Production of alcohol reduced wines by means of microbial and
biochemical procedures
Jessica Röcker and Manfred Grossmann
Hochschule Geisenheim University, Von-Lade-Str. 1, 65366 Geisenheim, Germany
Abstract: Alcohol reduction of wine is a subject of growing interest due to rising sugar levels in wine grapes.
Different tools for lowering the alcohol content of wine exist such as viticultural, microbiological, biochemical
and physical approaches. The presented two methods to reduce must sugar in the presence of oxygen deal with
a biochemical treatment using Glucose-Oxidase (GOX) for the oxidation of must sugar and a microbiological
approach with non-Saccharomyces yeasts for the assimilation of must sugar. The GOX approach led to an alcohol reduction of 1.72/1.52 vol% in the resulting wine within 43 h of aeration and greatly increased the gluconic
acid level. The non-Saccharomyces yeast Metschnikowia pulcherrima stood out most positive and with its application an alcohol reduction of 3.76 vol% within 2-3 days could be achieved.
1 Introduction
During the last years the interest in alcohol reduction of
wine increased steadily. By now there are already
several known approaches to reduce the alcohol content
in wine [1-4]. The biochemical and the microbiological
approaches have the potential of being a quick, low cost
method for lowering the alcohol content in wine without
compromising wine quality.
This work presents two different methods: the Glucose-Oxidase method and the treatment with nonSaccharomyces yeasts.
1.1 Glucose-Oxidase
The oxidation of ß-D-Glucose into D-Gluconolacton is
catalysed by the enzyme Glucose-Oxidase (GOX). Also
hydrogen peroxide (H2O2) is formed during this reaction.
Afterwards D-Gluconolacton is converted into gluconic
acid. The sugar oxidation in the must stadium prevents
elevated alcohol levels. GOX has a pH-optimum of pH
5.5 to pH 6.0 [5]. This oxygen dependent enzyme is
inactivated by high H2O2 concentrations [6].
The GOX/Catalase system was first used for the
removal of oxygen from wine before bottling. During
these trials also the GOX/Catalase-ratio on different wine
parameters was measured [7]. The idea to reduce sugar in
must by using Glucose-Oxidase was patented in 1986 by
Villettaz [8]. During the end of the last and beginning of
this century, several Glucose-Oxidase trials were
conducted [5, 9-12].
So far the disadvantages of the GOX-method have
been the high acidity of the resulting wines and their high
SO2 demand [11, 12].
1.2 Non-Saccharomyces yeast
The usage of Crabtree-negative non-Saccharomyces
yeasts in the presence of oxygen presents a promising
tool to lower the alcohol content in wine. The aerobic
phase in which the non-Saccharomyces yeasts assimilate
a desired amount of must sugar is followed by an anaerobic phase. At the beginning of this anaerobic phase a
sequential inoculation with Saccharomyces yeasts occurs
[2].
Over one decade ago preliminary work has been
done in this area and was not further developed [13-16].
More recently this topic became again a focus of interest
and different research groups are working in this field
[17-19]. However this research area is still barely explored and further investigations are necessary [2].
The biochemical and microbiological approach presented lowered the alcohol content in wine up to 1.72
and 3.76 %vol. respectively. The GOX-treatment took
place in a 220L-scale. The sugar oxidation and therefore
the aerobic phase lasted 43 h. The aeration phase of the
non-Saccharomyces trial took place in a 20L-bioreactor
and lasted 2-3 days. For more details see [20, 21].
2 Material and Methods
See [20, 21].
3 Results and Discussion
3.1 Glucose-Oxidase
These GOX-trials were conducted in 220L-scale with
two tanks for the GOX-treatments and two for the
controls. Together with the Glucose-Oxidase Gluzyme
Mono 10.000 BG (Novozymes) an adaquate amount of
catalase was added. The aeration phase with pure oxygen
lasted 43 h. An anaerobic fermentaion with a
Sccharomyces yeast followed afterwards. The GOXtreatment
oxidised
30.1/32.5 g/L
glucose
to
29.1/30.6 g/L gluconic acid. The pH dropped from 3.4 to
3.1 (Fig. 1). An alcohol reduction of 1.72/1.52 %vol.
accrued while the total acidity doubled. The
Web of Conferences
fermentations of the GOX-must lasted 14-15 days and
the fermentations of the control wines 4-5 days.
The total SO2 concentration after distillation was
140/124 mg/L for the GOX-wines and 155/145 mg/L for
the controls. Therefore the SO2 concentration of the
GOX-wine was not above the legal threshold as reported
in previous work [11]. Here are two possible explanations presented why a lack of catalase could have lead to
an elevated SO2 demand: (1) The H2O2 which was
formed during must oxidation was removed by SO2 instead of catalase [7]. (2) H2O2 oxidised the ethanol leading to acetaldehyde – a known SO2 binding partner [22,
23].
3.1.1 Double-salt deacidification
The high gluconic acid concentration and the low pH in
the resulting GOX-wines made the need of
deacidification before consumption obvious. Out of that
reason the GOX-wines were deacidified with a double
salt deacidification (DSD).
The high amounts of gluconic acid made the DSD
difficult. Overal 23/34 % tartaric acid participated. The
pH value of the GOX-wine (pH 3.1) could be elevated to
pH 3.3 and was again closer to the original must (pH
3.4), the total acidity was reduced by 20/21 % and the
malic acid was reduced by 9/12 %. No Gluconic acid
was removed.
3.1.2 Sensory evaluation
The sensory analyses of the wines was attended by 12-13
panellists (Fig. 2). The tasting was analysed with an
ANOVA and a Tuckey-Test as follow up test. The GOXand DSD-wine were both significantly (p>0.05) more
acidic than the control. This could already be observed in
a previous tasting were the GOX-wines (~60 g/L
gluconic acid) also were percieved as being significantly
more acidic than the control (p>0.05) [12]. In the present
work a significant difference of the GOX-and DSD-wine
compared to the control wine could be observed whereas
there was no such differnece in the tasting of Pickering
et al. [12].
The results show that the DSD needs to be developed
and adapted further to achieve a higher deacidification
range and to be able to provide an easy protocol for
winemakers.
Fig. 1: Impact of GOX treatment on pH, glucose and
gluconic acid concentrations of the two repetitions of
the 220L-tiral [20].
Fig. 2: Sensory analyses of the wines from the Glucose-Oxidase trials (n = 12-13). NS: not significant at
5%; **significant at 1% [20].
3.2 Non-Saccharomyces yeast
The aeration phase of the non-Saccharomyces trials took
place in a 20L-bioreactor (Proreact 5B, Nr.1371, Heinrich Frings GmbH & Co. KG). Four different yeast
strains were used (Candida zemplinina (CZ) V-260 from
LWG Veitshöchheim, Metschnikowia pulcherrima (MP)
V-131 from LWG Veitshöchheim, Pichia guilliermondii
(PG) 10-S6-L-Trester-82 and Pichia kluyveri (PK)
Frootzen from Chr. Hansen) – two repetitions each. They
were tested for their ability to assimilate must sugar in
the presence of oxygen without producing high amounts
of off-flavours. The aeration phase lasted until at least
35 g/L of the sugar was assimilated which could be
achieved within 2-3 days. The oxygen content mostly
oscillated around 20 % (1.6 mg/L at 20 °C and 1.4 mg/L
at 25 °C). After the aeration phase followed an anaerobic
fermentation with a Saccharomyces yeast, which lasted
12-15 days.
The wine with the M. pulcherrima strain had the
highest alcohol reductions (3.76 +/- 0.08 vol%) followed
by the P. kluyveri (3.03 +/- 1.02 %vol) and the P.
guilliermondii wines (2.00 +/- 0.93). The C. zemplinina
wine had by far the lowest alcohol reduction (0.84 +/0.17 vol%). Also other researchers found that the M.
pulcherrima species is aerobically as well as anaerobically especially good in reducing the alcohol content in
wine [18, 24, 25].
The acetic acid concentration of all the nonSaccharomyces strains was at the end of the aeration
phase lower than at the end of the anaerobic fermentation. This could be in correlation with the small amounts
of yeast assimilable nitrogen (YAN) at the end of the
aeration phase (1–24 mg N/L), despite the daily addition
during the aeration phase. The effect of nitrogen addition
during the anaerobic phase has to be checked. Difficult
fermentation conditions like lack of yeast nutrition can
lead to a higher acetic acid concentration [26, 27]. The
acetic acid content of the M. pulcherrima wine was 0,68
39th OIV Congress, Brazil 2016
+/- 0,06 g/L and therefore had besides the control (0.53
+/- 0.04 g/L) the lowest acetic acid content. Also the
wine of the P. guilliermondii and C. zemplinina strain
staid beneath the legal threshold of 1.08 g/L while the
acetic acid content of the P. kluyveri wine was significantly higher (2.02 +/- 0.21 g/L).
Sensory evaluation: The wines of the 20L-fermenter trial
were sensorially evaluated after 6 to 9 months. Because
the triangle test (15 panellists) indicated significant
differences between the two repetitions of the P. kluyveri
wines both repetitions were evaluated in the profile
tasting (13 panellists) while from the other wines just
one repetition was used.
The C. zemplinina wine with the least alcohol reduction was only perceived as being significantly different
to the control wine in regard to purity (p < 0.01). Thus it
received the best evaluation apart from the control wine.
Compared to the control all the other nonSaccharomyces wines were evaluated as being significantly less fruity and pure (p < 0.01). The P. kluyveri
wines had by far the highest scores regarding the descriptor solvent (p < 0.01) (Fig. 3).
The M. pulcherrima strain was most promising with
alcohol reductions up to 3.76 %vol. However the fermentation conditions need further optimization to make
the wines more appealing for the consumer.
4 Conclusion
This work presented a biochemical and a microbiological tool for partial alcohol reduction in wine. The aim
was to produce wines with lower alcohol levels without
compromising wine quality.
The Glucose-Oxidase trial is a quick tool for reducing the alcohol content in the resulting wine. In the present work within 43 h enough sugar could be oxidised to
achieve an alcohol reduction of 1.72/1.52 %vol. Because
of the high gluconic acid concentration a deacidification
of the resulting wine was necessary. The DSD still needs
further development and adaptation to provide a better
deacidification range and an easier protocol for winemakers. As a result of climate change high pH values of
grape musts often cause growth of unwanted microorganisms with negative effects on wine aroma. Lowering
the pH at the very beginning hinders growth of bacteria
and also fosters complexity of wine. Under this aspect,
the need for DSD application should be low.
The application of non-Saccharomyces yeast strains
is a versatile approach for reducing the alcohol content
in the resulting wine. In the present work the highest alcohol reduction (3.76 %vol) could be achieved with a M.
pulcherrima strain. For enhancing the quality of the resulting wines further investigations for the optimisation
of the experimental set-up are necessary.
5 Acknowledgements
The study was financially supported by the German Federal Ministry of Food and Agriculture (BMEL) through
the Federal Office for Agriculture and Food (BLE), grant
number 2810HS019. I wish to thank those staff and students of the Geisenheim University who helped me with
this study. My special thanks go to Michael Ludwig and
staff, Anja Giehl and Anja Rheinberger, Institute of
Wine Chemistry and Beverage Research and to Matthias
Schmitt and Maximilian Freund Institute of Oenology.
My thanks also go to Josef Valentin Herrmann and Erna
Schindler, Department of Microbiology of the Bavarian
State Institute LWG (Landesanstalt für Weinbau und
Gartenbau) Veitshöchheim, for providing two selected
yeast strains for one of the trials.
Fig. 3: Sensory analyses of the wines from the nonSaccharomyces trials (n = 13). CZ2: Candida
zemplinina V-260 (rep. 2); MP1: Metschnikwia pulcherrima V-131 (rep. 1); PG2: Pichia guilliermondii
10-S6-L-Trester-82 (rep. 2); PK1–2: Pichia kluyveri
Frootzen (Chr. Hansen; rep. 1–2) and SC1: Saccharomyces cerevisiae var. bayanus (control, rep. 1); NS:
not significant at 5%; **significant at 1%;
***significant at 0.1% [21].
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