to be the same (RSV.73, RSV.45, V.22), could now be considered to be closely related. This method is not, therefore, adequate to distinguish bet- ween many strains, which is necessary for the monitoring of wine fermentations undertaken using selected yeast strains.
3.2 Mitochondrial DNA restriction analysis
The high intraspecific variability occurring in the mitochondrial DNA of Sac- charomyces cerevisiae, shown by restriction analysis, has been used for cha- racterizing the different strains of that species. For the restrictions analysis, we used five different restriction enzymes (Alu I, Hinf I, Eco RI, Eco RV, Rsa I) that have been previously used by other authors (QUEROL, 1992; GUILLAMON, 1996).
Two enzymes, Alu 1 and Hinf 1, showed the highest number of patterns, that is, Figure 3
Grouping of twenty strains of Saccharomyces cerevisiae according to their fatty acid composition (C6-C18)
the highest discriminatory ability. Considering the patterns revealed by these two enzymes, we grouped the seven strains into three types (figure 4). Strains V.22, RSV.45, RSV.73 and TH.44 belong to the same type (pattern B); strains 3.22 and 3.53 make a second different type (pattern A), and finally, strain TH.54 makes a third type (pattern C).
Complete agreement was observed between the characterization obtained by both methods, which indicates that both are valid for these seven strains.
However, the results obtained with the mitochondrial DNA restriction analysis, were more clear-cut and easier to interpret. With this technique, four of our strains fell into the same group, although their cellular fatty acids pattern are not totally coincident. This analysis indicated that strain TH.44 was identical to the other three (RSV.45, RSV.73 and V.22) whereas fatty acid analysis did not show the same clear result.
The small variability found among the strains is due to the fact that the strains which succeeded in the selection process were isolated from the same winery or from wineries very close to it.
Figure 4
Mitochondrial DNA restriction patterns of seven strains of Saccharomyces cerevisiae using two different restriction enzymes, Alu I and Hinf I. The MW markers in the left-
hand lane (0) correspond to lambda phage treated with Eco RI and Hinf III
* Strains (1) V.22; (2) RSV.45; (3) RSV.73; (4) TH.44; (5) TH.55; (6) 3.22; (7) 3.53
4 - CONCLUSION
Our results are in agreement with previous works carried out on the intraspe- cific identification of oenological strains of Saccharomyces cerevisiae. DNA res- triction and other techniques based on the analysis of the polymorphism of nucleic acids are powerful and reliables methods for the microbiological control in inoculated fermentations of grape must. Cellular fatty acids analysis is useful only when a small number of strains is studied.
Received 19 November 1998, revised 13 January 2000, accepted 9 March 2000.
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