The effects of freezing and freeze-drying of Oenococcus oeni upon induction of malolactic fermentation in red wine

International Journal of Food Science and Technology 2000, 35, 75–79
75
The effects of freezing and freeze-drying of Oenococcus
oeni upon induction of malolactic fermentation in red
wine
Sergi Maicas1,2,*, Isabel Pardo1 & Sergi Ferrer1
1 Departament de Microbiologia i Ecologia, Facultat de Biologia, Universitat de València, Burjassot, Spain
2 Present address: Departament de Biotecnologia, Institut d’Agroquímica i Tecnologia d’Aliments, CSIC, Paterna, Spain
(Received 12 July 1999; Accepted in revised form 21 September 1999)
Summary
The use of Oenococcus oeni starter cultures for the induction of malolactic fermentation
(MLF) in wine permits control over the timing of the process and the quality of the wine.
Successful inoculation of bacterial starter cultures into wine depends on the selection of
suitable strains and on the preparation and conservation of those cultures. Medium for
Leuconostoc oenos (MLO) is the best medium for easy and rapid growth of O. oeni cul-
tures under laboratory controlled conditions for isolation and identi?cation. However,
this study showed that O. oeni cells inoculated in MLO failed to induce MLF in wine
while cells grown in Medium of Preculture (MP) or wine, stored at 20 °C or freeze-
dried retained the ability to induce MLF when inoculated in wine. Our results suggest
that the use of freeze-dried cultures of O. oeni previously grown in MP is the best choice
for industrial application.
Keywords
Lactic acid bacteria, Leuconostoc oenos, malic acid, starter culture.
Oenococcus oeni (Dicks et al., 1995), is adapted to
Introduction
high ethanol concentrations and low pH values in
The production of wine with low levels of malic
wine and is responsible for MLF (Davis et al.,
acid is considered a prerequisite for the commer-
1985). In recent years, starter cultures technolo-
cialization of this beverage. Traditionally, the
gies, involving the inoculation of O. oeni into
way to reduce the quantities of this acid has been
wine, have been developed for inducing MLF
the spontaneous growth of lactic acid bacteria
(Edwards et al., 1991; Nielsen et al., 1996).
naturally present in wine, which develop the mal-
However, failures usually occur because of the
olactic fermentation (MLF) i.e. the bacterial con-
lack of adaptation of cultures to wine or because
version of L-malic into L-lactic acid and CO
of cellular damage during storage. As the loss of
2
(Kunkee, 1967; Wibowo et al., 1985). In addition
viability is very high when the cells are directly
to deacidi?cation, the MLF is considered to con-
inoculated into wine, starter cultures need one or
tribute to the complexity of wine ?avour and to
more steps of reactivation and adaptation to wine
confer a degree of microbiological stability to the
before use in order to enhance the survival of the
wine (Gao & Fleet, 1994; Henick-Kling, 1995;
bacteria (Naouri et al., 1989; Nielsen et al., 1996).
Maicas et al., 1999c). Several studies have shown
Our aim is to enhance physiological adaptation
that Leuconostoc oenos, recently reclassi?ed as
of living cells by the incubation in preculture
media such as Medium for Leuconostoc oenos
*Correspondent: Fax: +34 96 3636301;
(MLO), Medium of Preculture (MP) or wine
email: sergi.maicas@iata.csic.es
before wine inoculation. The conditions of a pro-
© 2000 Blackwell Science Ltd

---

76
Ef?cient storage of Oenococcus oeni S. Maicas et al.
longed storage by freezing or by freeze-drying, on
with a culture grown in wine, to provide a con-
the survival rates and viability of O. oeni cells in
trol.
the preculture media, were also investigated.
Results
Materials and methods
In?uence of the culture medium on the
Strains and culture conditions
production of Oenococcus oeni biomass
Oenococcus oeni strains were collected from
The composition of the culture medium in?uences
Requena (Eastern Spain), isolated and identi?ed
the adaptation of the malolactic bacteria to the
as described by Pardo & Zúñiga (1992). Cells
stress conditions in wine (Hayman & Monk,
were cultured in Medium for Leuconostoc oenos
1982). The most favourable growth conditions for
(MLO) (Caspritz & Radler, 1983) or in Medium
nutritionally fastidious microorganisms such as
of Preculture (MP) (Maicas et al., 1999b) at 28 °C
O. oeni occurs when a strain is inoculated in a
without stirring.
synthetic medium (MLO) including nutritional
compounds, e.g. yeast extract, tween-80 or toma-
to juice (Beelman et al., 1977; Hayman & Monk,
Conservation and determination of stability
1982) (Fig. 1). The presence of suf?cient nutrients
of malolactic activity of cells during storage
and an appropriate pH permits developments of
Suspensions of O. oeni strains were stored at
high levels of biomass, approx 109 cfu mL 1 in 2–3
20 °C in glycerol (40% v/v) or freeze-dried in
days (Maicas et al., 1999a). A recently described
0.067 M glutamic acid and maintained at 4 °C.
complex medium to preculture O. oeni (MP) has
After one year, the vials were opened and per-
been shown to give better adaptation of bacteria
centages of surviving cells in the vials were deter-
to wine conditions (Maicas et al., 1999b). In the
mined by spreading onto the MLO plates. Then,
present work we produced 1010 cfu mL 1 in 2–3
cultures were diluted to reach 105 cfu mL 1, inoc-
days, which is a ten-fold increase in the biomass
ulated in Monastrell red wine (pH 3.5) containing
production compared with MLO, the optimum
11.1 % (v/v) ethanol and in g L 1: malic acid, 3.5;
medium to grow O. oeni strains up to date
glucose, 0.06 and fructose, 0.15, and incubated 7
(Fig. 1). However, red wine was not suitable for
weeks at 20 °C. The remaining malic acid quan-
direct inoculation with O. oeni strains since many
tities were quanti?ed weekly as described by
cells died. Cells should be grown in MLO or MP
Maicas et al. (1999a). Wines were also inoculated
before inoculation in wine in order to avoid loss
Figure 1 Effect of different
1e+11
culture media on Oenococcus oeni
1e+10
MA4 survival. Cells were
inoculated in MLO (), MP ()
1e+9
or wine (). Cells were incubated
1e+8
at 28 °C (MLO and MP) or
20 °C (wine). The data presented
c.f.u.)/mL
1e+7
are mean values from three
10
1e+6
separated experiments.
(log
1e+5
cells
1e+4
of
1e+3
N¼
1e+2
1e+1
0
12
24
36
48
60
72
Time (h)
International Journal of Food Science and Technology 2000, 35, 75–79
© 2000 Blackwell Science Ltd

---

Ef?cient storage of Oenococcus oeni S. Maicas et al.
77
Figure 2 Time course of four
Batch 1
Batch 2
Batch 3
Batch 4
1e+8
4
successive malolactic
fermentations in wine with
Oenococcus oeni MA4. , cfu
ml 1; , residual malic acid. Cells
3
(g/l)
used in batch 1 were precultured
in MP. Cells grown in a previous
c.f.u.)/mL
1e+7
acid
batch were diluted to give a
10
concentration of about 1
105
cfu mL 1 in fresh wine (batches
(log
2
malic
2–4). The data presented are
mean values from three separated
cells
1e+6
experiments.
of
1
N¼
Residual
1e+5
0
0
7
14
21
28
35
42
49
56
63
Time (days)
of viability of the organisms. Successive reinocu-
(Table 2), providing an acceptable method for
lations in wine were done without any loss of via-
storage of cultures. Freeze-dried suspensions of
bility although the nutritional shortage in wine
O. oeni cells were also stored for one year to
permits development of quantities of biomass of
check the survival rate and the retention of their
only about 107 cfu mL 1 and requires at least 2–3
ability to degrade the malic acid. The cells
weeks for each batch (Fig. 2).
showed a similar performance to that was previ-
ously described for storage at 20 °C (Table 2).
When cells were grown in MP, less than 10% of
Storage of cultures: survival and maintenance
the initial cells survived (with the only exception
of malolactic activity
of the O. oeni strain MA4). Stability was
The rates of survival of O. oeni cells in MLO or
improved for some strains grown in MLO or
in wine were higher than those recorded in MP,
wine (Table 1). However, mirroring the results for
after the storage for one year at –20 °C (Table 1).
storage at 20 °C, only cells that have gained
Nevertheless, when the living cells were inoculat-
adaptation by inoculation in MP or wine retained
ed in fresh wine (about 105 cfu mL 1) only cells
the ability to induce MLF when inoculated in
previously grown and stored in MP or wine were
wine (Table 2). O. oeni strain VV5 was able to
able to perform the MLF in 3–4 weeks. These
degrade L-malic acid, but it took almost two
results were similar to those obtained by direct
months. As part of this project to optimize the
inoculation to wine of cells growing in wine
conditions of storage for the retention of malo-
Table 1 Survival rate (%) for Oenococcus oeni strains, grown in different culture media, after one year storage at 4 °C
(freezed-dried cultures) or at 20 °C. The data presented are mean values from three separated experiments
Storage conditions
20 °C
Freeze-drying
O. oeni strain
MLO
MP
Wine
MLO
MP
Wine
MA4
78.9
20.0
87.5
57.9
62.5
29.1
TE3
95.2
26.9
93.3
27.4
5.2
20.8
VV5
92.3
10.5
73.3
23.3
5.8
26.0
BM3
72.0
13.3
88.9
7.7
2.1
8.5
TV3
82.0
10.4
80.0
3.3
1.5
8.3
© 2000 Blackwell Science Ltd
International Journal of Food Science and Technology 2000, 35, 75–79

---

78
Ef?cient storage of Oenococcus oeni S. Maicas et al.
Table 2 Time required to detect MLF in wine (weeks) with different Oenococcus oeni strains after storage (a direct
inoculation control was also included). The data presented are mean values from three separate experiments
Storage conditions
Direct inoculation
20 °C
Freeze-drying
O. oeni strain
MLO
MP
Wine
MLO
MP
Wine
MLO
MP
Wine
MA4
a
3
3
a
3
3
a
3
3
TE3
a
3
3
a
4
3
a
3
3
VV5
5
3
3
a
3
3
7
3
3
BM3
a
3
3
a
3
4
a
3
3
TV3
a
4
3
a
4
3
a
3
3
a After 7 weeks, malic acid degradation in wine was not detected.
lactic ability, we compared the survival rates for
tion problems often linked to starter cultures. The
the same O. oeni strain grown in a culture medi-
results obtained in freeze-drying experiments were
um and stored by freezing or freeze-drying.
similar to those described for storage at 20 °C.
Results showed higher retention of malolactic
The use of freeze-dried cultures is easy and inex-
activity for storage of cells at 20 °C for one year
pensive in industry.
than for freeze-drying, in agreement with results
reported by Henick-Kling (1991). However, mal-
Acknowledgments
olactic activity and time required to detect MLF
were very similar irrespective of cell storage con-
This work has been partially supported by grants
ditions.
from the Comisión Interministerial de Ciencia y
Tecnología (ALI93–0246) and by a grant from
the M.E.C. (Spanish Government) to S.M. The
Discussion
authors are grateful to À. Natividad and P.
MLO is the best medium for easy and rapid
González-Cabo for technical assistance.
growth of Oenococcus oeni cultures under labora-
tory controlled conditions for isolation and iden-
References
ti?cation. However, cells lose their natural
resistance to the adverse environmental condi-
Beelman, R.B., Gavin, A. & Keen, R.M. (1977). A new
strain of Leuconostoc oenos for induced malo-lactic
tions in wine and fail as starter to induce MLF
fermentation in eastern wines. American Journal of
(Henick-Kling, 1995). The growth of the starter
Enology and Viticulture, 28, 159–165.
bacteria in an appropriate preculture medium
Caspritz, G. & Radler, F. (1983). Malolactic enzyme of
such as MP (Maicas et al., 1999b) reduces the
Lactobacillus plantarum. Journal of Biological
preparation time of starter cultures, prepares the
Chemistry, 258, 4907–4910.
Davis, C.R., Wibowo, D., Eschenbruch, R., Lee, T.H. &
cells to survive after storage and allows growth in
Fleet, G.H. (1985). Practical implications of malolactic
wine. Moreover, cells ef?ciently perform MLF in
fermentation: a review. American Journal of Enology
wine. Storage at 20 °C is an effective procedure
and Viticulture, 35, 290–301.
too, but it takes 2–3 months to establish growth
Dicks, L.M.T., Dellaglio, F. & Collins, M.D. (1995).
in wine. However, this technique also requires
Proposal to reclassify Leuconostoc oenos as Oenococcus
oeni [corrig.] gen. nov., comb. nov. International
growth of cells in adaptation media prior to inoc-
Journal of Systematic Bacteriology, 45, 395–397.
ulation to ensure an adequate survival rate.
Edwards, C.G., Jensen, K.A., Spayd, S.E. & Seymour,
Despite loss of viable cells when stored at
B.J. (1991). Isolation and characterization of native
20 °C, MP is a better choice compared with
strains of Leuconostoc oenos from Washington state
rates recorded in wine, since adapted surviving
wines. American Journal of Enology and Viticulture, 42,
219–226.
cells are able to perform MLF immediately after
Gao, C. & Fleet, G.H. (1994). The degradation of malic
being inoculated in wine. This also involves a
by high density cell suspensions of Leuconostoc oenos.
reduction of production costs and of contamina-
Journal of Applied Bacteriology, 76, 632–637.
International Journal of Food Science and Technology 2000, 35, 75–79
© 2000 Blackwell Science Ltd

---

Ef?cient storage of Oenococcus oeni S. Maicas et al.
79
Hayman, D.C. & Monk, P. (1982). Starter culture
malolactic fermentation in red wine using free
preparation for the induction of malolactic
Oenococcus oeni. World Journal of Microbiology and
fermentation in wine. Food Technology in Australia, 34,
Biotechnology, in press.
16–18.
Maicas, S., Gil, J.V., Pardo, I. & Ferrer, S. (1999c).
Henick-Kling, T. (1991). Use of malolactic starter
Improvement of volatile composition of wines by
cultures in the winery. Proceedings of the Focus Eastern
controlled addition of malolactic bacteria. Food
US Chardonnay Symposium (edited by T. Henick-
Research International, in press.
Kling). Pp. 112–117. Front Royal: University of Davis.
Naouri, P., Chagnaud, P., Aranaud, A., Galzy, P. &
Virginia: Virginia Polytechnic Institute and State
Mathieu, J. (1989). Optimization of the conditions for
University.
preparing bacterial cultures for malolactic fermentation.
Henick-Kling, T. (1995). Control of malo-lactic
Journal of Biotechnology, 10, 135–150.
fermentation in wine: energetics, ?avour modi?cation
Nielsen, J.C., Prahl, C. & Lonvaud-Funel, A. (1996).
and methods of starter culture preparation. Journal of
Malolactic fermentation in wine by direct inoculation
Applied Bacteriology Supplement, 79, 29S–37S.
with freeze-dried Leuconostoc oenos cultures. American
Kunkee, R.E. (1967). Malo-lactic fermentation. Advances
Journal of Enology and Viticulture, 47, 42–48.
in Applied Microbiology, 9, 235–279.
Pardo, I. & Zúñiga, M. (1992). Lactic acid bacteria in
Maicas, S., González-Cabo, P., Ferrer, S. & Pardo, I.
Spanish red rosé and white musts and wines under
(1999a). Production of Oenococcus oeni biomass to
cellar conditions. Journal of Food Science, 57, 392–405.
induce malolactic fermentation in wine by control of
Wibowo, D., Eschenbruch, R., Davis, C.R. & Lee, T.H.
pH and substrate addition. Biotechnology Letters, 21,
(1985). Occurrence and growth of lactic acid bacteria in
349–353.
wine: a review. American Journal of Enology and
Maicas, S., Pardo, I. & Ferrer, S. (1999b). Continuous
Viticulture, 36, 302–313.
© 2000 Blackwell Science Ltd
International Journal of Food Science and Technology 2000, 35, 75–79

---