J Sci Food Agric 1989,49, 249-258

Differences between Wines Fermented with and without Sulphur Dioxide Using Various Selected Yeasts

Tomas Herraiz, Pedro J Martin-Alvarez," Guillermo Reglero, Marta Herraiz and Maria D Cabezudo

Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain

(Received 11 July 1988; revised version received 1 December 1988; accepted 4 January 1989)

ABSTRACT

Comparisons were made between wines resulting from the fermentation of a single grape must of the Verdejo cultivar with various yeasts, namely the indigenous must . microflora, pure cultures of Kloeckera apiculata, Torulaspora delbrueckii and Saccharomyces cerevisiae, and mixtures of these cultures, all with or without the addition of SOz. There were major differences in both volatile and non-volatile compounds. Principal component analysis and cluster analysis showed that these differences were due mainly to the effects of the sulphur dioxide upon the yeasts. Analytical data on the wines demonstrate that the nature of a wine depends

upon the particular yeasts present during the fermentation in the absence of SO,. On the other hand, if the fermentation is carried out in presence of SO,, similar wines are produced despite differences in the microflora.

Key words: Wine, vinification without SO,, flavour components, multivariate techniques.

INTRODUCTION

Sulphur dioxide fulfils various functions in vinification: its bactereostatic properties prevent development of undesirable organisms; as an antioxidant it protects the substrate from atmospheric oxygen; it inhibits must enzymes (tyrosinase and laccase); it assists extraction of the skin pigments; and facilitates fining of the must.

* To whom correspondence should be addressed.

249

J Sci Food Agric 0022-5142/89/$03.50 0 1989 Society of Chemical Industry. Printed in Great Britain

T Herraiz et a1 250

The use of SO, has hitherto been considered absolutely necessary; however, several authors (Aerny 1986a,b,c; Sozzi 1986) have considered the possibility of reducing its use in accordance with the main thrust from international bodies whose recommendations extend to total elimination. To this end there are proposals to use massive seeding with yeasts in order to provide the adequate physical and chemical stability to the wine (Iiiigo 1986; Tini et a1 1987) and to use modern vinification technology to achieve adequate microbiological stability (Galassi and Mancini 1985; Usseglio-Tomasset 1985).

Several effects of sulphur dioxide on the composition of wines have been described (Daudt and Ough 1973), and it is known that some yeasts are more resistant to its action (Saccharomyces cerevisiae, Saccharomycodes ludwigii) whereas others are more sensitive (Kloeckera apiculata) (Beech and Thomas 1985; Rose 1987). Other authors note that the volatile compounds in wines differ according to the yeast used (Suomalainen and Lehtonen 1979; Soufleros and Bertrand 1979; Soles et a1 1982). In addition, if the SO2 is absent, the indigenous must microflora predominates (Kunkee and Amerine 1970).

In this work, wines produced by fermentation of a must of the Verdejo cultivar with the most abundant yeast among the indigenous microflora, in the presence and absence of SO,, were compared. The data were subjected to multivariate analysis techniques, already used in the characterisation of wines (Cabezudo et a1 1986; Martin-Alvarez et a1 1987). Results which clearly establish the compositional differences between the wines according to the method of production have been obtained.

EXPERIMENTAL

Wine samples

Yeasts Three species of yeasts were used, namely: Kloeckera apiculata (A), Torulaspora delbrueckii (formerly Saccharomyces rosei) (B) and Saccharomyces cerevisiae (C). These yeasts were classified according to Kreger Van Rij (1984) and they are representative of the viticulture area yeast microflora, which has a cold and dry climate; they were isolated from fresh must and cultured on agar slices. These cultured yeasts exhibit a behaviour similar to that of recent indigenous yeasts.

Fermentation Aliquots (500 ml) of a fresh must (pH = 3.2) of the white Verdejo cultivar were fermented in 1-litre flasks at 21°C in two series, one with SO, (1 20 mg litre- ',added as NaHSO,) and the other without. The two series differed only in regard to the yeasts involved in the fermentation. One consisted of spontaneous fermentations by the yeasts indigenous to the musts (E); in the other series inocula (20mllitre-') consisted of 48-h cultures of the yeasts listed above in the following combinations: A, B, C, A + B (AB), A + C (AC), B + C (BC) and A + B + C (ABC). All the samples were prepared in duplicate, but three of them were considered as outliers and consequently were excluded from further study. Once the fermentation was finished,

Comparison of wines fermented with and without sulphur dioxide 25 1

the resulting wines (13 made without SO, and 16 with SOz), were centrifuged and maintained at low temperature until analysed.

Analytical procedure

The volatiles of boiling point < 145°C were analysed by direct injection of 2 pl of wine in a 5-m micropacked GC column made in this laboratory from 0 8 mm id deactivated Pyrex glass. Desilanised Volaspher A-2 (100-120 pm from Merck, Darmstadt, FRG) was used as solid support with 40 mg g-' of Carbowax 300+ bis- 2-ethylhexylsebacate (92:8 w) (Reglero et a1 1986). In all the cases pentan-3-01 was used as internal standard.

Freon 11 extracts were prepared for analysis of the medium volatility fraction components (boiling point > 145°C) (Rapp et a1 1976). Analyses of the concentrated extracts were accomplished by GC in a 10-m Volaspher A-2 (100-120 pm) was 0.8 mm id deactivated Pyrex glass. Desilanised Volaspher A-2 (100-120 pm) was used as solid support with 40mgg-' of Igepal Co-880 and Carbowax 20 M (80:20 w). 2-Phenylethanol was also analysed in this column by direct injection of 2 p1 of wine. All the compounds of this fraction were previously identified through GC-MS by comparison with the spectra obtained from standard substances analysed in the same conditions. The GC equipment used was Shimadzu Model GC R1A (Chemicontrol SA, Madrid, Spain).

L-Lactic acid, L-malic acid and glycerol were analysed according to the enzymic methods of Boehringer (1975).

Statistical methods

Factor analysis (principal component method) and cluster analysis were applied to the data obtained. The BMDP (Dixon 1983) package was used for factor analysis (BMDP4M program), and cluster analysis was carried out by using the CLUSTAN program (Wishart 1978). All these programs were run in a CDC CYBER 180/855 computer.

RESULTS AND DISCUSSION

The application of principal component analysis to the samples has shown that the main cause of variation was the presence or absence of SO,, as can be observed in Fig 1 in which the 29 wines are represented in the plane defined by the two first components. The wines fermented with SO, are grouped in a compact form whereas those that have been 'fermented without SO, constitute a more dispersed group.

Table 1 shows the mean values and standard deviations of each variable in the two groups considered. One-way analysis of variance shows that 23 of the 37 variables given are significant for distinguishing between the wines studied. A classification of all the variables analysed according to the value of the F statistic is also included in Table 1.

As can be seen the largest amounts of 2-methylpropan-1-01 and 1-propanol are obtained in wines fermented without SO, whereas 2-methylbutan-1-01, 3- methylbutan-1-01 and 2-phenylethanol are more abundant in wines fermented with

252 T Herraiz et a1

m m m

E

AC Cl AC \ \ \ ABC

-2 00 - f 00 0 0 0 I 043 2 00 P R I N C I P A L COMPONENT 2

1

00

Fig 1. Representation of the 29 wines in the plane defined by the first two principal components. With SO, (0) and without SO, (O), A (Kloeckera apiculata), B (Torulaspora delbrueckii) and C

(Saccharomyces cereuisiae).

SO,. The amounts of other alcohols such as l-hexanol, 3-ethoxypropan-1-01, (2)-3- hexen-1-01, linalool and 1 -heptanol are different in the wines of both groups and are consistently larger in wines fermented without sulphur dioxide. Benzyl alcohol, E- terpineol, methanol, ethanol and glycerol, as well as the ratio of 3-methylbutan-1-01 to 2-methylbutan-1-01 (3MlB/2MlB) are not significantly different.

As far as the esters are concerned, the wines with SO, have higher levels of ethyl octanoate and diethyl succinate, as has also been reported by Brugerolle et at (1978) and Margheri and Versini (1986). The wines without SO, have higher concentrations of ethyl acetate, ethyl lactate and isoamyl lactate. There are no significant differences in the other esters.

Significantly higher concentrations of acetaldehyde are obtained in wines which were fermented with sulphur dioxide. In the same way relatively high values of 1,l- diethoxyethane (diethylacetal acetaldehyde) are found, as is to be expected because of the higher levels of acetaldehyde. The occurrence of benzaldehyde in the wines is not significantly affected by the presence of sulphur dioxide.

The concentrations of tartaric acid, malic acid, octanoic acid and y-butyrolactone are significantly hgher in the wines fermented with SO,, and the concentration of lactic acid is higher in wines without SO,.

The dispersion of the data (Table 1) within each group arises from the use of several different cultures of single or mixed yeasts. In most cases, the values of the standard deviation are significantly lower (Levene’s test for equality of variances at

Comparison of wines fermented with and without sulphur dioxide 253

TABLE 1 Composition of the wines obtained when a single must of Verdejo cultivar is fermented, with or without the addition of SO,, using different yeasts. The variables are ordered according to

the value of the F obtained when both groups are compared (F,,,,,,,=4.21)

Compounds Without SO, With SO, (mg litre-') ( n = 13) (n= 16)

- - X SD X SD F

Significant variables 1-Propanol Malic acid Ethyl acetate 2-Methylpropan-1 -01 3-Ethoxypropan-1-01 2-Methylbutan-1-01 Octanoic acid y-But yrolactone Ethyl octanoate Linalool 3-Methylbutan-1 -01 Diethyl succinate 1 -Hexanol 2-Phenylethanol 1-Heptanol Tartaric acid Isoamyl lactate (Z)-3-hexen-l-o1 1,l -Diethoxyethane Acetaldehyde Ethyl lactate Lactic acid

PH

24.33 890.00 127.06 65.81

4.56 43.46

1.34 3.45 0.14 0.07

138.36 0.26 2.42

52.89 0.033

3 830.00 0.23 0.07 1.59

26.59 14.44

970.00

3.06

Non-significant variables

Ethyl hexanoate 0.17 Isoamyl acetate 0.35 Benzyl alcohol 0.26 a-Terpineol 0.41 Benzaldehyde 0.19 Methanol 68.5 1 Lactic + malic acids 1860.00 Glycerol 6740.00 Hexyl acetate 0.005 Ethyl formate 5.13 3M 1 B/2M 1 B" 3.15 Ethanol (ml litre-') 105.4 Nitrogen (mg litre-') 85.06

a 3-Methylbutan-1 -01/2-methylbutan-l-01.

Ethyl propionate 3.55

6.03 180.00 63.39 22.49

3.80 10.57 0.87 1.43 0.14 0.01

44.19 0.16 0.89

12.80 0.01

530.00 0.26 0.02 2.00

39.38 16.8 1

166.00

0.08

3.1 1 0.14 0.13 0.09 0.19 0.16

13.04 173.00

710.00 0.008 2.08 0.82

15.0 21.18

12.8 1 1450.00

26.70 36.05 0.3 1

55.72 2.6 1 5.53 0.28 0.05

182.16 0.50 1.69

66.80 0.019

4400.00 0.05 0.05 4.70

86.61 5.04

80.00

3.00

6.02 0.23 0.30 0.30 0.47 0.23

64.85 1530.00 66 10.00

0.005 5.17 3.27

88.28 105.6

1.72 240.00

7.03 3.73 0.21 6.73 0.92 1.54 0.06 0.01

20.05 0.22 0.10

11.00 0.010

480.00 0.03 0.02 3.84

80.06 1.79

80.00

0.05

4.87 0.05 0.12 0.08 0.12 0.14

17.3 1 280.00 670.00

0.004 3.1 1 0.15

11.0 10.58

53.48 4685 39.83 27.30 2007 14.40 1418 13.94 13.52 13.28 12.61 10.81 10.45 9.86 9-72 9-42 7.83 751 6.95 6-08 4.97 465

5.53

2-5 1 2.23 1.44 1.08 1.00 0.42 0.39 0-37 0.24 0.004 0.002 0.32 0.01 0.28

T Herraiz et a1 254

0.05 significance level) in the group of wines fermented with SO,, demonstrating greater homogeneity in the composition of these wines as a result of the inactivation of some yeasts by the SO,. This inactivation has been described previously (Beech and Thomas 1985; Mateos et al 1985; Rose 1987).

Additional results of the application of principal component analysis to the 29 samples are shown in Table 2. The first principal component, highly correlated with 1-propanol, 2-methylpropan-1-01, ethyl acetate and malic acid, shows a different performance during the alcoholic fermentation as well as the possible influence of SO, on the associated malolactic fermentation. The second principal component is probably related to the malolactic fermentation which does not take place in the presence of SO,. The variables most correlated with the third factor are compounds that depend upon yeasts and SO, (such as 2-methylbutan-1-01 and 3-methylbutan- 1-01). The fourth principal component groups the variables which are traditionally known because their concentration increases during the fermentation with sulphur dioxide: acetaldehyde and 1,l -diethoxyethane. The correlation coefficients between the compounds which define the factors are listed in Table 3.

TABLE 2 Results of the principal component analysis applied to the 29 wines

Principal Variance Cumulative Variables more component explained ( %) proportion (%) correlated

1 23.15 23.15 2-Methylpropan-1-01, ethyl acetate,

2 19.71 42.86 Ethyl lactate, isoamyl lactate,

3 17.97 60.83 2-Methylbutan-1-01, 3-methyl-

4 12.36 73.19 Acetaldehyde, 1,l-diethoxyethane

1-propanol, malic acid

lactic acid, 3-ethoxypropan-1-01

butan-1-01, 1-hexanol

TABLE 3 Some correlation coefficients between the variables that define the principal

components

Variables Correlation coefficient

2-Methylpropan-1-01 and ethyl acetate 2-Methylpropan-1 -01 and 1-propanol Ethyl lactate and 3-ethoxypropan-1-01 Ethyl lactate and isoamyl lactate Ethyl lactate and lactic acid Isoamyl lactate and lactic acid Isoamyl lactate and 3-ethoxypropan-1-01 2-Methylbutan-1-01 and 3-methylbutan-1-01 Acetaldehyde and 1 ,ldiethoxyethane

0.862 0.837 0-884 0887 0.821 0.885 0.799 0.844 0.839

Comparison of wines fermented with and without sulphur dioxide

zos-3av

70s-3 20s-v

20s-39 20s-38

20s-3 20s- 3

20s-3v 20s- a 20s-a

20s-av

2 o s - m

20s-3

20s-3v

20s-v

F zos-a: I 3

V

V

38 9 8

3V

38V 3av

3V

av 3

I : I

255

T Herraiz et a1 256

From Fig 1 can be observed the homogeneity of the wines obtained with SO,. Thus, the yeasts of the indigenous must microflora, Kloeckera apiculata and Torulaspora delbrueckii, prove to be not very active in the presence of the more resistant Saccharomyces cerevisiae. Among the group of wines fermented without SO, there is greater dispersion of the values of the variables correlated with the second principal component, the highest values corresponding to the wines obtained with Torulaspora delbrueckii in agreement with the results obtained by Di Stefan0 e f al (1981) for 3-ethoxypropan-1-01.

The results of the application of cluster analysis (Ward’s method) to the 29 wines, using the 23 variables already mentioned, are shown in Fig 2. The samples are grouped into two main areas, depending upon whether SO, is added or not. Only the wines obtained through fermentation with Saccharomyces cerevisiae without SO, are found in the SO, group. This confirms that, when must is fermented with sulphur dioxide, the wines obtained are more similar to those arising from fermentation with S cerevisiae. That is probably the reason why some authors who use this yeast have not observed effects due to the presence or absence of sulphur dioxide. Assuming that the fermentation is carried out without SO,, growth of and metabolism by yeasts other than S cerevisiae may occur, irrespective of their origin (indigenous to the must or from starter cultures). In consequence, compositional differences are obtained in the resulting wines.

When the cluster analysis is applied to the same wines using only the five higher alcohols, 1 -propanol, 2-met hylpropan- 1-01,2-met hylbutan- 1-01,3-methylbutan-l- 01 and 2-phenylethanol, the same results are obtained.

CONCLUSIONS

Controlled fermentation of the same grape must (Verdejo cultivar) with indigenous must microflora or pure cultures of three individual yeasts (Kloeckera apiculata, Torulaspora delbrueckii and Saccharomyces cerevisiae), as well as with mixtures of these cultures, in the presence and absence of SO, provides substantial information about the influence of SO, on the composition of the wines obtained. The following conclusions can be drawn:

(a) The main cause of variation between wines is the presence or absence of sulphur dioxide during the fermentation.

(b) Fermentation without SO, leads to wines whose composition depends substantially upon the yeast predominating during the fermentation.

(c) If the fermentation is carried out with SO,, the wines obtained are more uniform and are more similar to those obtained by fermentation using Saccharomyces cerevisiae in the absence of SO,.

ACKNOWLEDGEMENTS

The authors thank Dr B liiigo, Instituto de Fermentaciones Industriales, CSIC, for providing the yeasts and for helpful discussions. Thanks are also due to Mr J L

Comparison of wines fermented with and without sulphur dioxide 251

Andreu for his analyses. One of the authors (TH) is grateful to the Spanish Ministerio de Educacion y Ciencia for a grant. The work was made possible by financial assistance from the Comision Asesora de Investigacion Cientifica y Tecnica, Proyect 237184.

REFERENCES

Aerny J 1986a L‘anhydride sulfureux: est-il indispensable en oenologie? Bull Soc Frib Sc Nat

Aerny J 1986b Diminution de la teneur des vins en anhydride sulfureux. 1.-L’anhydride sulfureux et ses propietes utiles en vinification. Reu Suisse Vitic Arbor Hort 18 17-21.

Aerny J 1986c Diminution de la teneur des vins en anhydride sulfureux. 2.-Aspects pratiques. Rev Suisse Vitic Arbor Hort 18 143-146.

Beech F W, Thomas S 1985 Action antimicrobienne de I’anhydride sulfureux. Bull 1‘01V 652453 564-581.

Boehringer Mannheim 1975 Enzymatic Analysis of Food. Boehringer, Mannheim. Brugerolle C M, Sarre C, Bertrand A 1978 Influence du debourbage des mouts et du sulfitage

sur les teneurs en substances volatiles des vins et des eaux-de-vie. Conn Vigne Vin 12 11 1-120.

Cabezudo M D, Polo M C, Herraiz M, Reglero G, Gonzalez-Raurich M, Caceres I , Martin-Alvarez P 1986 Using discriminant analysis to characterize Spanish variety white wines. In: The Shelf Life of Foods and Beverages, ed Charalambous G. Elsevier, Amsterdam, pp 186-204.

Daudt C E, Ough C 1973 Variations in some volatile acetate esters formed during grape juice fermentation. Effects of fermentation temperature, SO,, yeast strain, and grape variety. Amer J Enol Vitic 24 130-135.

Di Stefan0 R, Ciolfi G, Delfini C 1981 Composti volatili prodotti dai lieviti. Rio Vitic En01 Conegliano 34 342-355.

Dixon W J 1983 (ed) BMDP: Biomedical Computer Programs. University of California Press, Los Angeles.

Galassi S, Mancini C 1985 Technologie per ridurre l’uso di SO, in enologia. Vignevini 5

Iiiigo B 1986 Elaboracion ecologica del vino. Enol Enotec 1 10-13. Kreger Van Rij N J W (ed) 1984 The Yeast, a Taxonomic Study (3rd edn). Elsevier,

Amsterdam. Kunkee E R, Amerine M A 1970 Yeast in wine making. In: The Yeast. Yeast Technology, Vol

3, eds Rose A H & Harrison J S. Academic Press, New York, pp 6-60. Margheri G, Versini G 1986 Aspectti tecnologici e biochemici connessi con la produzione dei

vini bianchi di qualita con basso tenore di anidride solforosa. Vignevini 5 61-65. Martin-Alvarez P J, Polo M C, Herraiz M, Caceres I, Gonzalez-Raurich M, Herraiz T,

Reglero G, Cabezudo M D, Sanz J 1987 Application of chemometrics to the characterization of Spanish wines. In: Flavour Science and Technology, eds Martens M, Dalen G A & Russwurm Jr H. John Wiley, New York, pp 489-499.

Mateos P L, Sanchez P, Arroyo V, E g o B 1985 Mostos de uva de la zona Utiel-Requena. Influencia del anhidrido sulfuroso en la microflora fermentativa. Alimentaria 164 29-36.

Rapp A, Hastrich H, Engel L 1976 Gaschromatographische Untersuchungen iiber die Aromastoffe von Weinbeeren I. Anreicherung und Kapillarchromatographische Auftrennung. Vitis 15 29-36.

Reglero G, Herraiz T, Herraiz M, Cabezudo M D 1986 Mixed micropacked columns designed for selective separation of fermentation products. Chromatographia 22 358-362.

Rose A H 1987 Responses to chemical environment. In: The Yeast. Yeasts and the Environment (2nd edn), Vol 2 eds Rose A H & Harrison J S. Academic Press, London,

75 15-16.

13-18.

pp 5-40.

258 T Herraiz et a1

Soles R M, Ough C S, Kunkee R E 1982 Ester concentration differences in wine fermented by

Soufleros E, Bertrand A 1979 Role de la ‘souche de levure’ dans la production des substances

Sozzi T 1986 L‘anidride solforosa in enologia e ancora indispensabile? Vigneuini 7-8 39-47. Suomalainen H, Lehtonen M 1979 The production of aroma compounds by yeast. J Inst

Tini V, Chiodini 0, Suzzi G, Romano P, Erbetti M, Zambonelli C 1987 Ricerca di lieviti

Usseglio-Tomasset L 1985 Les technologies de vinification permettant de diminuer les doses

Wishart D 1978 CLUSTAN. User Manual (3rd edn). Program Library Unit, Edinburgh

various species and strains of yeasts. Amer J Enol Vitic 33 94-98.

volatiles au cours de la fermentation du jus de raisin. Conn Vigne Vin 13 181-189.

Brew 85 149-156.

stabilizzanti producenti basse quantita di anidride solforosa. Vigneuini 9 45-49.

de SO,. Bull l’OIV 652453 606-616.

University.