FEMS Microbiology Letters 118 (1994) 213-218 213

© 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00

Published by Elsevier

FEMSLE 05938

Acetaldehyde production in Saccharomyces

cerevisiae wine yeasts

Downloaded from https://academic.oup.com/femsle/article/118/3/213/540165 by guest on 08 March 2022

Patrizia R o m a n o *'a G i o v a n n a Suzzi a L u c a T u r b a n t i b a n d M a r i o Polsinelli b

a Dipartimento di Protezione e Valorizzazione Agroalimentare, Sezione di Chimica e Tecnologia degli Alimenti, Universith di Bologna,
Via San Giacomo 7, 40126 Bologna, Italy, and b Dipartimento di Biologia Animale e Genetica, Universitgt di Firenze, Firenze, Italy

(Received 6 January 1994; revision received and accepted 25 February 1994)

Abstract: Eighty-six strains of Saccharomyces cerevisiae were investigated for their ability to produce acetaldehyde in synthetic
medium and in grape must. Acetaldehyde production did not differ significantly between the two media, ranging from a few m g / l
to about 60 mg/l, and was found to be a strain characteristic. The fermentation temperature of 30°C considerably increased the
acetaldehyde produced. This study allowed us to assign the strains to different phenotypes: low, medium and high acetaldehyde
producers. The low and high phenotypes differed considerably also in the production of acetic acid, acetoin and higher alcohols
and can be useful for studying acetaldehyde production in S. cerevisiae, both from the technological and genetic point of view.

Key words: Saccharomyces cerevisiae; Acetaldehyde production; Wine fermentation

Introduction that pyruvate and acetaldehyde should be consid-

Because of their low sensory threshold values
[1], aldehydes play an important role in the aroma
and bouquet of wine and, among these, acetalde-
hyde is the major component, constituting more
than 90% of total aldehyde content [2].
Acetaldehyde originates as an intermediary
product of yeast metabolism from pyruvate
through the glycolytic pathway enzymes [3] and it
is a precursor for acetate [4,5], acetoin [6], as well
as for ethanol. Strassman et al. [7,8] suggested
* Corresponding author. Tel: (051) 259785; Fax: (051) 259782.
ered as joint precursors for the formation of
valine and leucine. Other authors [9,10] showed
that pyruvate, acetaldehyde and acetic acid
metabolites can serve for the synthesis of amino
acids, from which fuel oil alcohols or their pre-
cursors originate.
Acetaldehyde is a normal product of alcoholic
fermentation and its amount can vary in wine
from 10 mg/1 up to 300 mg/1 [11]. In white wines
acetaldehyde is regarded as an indicator of the
wine oxidation state, whereas in red wines it
should be present in amounts up to 100 m g / l . At
amounts of 500 m g / l , wine is considered unmar-
ketable. However, Teixeira [12] reported that
wines of higher quality have a somewhat higher

SSDI 03 78 - 1 0 9 7 ( 9 4 ) 0 0 0 8 4 -5
214
acetaldehyde content. Sherry or Tokay wines, for
example, contain relatively high amounts of ac-
etaldehyde, due to their production under oxida-
tive conditions [11].
The formation of acetaldehyde depends on
various factors, such as extreme aerobic growth
conditions [13], the medium composition [14] or
the nature of insoluble materials used to clarify
musts [15]. The amount of acetaldehyde present
in wines increases during aging [16] owing to
oxidation to ethyl alcohol, or to the activity of
film yeasts or to aeration [17]. The use of high
concentrations of sulfur dioxide in grape must
fermentation causes a considerable increase in
acetaldehyde levels by yeast cells [18-21]. Casa-
lone et al. [22] found that sulphite-resistant mu-
tants of Saccharomyces cerevisiae accumulate
much more acetaldehyde in the medium than the
parental strain, both in the presence and in the
absence of sulphite. Fermentation temperature
can affect acetaldehyde content, but the reports
are controversial. Amerine and Ough [23] re-
ported that fermentation temperature does not
affect the final total aldehyde content greatly, if
at all, whereas other authors [17,24] found that
acetaldehyde content increased with increasing
fermentation temperature.
The ability to produce acetaldehyde is a prop-
erty of different wine yeasts and the data avail-
able indicate that S. cerevisiae strains produce
relatively high levels of acetaldehyde, from 50 to
120 mg/l, whereas other yeasts, such as Kloeck-
era apiculata, Candida krusei, Candida stellata,
Hansenula anomala and Metschnikowia pulcher-
rima, produce low levels, from non-detectable
amounts to 40 rag/1 (for a review see [25]). Longo
et al. [19], analyzing 14 strains of S. cerevisiae,
found variations in the production of acetalde-
hyde from 13.1 to 24.3 rag/1 in wine.
The present paper deals with acetaldehyde
production by 86 wine strains of S. cerevisiae in
synthetic medium and in grape must. This study
is part of an investigation concerning the influ-
ence of yeast strain on the composition and qual-
ity of wine. The chief aim is to select the most
suitable strains for use in industrial fermentations
and for the genetic study of acetaldehyde produc-
tion.
Materials and Methods
Organisms and media
A total of 86 strains of S. cerevisiae, belonging
to the collection of our Department, was used.
These strains were isolated from different grape
musts of different Italian regions. Each strain
represents the dominant yeast of a separate spon-
taneous fermentation. The strains were identified
as species of S. cerevisiae according to the official
Downloaded from https://academic.oup.com/femsle/article/118/3/213/540165 by guest on 08 March 2022
method of yeast classification described by
Kreger-van Rij [26].
To study the strain capacity to produce ac-
etaldehyde, a basal synthetic medium and grape
must were used. The composition of the synthetic
medium, similar to that recommended by Wicker-
ham [27], is reported by Kreger-van Rij [26]. The
grape must was from a Trebbiano cultivar of the
Emilia-Romagna region (18% fermentable sugar,
0.68 titrable acidity, pH 2.9).
To study the influence of the medium on the
production of acetaldehyde, eight different grape
musts were employed: Albana, Barbera, Char-
donnay, Montuni, Pinot, Riesling, Sangiovese and
Trebbiano.
Fermentation
Synthetic medium fermentation was carried out
in 50 ml of the medium inoculated with 106 cells.
The samples were incubated statically at 20°C for
20 days, then racked and stored at -20°C until
analysis. Must fermentations were carried out in
130-ml Erlenmeyer flasks containing 100 ml of
sterilised juice, inoculated with 5% of 48 h
precultures in the same must. The samples were
incubated at 20°C until CO 2 evolution ceased,
then refrigerated for 2 days at 2°C, racked and
stored at -20°C until analysis.
To study the influence of fermentation tem-
perature on acetaldehyde production, duplicate
fermentations were carried out in Trebbiano
grape must at four temperatures: 12°C, 18°C,
24°C and 30°C, until CO z evolution ceased, then
racked and stored as described above.
Analytical determinations
Acetaldehyde, acetoin and higher alcohols
were analysed by direct injection of 2 /.~1 of fer-
 215
45 T
/

4o ! I I chromatograph, equipped with a flame ionization

35 detector and linked to a Shimadzu C-R3A Chro-
. 30
matopac integrator and recorder, was used. The
"~ ~s oven temperature was programmed from 60°C to
o 20

15
198°C at a rise rate of 6°/min. The carrier gas
10 was nitrogen at a flow rate of 20 ml/min. N-
5 butanol (100 m g / l ) was used as the internal stan-
0 dard.
< 15 15-30 30-45 45-60 > 60
Acetaldehyde (rag/I) Ethanol was determined by direct injection of

Downloaded from https://academic.oup.com/femsle/article/118/3/213/540165 by guest on 08 March 2022

1 /zl of diluted wine (1:9) into the same glass
Fig. 1. Histograms of acetaldehyde production by 86 strains of
column. Aceton (10 g / l ) was used as the internal
Saccharomyces cerevisiae in synthetic medium ( I ) and in
grape must ( [] ). standard, and nitrogen (20 m l / m i n ) as the carrier
gas. The oven temperature was maintained at
mented medium into a 180 c m x 2 mm glass 80°C.
column packed with 8 0 / 1 2 0 Carbopack B / 5 % Acetic acid production was determined by a
Carbowax 20M (Supelco Inc.). A Packard 427 gas biochemical test combination (Boehringer).

Table 1
Acetaldehyde production (mg/l) by four strains of S. cerecisiae in eight different grape musts
Strain Phenotype Variety of grape juice Mean
1 2 3 4 5 6 7 8
Ba28 low 14.7 15.5 17.3 24.7 19.8 24.7 38.6 18.3 21.7
Ba35 medium 47.5 50.3 30.5 30.0 36.6 50.3 40.9 40.8 40.9
Bal70 medium 49.4 52.8 34.6 43.5 43.7 44.4 41.3 48.5 44.8
Ba145 high 87.6 68.6 64.8 74.6 58.3 115.4 65.2 75.6 76.3
Mean 49.8 46.8 36.8 43.2 39.6 58.7 46.5 45.8

40

35

-- 30

25 / :SJ /
Q / f•-
"0
~ 20
J

o
'~ 10

I I I I
12°C 18°C 24oc 30oc
Fermentation temperature
Fig. 2. Influence of different temperatures (12°C, 18°C, 24°C and 30°C) on acetaldehyde production in five Saccharomyces cerevisiae
strains producing medium quantities of the compound.
216

Results teristic to be low, medium or high acetaldehyde

Acetaldehyde production by 86 strains of S. cere-
visiae
Eighty-six strains of S. cerevisiae were tested
for the production of acetaldehyde in synthetic
medium and in grape must. Figure 1 shows the
distribution of the strains in relation to acetalde-
hyde produced in the two media.
producer also in the different grape musts.
Influence of fermentation temperature
For this study, we chose five strains belonging
to the same medium class of acetaldehyde pro-
duction, i.e. strains producing from 15 to 30 mg/1
in synthetic medium. The fermentation was car-
ried out in Trebbiano white must at different

Downloaded from https://academic.oup.com/femsle/article/118/3/213/540165 by guest on 08 March 2022

Acetaldehyde production did not differ signifi- temperatures: 12°C, 18°C, 24°C and 30°C. The
cantly between the two media, varying from 4 strains showed a similar behaviour, as illustrated
mg/1 to 59 m g / l in synthetic medium and from in Fig. 2. Acetaldehyde production was not af-
6.5 to 88 mg/1 in grape must. Only two strains fected by the temperatures of 12°C, 18°C and
produced more than 60 mg/1 of acetaldehyde in 24°C, whereas it increased considerably at 30°C.
grape must, 64 and 88 m g / l respectively. In both
media the majority of the strains produced quan- Secondary products of fermentation by high and
tities ranging from 15 to 30 m g / l . low acetaldehyde producing strains
To verify whether the different production of
Influence of the medium acetaldehyde was correlated with other secondary
To establish whether the differences found products of fermentation, five low and five high
were reproducible, we chose four strains belong- acetaldehyde producers were tested in grape must
ing to different classes of acetaldehyde produc- for the production of acetic acid, acetoin, higher
tion: low (Ba28), medium (Ba35, Bal70) and high alcohols and ethanol. The results, reported in
(Ba145) producers in synthetic medium. The ex- Table 2, showed that the two different pheno-
periment was carried out in eight different grape types behaved differently. Wines obtained with
musts of our region. The results, reported in the low acetaldehyde producers had traces of
Table 1, showed that each strain kept its charac- acetoin, lower amounts of acetic acid not exceed-
ing 500 mg/1, and a higher total content of higher
alcohols more than 300 mg/1. Wines obtained
with the high producers showed a different pat-
Table 2 tern, containing detectable amounts of acetoin,
By-products formation in Trebbiano must by low and high elevated amounts of acetic acid, from 528 to 1185
acetaldehyde producing strains
mg/1, and a lower total content of higher alco-
Strain Acetal- Acetic Acetoin Higher Ethanol hols, from 256.2 to 270.1 mg/1. Conversely, no
dehyde acid mg/l alcohols (% vol) relationship was found between the acetaldehyde
mg/l mg/l (mg/l)
produced and the specific rate of ethanol yielded.
Low
producers:
Ba2 17.4 485 traces 342.6 9.08
Bal9 19.6 267 traces 324.5 8.50 Discussion
Ba28 14.7 160 traces 321.8 8.50
Ba128 18.5 497 traces 301.6 10.80 It is well established and documented by the
Ba137 10.2 378 traces 303.8 7.46 work of various authors that differences exist in
High
producers: the amounts of acetaldehyde formed by various
Bal20 56.7 960 5.6 270.1 8.99 yeasts during fermentation [25,28,29], while it is
Ba145 87.6 810 16.9 268.8 9.27 reported that strains of the same species tend not
Ba167 53.4 873 12.1 256.2 8.41 to differ significantly in the formation of some
Bal70 57.0 528 5.2 256.3 9.23 secondary products of fermentation [30]. At vari-
Ba176 52.3 1185 11.3 267.3 8.79
ance, in our work with 86 S. cerevisiae strains, a

c o n s i d e r a b l e strain v a r i a t i o n in a c e t a l d e h y d e p r o -
d u c t i o n was o b s e r v e d , showing t h a t y e a s t strain
r e p r e s e n t s a p r o m i n e n t factor in d e t e r m i n i n g the
c o n t e n t o f a c e t a l d e h y d e in wine. It is w o r t h not-
ing t h a t t h e f e r m e n t a t i o n t e m p e r a t u r e o f 30°C
e x e r t e d a c l e a r effect on S. cerevisiae, causing a
n o t a b l e i n c r e a s e in a c e t a l d e h y d e by all t h e strains.
T h e a c c u m u l a t i o n o f a c e t a l d e h y d e d u r i n g fer-
m e n t a t i o n is r e p o r t e d to be d e p e n d e n t on the
e q u i l i b r i u m b e t w e e n t h e e n z y m e s alcohol, dehy-
d r o g e n a s e a n d a l d e h y d e d e h y d r o g e n a s e [23-28].
C o n s e q u e n t l y , t h e i n c r e a s e d level o f a c e t a l d e h y d e
at 30°C could b e d u e to an inhibitory effect o f t h e
t e m p e r a t u r e on t h e activity o f alcohol d e h y d r o g e -
nase, t h e e n z y m e r e d u c i n g a c e t a l d e h y d e to
e t h a n o l [31,32].
T h e g r e a t n u m b e r o f strains t e s t e d led us to
distinguish two d i f f e r e n t p h e n o t y p e s , high a n d
low a c e t a l d e h y d e p r o d u c e r s , which b e h a v e d dif-
f e r e n t l y also with r e g a r d to o t h e r s e c o n d a r y p r o d -
ucts of f e r m e n t a t i o n , such as acetoin, acetic acid
a n d h i g h e r alcohols. This c o n f i r m s t h e existence
o f a r e l a t i o n b e t w e e n t h e flavour of alcoholic
b e v e r a g e s a n d t h e yeast strain p e r f o r m i n g fer-
mentation. Moreover, the consistent relationship
f o u n d b e t w e e n a c e t a l d e h y d e a n d t h e total con-
t e n t in h i g h e r alcohols s e e m s to i n d i c a t e a key
role of a c e t a l d e h y d e in t h e biosynthesis o f h i g h e r
alcohols, p r o b a b l y as a p r e c u r s o r for t h e f o r m a -
tion o f valine a n d leucine, f r o m which i s o b u t a n o l
a n d isoamyl alcohol derive.
C o n s i d e r i n g that t h e absence, t h e p r e s e n c e o r
varying c o n c e n t r a t i o n s o f a c e t a l d e h y d e m a y rep-
r e s e n t a n i m p o r t a n t f a c t o r in t h e u l t i m a t e b o u -
q u e t of a wine, t h e p o s s i b l e choice b e t w e e n high
a n d low a c e t a l d e h y d e p r o d u c e r s b e c o m e s of p a r -
ticular i n t e r e s t f r o m t h e t e c h n o l o g i c a l p o i n t o f
view. In fact, a c e t a l d e h y d e is a w e l l - k n o w n a n d
d e s i r a b l e c o n s t i t u e n t of sherry, d e s s e r t a n d p o r t
wines [12,33-35], w h e r e a s in o r d i n a r y t a b l e wines
a c e t a l d e h y d e causes an u n d e s i r a b l e o x i d i z e d taste
[36]. T h e r e f o r e , t h e p h e n o t y p e ' a c e t a l d e h y d e p r o -
duction' assumes a technological importance and
its i n t r o d u c t i o n as a feasible c h a r a c t e r in t h e
strain selection p r o g r a m m e for i n d u s t r i a l a p p l i c a -
tions is suggested.
A s t h e d i f f e r e n c e s f o u n d in a c e t a l d e h y d e p r o -
d u c t i o n a r e r e p r o d u c i b l e a n d consistent, high a n d
217
low p r o d u c i n g strains will b e u s e d to study t h e
g e n e t i c basis of a c e t a l d e h y d e p r o d u c t i o n in S.
cerevisiae.
Acknowledgements
This r e s e a r c h was s u p p o r t e d in p a r t by t h e
N a t i o n a l R e s e a r c h C o u n c i l of Italy, special Pro-
j e c t R A I S A , s u b - p r o j e c t No. 4, P a p e r No. 1446,
Downloaded from https://academic.oup.com/femsle/article/118/3/213/540165 by guest on 08 March 2022
a n d by a g r a n t f r o m the N a t i o n a l R e s e a r c h C o u n -
cil.
References
1 Suomalainen, H. and Lehtonen, M. (1979) The production
of aroma compounds by yeast. J. Inst. Brew. 85, 149-156.
2 Nykanen, L. (1986) Formation and occurrence of flavour
compounds in wine and distilled alcoholic beverages. Am.
J. Enol. Vitic. 37, 84-96.
3 Lehninger, A.L. (1970) Biochemistry. The Molecular Basis
of Cell Structure and Function. Worth, New York.
4 Nordstrom, K. (1966) Metabolism of acetaldehyde - a
metabolic branching point. Acta Chem. Scand. 20, 474-
478.
5 Nordstrom, K. (1968) Metabolism of acetaldehyde - a
metabolic branching point. If. Effect of ethanol. J. Inst.
Brew. 74, 192-195.
6 Collins, E.B. (1972) Biosynthesis of flavor compounds by
microorganisms. J. Dairy Sci. 55, 1022-1028.
7 Strassman, M., Thomas, A., Locke, A. and Weinhouse, S.
(1954) Intramolecular migration and isoleucine biosynthe-
sis. J. Am. Chem. Soc. 76, 4241-4242.
8 Strassman, M., Thomas, A. and Weinhouse, S. (1955) The
biosynthesis of valine. J. Am. Chem. Soc. 77, 1261-1265.
9 Ingraham, J.L. and Guymon, J.F. (1960) The formation of
higher aliphatic alcohols by mutant strains of S. cereuisiae.
Arch. Biochem. Biophys. 88, 157-166.
10 Thoukis, G. (1958) The mechanism of isoamyl alcohol
formation using tracer techniques. Am. J. Enol. Vitic. 9,
161.
11 Schreier, P. (1979) Flavor composition of wines: a review.
CRC Crit. Rev. Food Sci. Nutr. 12, 59-111.
12 Teixeira Junior, E. do V. (1940) O etanal no vinho do
Porto. Anais Inst. Vinho Porto 1,295-303.
13 Bennetzen, J. and Hall, B.D. (1982) The primary structure
of S. cereeisiae gene for alcohol dehydrogenase I. J. Biol.
Chem. 257, 3018-3025.
14 Denis, C.L., Ferguson, J. and Young, E.T. (1983) mRNA
levels for the fermentative alcohol dehydrogenase of S.
cerevisiae decrease upon growth on a non-fermentable
carbon source. J. Biol. Chem. 258, 1165-1171.
15 Delfini, C. and Costa A. (1993) Effects of the grape must
lees and insoluble materials on the alcoholic fermentation
218
rate and the production of acetic acid, pyruvic acid, and
acetaldehyde. Am. J. Enol. Vitic. 44, 86-92.
16 Bayer, E. (1966) Quality and flavor by gas chromatogra-
phy. J. Gas Chromato. 4, 67-73.
17 Wucherpfenning, K. and Semmeler, G. (1972) Acetalde-
hydbildung im Verlauf der Garung in Abhangigkeit vom
Wuchsstoffgehalt des Garsubstrates. I. Milleilung. Z.
Lebensm.-Unters.-Forsch. 148, 77-82.
18 Lafon-Lafourcade, S. (1985) Role des microorganismes
dans la formation de substances combinant le SO 2. Bull.
Off. Int. Vigne Vin 58, 590-604.
19 Longo, E., Velazquez, J.B., Sieiro, C., Cansado, J., Calo P.
and Villa, T.G. (1992) Production of higher alcohols, ethyl
acetate, acetaldehyde and other compounds by 14 Saccha-
romyces cerevisiae wine strains isolated from the same
region (Saln6s, N.W. Spain). World J. Appl. Microbiol.
Biotechnol. 8, 539-541.
20 Schopfer, J.F. and Aerny, J. (1985) Le role de l'anhydride
sulfureux en vinification. Bull. Off. Int. Vigne Vin 58,
515-542.
21 Weeks, C. (1969) Production of sulfur dioxide-binding
compounds and of sulfur dioxide by two Saccharomyces
yeasts. Am. J. Enol. Vitic. 20, 32-39.
22 Casalone, E., Colella, C.M., Daly, S., Gallori, E., Moriani,
L. and Polsinelli, M. (1992) Mechanism of resistance to
sulphite in Saccharomyces cerevisiae. Curr. Genet. 22,
435 -440.
23 Amerine, M.A. and Ough, C.S. (1964) Studies with con-
trolled fermentation. VIII. Factors affecting aldehyde ac-
cumulation. Am. J. Enol Vitic. 15, 23-33.
24 Karklinya, D.Ya. and Liyelpetere, A.Ya. (1985) Dispersion
analysis of productivity of different yeast strains. Izv. Akad.
Nauk latv. SSR 11, 84-86.
25 Fleet, G.H. and Heard, G.M. (1993) Yeasts: growth during
fermentation. In: Wine Microbiology and Biotechnology
(Fleet, G.H., Ed.), pp. 27-54. Harwood Academic Publish-
ers.
26 Kreger-van Rij, N. (1984) The Yeasts, a Taxonomic Study.
Amsterdam, Elsevier.
27 Wickerham, L.J. (1951) In: U.S. Department of Agricul-
ture, Technical Bulletin No. 1029.
28 Millan, C. and Ortega, J.M. (1988) Production of ethanol,
acetaldehyde, and acetic acid in wine by various yeast
races: role of alcohol and aldehyde dehydrogenase. Am. J.
Enol. Vitic. 39, 107-112.
29 Van Zyl, J.A., De Vries, M.J. and Zeeman, A.S. (1963)
Downloaded from https://academic.oup.com/femsle/article/118/3/213/540165 by guest on 08 March 2022
The microbiology of south African winemaking. III. The
effect of different yeasts on the composition of fermented
musts. S. Afr. Agric. Sci. 6, 165-180.
30 Cabrera, M.J., Moreno, J., Ortega, J.M. and Medina, M.
(1988) Formation of ethanol, higher alcohols, esters, and
terpenes by five yeast strains in musts from Pedro Xim~nez
grapes in various degrees of ripeness. Am. J. Enol. Vitic.
39, 283-287.
31 Lutstorf, U. and Megnet, R. (1968) Multiple forms of
alcohol dehydrogenase in S. cerevisiae. I. Physiological
control of ADH-2 and properties of ADH-2 and ADH-4.
Arch. Biochem. Biophys. 126, 933-944.
32 Schimpfessel, L. (1968) Pr6sence et r6gulation de la syn-
thes6 de deux alcool deshydrogenases chez la levure S.
cerevisiae. Biochim. Biophys. Acta 151,317-329.
33 Amerine, M.A. (1954) Composition of wines. I. Organic
constituents. Ad. Food Res. 5, 353-510.
34 Owades, J.L. and Dono, J.M. (1968) A new direct colori-
metric method for determining aldehydes in alcoholic bev-
erages. J. Assoc. Off. Anal. Chem. 51, 148-151.
35 Peynaud, E. (1950) Analyses completes de huit vins doux
naturels. Ann. Agron. Ser. A 1,382-388.
36 Paul, F. (1958) Der Acetaldehyde als Ursache fiir den
Luftgeschmack eines Weines. Kloster. Mitteil. 8, 256-261.