International Journal of Food MicrobiOlogy', 14 (1991) 153-160 153 © 1991 Elsevier Science Publishers B.V. All rights reserved 0168-1605/91/$03.50

FOOD 00444

Contribution of different yeasts isolated from musts of monastrell grapes to the aroma of wine

J.J. M a t e o 1, M. J i m e n e z 1, T. H u e r t a 1 a n d A. P a s t o r : J Departamento de Microbiologfa, Facultad CC Biol6gicas, and 2 Departamento de Qulmica Analitica,

Facultad CC Odmicas, Burjasot, Valencia, Spare

(Received 13 March 1991; accepted 30 August 1991)

Volatile substances of wines obtained by fermentation of musts from 'Monastrelr grapes (Alicante, Spain) was studied for yeast isolated from such musts. The results of the statistical treatment performed show the importance of yeasts of low fermentative power, particularly Kloeckera apiculata, in the production of volatile substances. Saccharomyces cereuisiae var. chevalieri was found to be the most important yeast of high fermentative power.

Key words: Yeast; Volatile; Gas chromatography; Fermentation; Wine

Introduction

Grape must, because of its composition (Amerine et al., 1982), is a highly suitable medium for the growth of a large number of microorganisms. Thus, fresh musts usually contain a wide variety of fungi, yeasts and bacteria (Kunkee et al., 1977). However, they diminish throughout fermentation as a result of the increas- ing concentration of ethanol (Mareca, 1983);, by the end of the process, various strains of Saccharomyces cerevisiae are virtually the only microbial species found in wine (Lafon Laforcade, 1983), which led many authors (Domerq, 1957; Kunkee and Amerine, 1970) to regard this yeast as the one organism responsible for the fermentation process.

Despite the apparent simplicity of fermentation, some sensory properties of wine, particularly the aroma, reveal the occurrence of complex changes during the process. Even though it seems to be the result of various factors including the grape variety used, the transport, storage and fermentation conditions (Cordon- nier, 1970; Cordonnier and Bayonove, 1981), the aroma of wine is mainly deter- mined by the physico-chemical processes involved in fermentation, particularly those occurring in the must or wine at any stage during fermentation play the most

Correspondence address: J.J. Mateo, Departamemo de Microbiologia, Facultad CC Biol6gicas, Dr. Moliner 50, E-46100 Bu~asot, Valencia, Spain.

154

significant role in the production of volatile compounds. Some authors (Di Stefano et al., 1981; Alvarez et al., 1984: Cabrera et al., 1988) assign a quite significant role to apiculate yeasts such as Kloeckera apiculata and Hanseniospora uL'arurn, which occur in the fermentation medium in the early stages of the process, while others (Zeeman et al., 1982) believe the aroma of wine is basically determined by the metabolism of S. cerevisiae.

The aim of this work was clarifying the role of different yeasts isolated in musts from 'Monastrell' grapes in the formation of the aroma of wine in order to determine the relative significance of such microorganisms to the production of volatile compounds during fermentation.

Materials and Methods

Yeasts All yeasts species were isolated from musts and wines obtained from grapes

harvested in Sax, Alicante (Spain). Yeasts species were: Candida valida, Bret- tanomyces bruxellensis, Rhodotorula aurantica, Hanseniospora ut'arum, Kloeckera apiculata, Deckera intermedia (low fermentative power yeasts), Saccharomyces cerevisiae var. capensis, S. cerevisiae var. chevalieri, S. cerevisiae var. bayanus, S. cerevisiae var. cerevisiae (high fermentative power yeasts) and their identification was based on Lodder's "The Yeasts" (1970). The occurrence of such yeasts in these musts was previously reported (Querol et al., 1990). A small amount of cells of each species was transferred from the original culture to a tube with malt-agar medium (Glucose (Panreac) 20 g, malt extract (Oxoid) 20 g, mycological peptone (Oxoid) 1 g, European Bacteriological Agar (Pronadisa) 20 g, distilled water 1000 ml) and then incubated at 28 °C during 2 days.

Fermentation 1000-ml Erlenmeyer flasks were sterilized by autoclaving, filled with 500 ml of

sterilized Sax must and inoculated with 5 • 105 cells/ml of each yeast, respectively. Fermentations were carried out at 28 °C and the end of CO2 formation was used as criterion for the end of fermentation (Hock et al., 1984). All operations were conducted under strictly sterile conditions. After fermentations, flasks were stored at 4 °C during 7 days to precipitate insoluble bitartrate. Yeast cells were removed by centrifugation at 15 000 x g.

Isolation of t'olatiles The centrifuged fermented samples were added 8% (w/v) NaCI (Lamikanra,

1987) and volatiles were extracted with 3:2 pentane/dichloromethane (Usseglio Tomasset, 1969) by continuous liquid-liquid extraction (Rapp et al., 1976; Usseglio Tomasset and Di Stefano, 1980). The extracting solvents were 99.9% pure and were used without further purification. The extracts were filtered after drying over anhydrous sodium sulfate and concentrated to a volume of 2 ml with a Vigreux column. The samples were stored in air-tight containers at - 2 0 °C until the gas chromatographic analysis.

155

Analysis of volatile compounds Volatiles were analysed using a Perkin Elmer model Sigma 3 gas chromatograph

furnished with F.I.D. and a stainless steel column (4 m x 2 mm i.d.) packed with 15% Carbowax 1500 on Chromosorb W-HP 80/100 mesh. The nitrogen carrier flow-rate was 30 ml/min. The detector and injector temperature was 250 * C, and the hydrogen and air flow-rate wave 30 and 500 ml/min, respectively. The original oven temperature (40 ° C) was kept for 6 min and then increased to 160 *C at a rate of 3 ° C/min and was finally held at 160 °C for 30 rain. 1 ~1 of the aroma concentrate was injected for each analysis. Chromatographic peaks were recorded and integrated with a Hewlett Packard model 3390-A recorder integrator.

Identification and quantification of volatiles Volatiles were identified from their relative retention times (RRT), which were

previously determined by injection of standards containing ethanol, 1-butanol and the compound concerned. Quantitation was accomplished by comparison with standard curves obtained by triplicate analysis of standard solutions of 'synthetic' wines (Mateo et al., 1990). Two fermentations were carried out with each yeast, and each sample was injected in triplicate. The average content and coefficient of variation of each compound was determined.

Data treatment The statistical methods used for data processing were multiple correlation

analysis and cluster analysis. The Lotus 1-2-3 v. 2.0 program (Lotus Development Co., 1986) was used for multiple correlation analysis. Cluster analysis was carried out using the BMDP package (BMDP 2M program) (Dixon et al., 1985).

Results and Discussion

Table I lists the concentrations and coefficients of variation of each volatile. The concentrations given for the unidentified components were estimated by the chromatographic response factor intermediate between those of adjacent known compounds (Mateo et al., 1990). As a rule, the concentrations found were always comparable to those reported by other authors (Schreier, 1979; Baumes et al., 1986).

The mean coefficients of variation obtained in the determination of each compound in most cases were less than 10%. This range of variation confirms the reproducibility of the analytical procedure used and is consistent with those reported by other authors who used packed columns (Romero, 1985).

As can be seen from Table I, the yeasts investigated varied in their aroma production.

Table II shows the overall production of volatile compounds by the different yeasts, as well as those of higher alcohols and esters and the ratios between them. Ethyl acetate production was dealt with individually as this ester is the major component and may mask the contribution from the other esters which are highly

156

TABLE I

Concentration. coefficient of variation and comparative study of major volatile compounds produced b~ different yeasts

PEAK ~ RRT b Compound Candida Brettanomyces Rhodotorula Hansentospora t'alida bru.xellensis aurantica ut'antm

mg/ l C.V. mg / I C.V. mg/'l C.V. mg/ I C.V.

8 0.364 Isopropyl acetate 15.0 1.82 9 0.399 Ethyl acetate 1360.0 3.80

10 0.461 Methanol 11 0.504 Ethyl propanoate 1.0 18.39 12 0.612 Isobuthyl acetate 0.3 0.18 13 0.653 Ethyl butanoate 0.1 5.85 14 0.717 2-Butanol +

buthyl acetate 0.10 3.02 15 0.768 1-Propanol 15.2 5.74 16 0.813 Compound 16 c 1.4 • 4.16 17 0.873 Isobutanol +

isoaeyl acetate 65.0 8.94 18 0.958 Amyl acetate 19 1.000 1-Butanol 1.3 13.59 20 1.083 Ethyl caproate 0.2 5.74 21 1.132 Isoamyl alcohol 86.0 3.12 22 1.198 Hexyl acetate 23 1.218 Amyl alcohol 0.2 11.59 24 1.243 Compound 24 c 0.2 = 9.14 25 1.344 Compound 25 c 0.6 • 6.08 26 1.374 Compound 26 c 0.1 = 0.96 27 1.424 1-Hexanol +

ethyl lactate 2.9 3.32 28 1.480 Compound 28 c 0.01 = 8.37 29 1.514 Ethyl caprilate 0.4 6.85 30 1.547 Compound 30 c 0.1 • 7.59 31 1.617 1-Heptanol 0.1 13.95 32 1.654 Compound 32 c 0.1 • 4.68 33 1.744 Benzaldehyde c 0.1 9.39 34 1.771 1-Octanol 0.3 5.89 35 1.867 Ethyl caprate 23.0 6.87 36 1.922 2,3-Butandiol 108.0 3.02 37 1.959 Diethyl succinate 2.0 2.16 38 2.014 3,-Bu .tyrolactone 44.0 8.08 39 2.063 Compound 39 c 2.3 = 1.09 40 2.095 Compound 40 c 1.0 • 17.62 41 2.164 1-Decanol 0.1 1.27 42 2.254 Compound 42 c 1.4 ¢ 3.16 43 2.361 Ethyl laurate +

2-phenetyl acetate 2.7 9.80 44 2.439 Compound 44 c 2.7 • 0.94 45 2.806 2-Phenylethanol 30.0 3.93

8.3 3.87 7.8 5.30 11.2 4.77 860.0 3.07 1140.0 6.75 441.0 2.16

33.0 4.87 19.0 7.37 0.3 6.78 0.5 4.08

0.2 6.07 0.03 3.33 0.4 5.24 0.5 1.77 0.1 6.63

0.03 5.37 0.1 0.47 0.01 3.35 2.4 7.50 4.4 2.76 17.3 5.69 2.1 • 3.36 0.9 • 7.65 0.34 9.97

19.2 2.34 31.4 1.75 24.5 3.60 0.2 • 1.82 0.4 e 3.95 0.1 ¢ 4.69 0.5 9.76 0.5 9.01 10.1 4.27 0.1 5.34 0.1 8.84 0.3 8.34

46.0 13.11 76.0 2.10 75.0 2.21 0.03 6.35 0.02 9.87

0.1 7.45 0.1 6.94 0.2 1.39 0.2 = 3.59 0.4e 8.88 0.9 c 3.22 44.4 c 3.65 0.2 • 1.23 0.5 c 6.54 0.2 ~ 9.02

3.0 5.44 3.8 6.08 4.0 7.56 0.01 ~ 0.73 0.2 = 8.62 0.8 " 3.28 0.1 6.70 0.3 11.70 2.0 3.22 0.04 e 6.66 0.1 = 4.74 0.1 ~ 6.28 0.I 15.24 0.03 8.36 0.3 13.41 0.2 • 9.22 0.2 • 14.39 0.2 e 8.34 0.1 2.81 0.6 6.41 1.0 5.04 0.4 0.75 0.8 1.28 0.5 1.52 2.6 5.76 5.5 1.19 8.2 1.71

24.0 15.60 51.0 6.48 166.0 2.88 1.9 15.71 2.7 13.10 3.3 7.23

25.0 11.35 25.0 5.7q 34.0 4.93 0.4 = 1.38 0.7 c 1.60 0.1 = 5.48 0.7 c 8.28 0.5 • 4.77 0.2 c 9.90 0.6 8.80 0.2 14.61 0.01 7.66 0.3 ¢ 13.63 0.5 ~ 10.89 1.3 ¢" 3.43

0.3 4.29 0.1 4.94 0. l 4.28 2.1 = 0.21 2.5 = 6.39 2.4 = 2.98

25.7 2.73 102.0 3.88 37.0 4.77

a Peak n u m b e r on the ch roma tog ram.

b R R T , re lat ive re ten t i on t ime.

157

i

Kioeckera D e c k e r a Saccharomyces Saccharomyce$ Saccharomyces Saccharomyces apiculata i n t e rmed ia cerev is iae cerev i s iae cerev i s iae cerecisiae

var. capens/s var. chevalieri var. bayanus var. cere~'isiae

rag/! C.V. mg/ l C.V. mg/ l C.V. mg/I C.V. mg/I C.V. mg/I C.V.

69.0 6.23 5.5 870.0 3.55 1360.0

39.0 4.38 0.7 4.84 1.3 0.2 9.21 0.3 10.73 0.1

8.69 28.4 1.70 0.9 5.76 11.7 2.46 2.35 640.0 4.85 970.0 7.41 490.0 4.31 940.0 0.90

74.0 12.85 58.0 1.95 7.37 0.4 8.63 0.4 2.01

0.1 6.06 0.1 11.56 0.1 10.67 4.09 0.03 6.63 0.5 7.61 0.02 6.63 0.4 1.21

0.03 3.35 0.02 7.37 7.5 2.30 5.2 16.10 0.5 c 7.50 2.8 • 4.19

0.04 0.19 0.1 5.64 6.7 10.41 14.1 1.41 0.4 4.73 3.3 12.69 1.0 e 6.01 1.69 • 5.22 0.1 • 6.01 0.6 • 8.45

60.0 7.11 0.2 • 4.16 1.2 5.79 1.0 5.80

117.0 5.85 0.04 6.35 0.1 6.08 0.1 • 6.37 5.0 • 7.84 0.1 e 3.81

28.0

0.8 0.2

65.0

0.1 0.2 • 18.61 0.8 • 21.09 0.04 • 4.01

4.98 23.2 1.16 63.60 1.29 5.5 11.43 27.6 3.41 0.3 • 8.10 0.1 c 3.70

7.14 0.4 7.06 1.9 11.94 0.3 3.23 0.9 2.49 4.99 0.4 9.86 0.1 6.99 0.1 12.03 4.53 59.0 7.06 153.0 3.22 60.0 2.48 56.2 1.40

0.03 10.14 1.37 0.03 6.94 0.2 13.74 0.02 6.94 0.1 3.71

0.5 • 6.37 0.4 * 3.69 3.2 • 6.3 • 0.3 c 13.77 0.6 • 2.41 0.04 * 3.81 0.1 • 3.81 0.03 • 9.54 0.3 • 4.35

7.0 4.04 2.8 3.78 0.03 • 3.61 0.02 • 1.96 0.8 7.03 0.1 2.90 0.1 • 19.06 0.1 • 8.34 0.4 3.87 0.1 9.07 0.1 • 8.34 0.04 ¢ 4.78 0.3 3.36 0.3 15.38 1.2 2.86 6.6 2.31 17.0 13.67

1.6 1.30 3.8 4.22 0.2 c 7.05

0.1 4.30 0.3 7.37 0.03 c 8.84 0.2 • 9.70 0.1 16.55 0.3 15.39 0.1 • 6.67 0.1 ® 16.37 0.1 17.15 0.1 6.41 0.1 9.87 0.2 2.6 8.53 45.0

350.0 8.15 190.0 14.95 16.0 8.15 70.0 8.0 12.12 8.1 1.69 0.5 2.39 3.0

32.0 13.54 14.9 3.18 4.1 2.80 35.6 1.0 c 14.30 1.2 e 5.48 0.8 c 5.72 0.8 c 1.2 • 11.17 2.0 12.42 1.4 e 8.71

2.5 • 2.40 0.6 • 9.27 0.6 • 10.75 0.7 3 .92 0.5 7.64 0.3 16.77 0.6 • 7.96 0.I • 9.27 0.8 • 8.80

1.5 11.31 2.6 3.53 0.04 • 7.86

0.1 1.78 0.2 5.61 0.1 • 12.76

0.1 8.81 0.03 c 8.34 0.3 • 7.39 0.1 6.41 0.4 5.98

5.68 0.2 12.62 0.6 8.85 5.61 3.1 4.03 8.1 0.22 7.18 36.0 4.72 59.0 5.11 7.77 0.8 7.77 5.0 a.66 1.74 9.2 7.25 29.1 1.39 5.48 0.2 c 5.48 1.2 • 0.80

0.4 • 9.27 2.3 ~ 5.76 0.9 10.19 0.7 7.70 0.5 • 12.96 1.1 c 3.32

2.1 1.10 2.5 • 11.3

35.0 3.53

0.4 8.97 0.1 4.94 1.4 0.5 c 9.26 0.5 c 9.73 6.5 c

38.1 2.74 16.3 3.25 37.0

7.13 0.2 9.49 1.2 6.16 6.22 2.2 c 9.17 2.8 • 2.67 4.17 31.0 6.07 30.0 4.79

Unindentified compounds. d C.V., coefficient of variation. • Estimated.

158

TABLE II

Overall and relative production of volatile compounds b'~' yeasts isolated from "Monastrell ' musts

Yeast Volatiles Higher alcohols Ethyl acetate Other esters ( B + C ) C/ 'A concentrat ion A % B % C % / A

(rag/ l ) (mg/ I ) ( rag/ l ) (mg/ I )

Candida calida 1 768 275 15.6 1 360 77.1 77 4.4 5.23 0.28 Brettanom~'ces bruxellensis 1064 144 13.5 860 81.2 25 2.4 6.19 0.18 Rhodotorula aurantica 1463 2.94 20.1 l t40 78.0 36 2.5 4.00 0.12 Hanseniospora ucarum 906 340 37.5 441 48.7 40 4.4 1.41 0.12 Kloeckera apiculata 1626 584 35.9 870 53.8 123 7.6 1.71 0.21 Deckera intermedia 1 747 319 18.3 1360 77.7 47 2.7 4.40 0.15 Saccharomyces cerecisiae

vat. capensis 775 112 14.4 640 82.6 16 2.0 5.87 0.14 Saccharomyces cerecisiae

var. chet'alieri 1520 384 25.3 970 63.9 114 7.5 2.82 0.30 Saccharomyces ceret'isiae

vat. bayanus 646 132 20.5 490 76.1 9 1.4 3.78 0.07 Saccharomyces cerecisiae

var. cerecisiae 1 249 224 18.0 940 75.5 42 3.4 4.38 0.19

significant to the sensory properties of wine (Wegener et al., 1968). Ethyl acetate production excluded, Kloeckera apiculata and Saccharomyces cerecisiae var. checa- lieri were the major producers of volatiles throughout fermentation. Based on Wegener et al. (1968), we believe the ratio between the ester and alcohol contents (C/A) can be a good measure of the significance of the contribution of a given yeast to the aroma of wine. Judging by this indicator, the two above-mentioned yeasts showed high values, in addition to Candida valida on account of its increased production of esters other than ethyl acetate.

The analysis of relative data (Table II) provides new information. Thus, Hanse- niospora ucarum and Kloeckera apiculata were the main relative producers of higher alcohols (A) and the smallest producers of ethyl acetate (B). Although they different amounts of the other esters (C) they showed a degree of similarity which may be ascribed to the fact that they are the perfect and imperfect form respec- tively of the same species. They differ in their resistance to ethanol and the methabolic activity of the perfect form is reported to stop before that of its perfect counterpart (Lafon Laforcade, 1983). This is also the case with Brettanomyces bruxellensis (imperfect form) and Deckera intermedia (perfect form). Saccha- rom)'ces cerecisiae vat. bayanus produced average relative amounts of higher alcohols but very small amounts of esters, consistent with literature reports (Amerine et al., 1982).

Coefficients of correlation between the amounts of different compounds are also calculated. We shall emphasize the high degree of correlation between the production of amyl acetate and compound 24, isopropyl acetate and ethyl caproate, 1-butanol and compound 25, hexyl acetate and compound 28 and hexyl acetate and compound 39 (results not shown). These correlations appear to be typical of the

159

Z 0

4( : i 4:

4: a; 4C

1 0 -

8 -

6 - -

4 - -

2--

4 5 8 2 10 7 9 3 6 i

YEAST Fig. 1. Result of the cluster analysis between the amount of volatiles listed in Table I. 1. Candida t'alida; 2, Brettanomyces bruxellensis; 3, Rhodotorula aurantica; 4, Hanseniospora uvarum; 5, Kloeckera apicu- lata; 6, Deckera intermedia; 7, Saccharomyces cerevisiae var. capensis; 8, Saccharomyces ceret'isiae var.

bayanus; 10, Saccharomyces cerevisiae var. ceret'isiae

fermentations of Monastrell musts and distinctive from those of other grape varieties (Gassiot et al., 1983).

Figure 1 shows the dendogram constructed from the data listed in Table If. The results of the cluster analysis are consistent with those discussed above, except perhaps for Saccharomyces cerevisiae var. cerevisiae, which appears to be more closely related to those yeasts producing low concentrations of volatiles than was apparent from the analysis of the data in Tables I and II.

From these investigations we conclude that the yeasts with low fermentation power produce significant overall amounts of volatiles, This is particularly applica- ble for Kloeckera apiculata, which is the prevalent yeast in the early stages of the fermentation process. Among the yeasts with high fermentative power, Saccha- romyces cerevisiae var. chevalieri produces large amounts of aroma compounds. According to the present results, current winemaking trends with use of starters of pure yeasts with high fermentation power, usually Saccharomyces cerevisiae var. cerevisiae or Saccharomyces cerevisiae var. bayanus, may be detrimental to the sensory properties of the wine. For reasons of ecological competition, yeasts with low fermentative capacity will have little opportunity to proliferate. Therefore, it seems more practical to use mixed yeast starters including yeasts responsible for the final aroma of wine and others acting chiefly during the fermentation process.

Acknowledgements

This work was supported by financial assistance from the Comision Interminis- terial de Ciencia y Tecnologia (I + D ALl 90-0949). We wish to acknowledge R. Mateo for support in the statistical treatment of the data.

160

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