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LWT - Food Science and Technology 64 (2015) 25e31

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LWT - Food Science and Technology


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A novel culture medium for Oenococcus oeni malolactic starter


production
Carmen Berbegal, Yaiza Benavent-Gil, Isabel Pardo, Sergi Ferrer*
Enolab, ERI BioTecMed/IViSoCa, University of Valencia, Dr. Moliner 50, Burjassot, Valencia 46100, Spain

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 9 January 2015
Received in revised form
12 April 2015
Accepted 13 May 2015
Available online 22 May 2015

Malolactic fermentation is essential in wine quality. One of the strategies used to control this fermentation involves the inoculation of selected lactic acid bacteria, mainly Oenococcus oeni. Laboratory media
usually produce large amounts of biomass, but with little or no adaptability to wine. We propose a
culture medium to grow and pre-adapt O. oeni cells, and the steps to scale-up production. To achieve this
objective, 27 different media were tested. All contained grape must and wine, and nutritional supplements in order to benet bacterial growth. Those media contained different ethanol levels, pH values,
and grape must concentrations. The optimized culture medium named Oenococcus Production Medium
(OPM) contained diluted commercial 4X concentrate white must (1:6), ASv 4%, and a pH of 3.8. The
total time to obtain 80 L of an O. oeni liquid starter culture from the stock culture at the laboratory, with a
nal population of CFU 1  109 mL1, was 22 d. The starter culture was efciently scaled-up, and
preserved at 4  C, 20  C, or freeze-dried. This new culture medium also allowed adaptation of bacteria
to the wine conditions, consuming all malic acid (3 g/L) in 7 d with an inoculum of CFU 1  106 mL1.
2015 Elsevier Ltd. All rights reserved.

Keywords:
Oenococcus oeni
Malolactic fermentation
Malolactic starter culture
Scale-up
Production medium

1. Introduction
The main value of malolactic fermentation (MLF) in vinication
is biological de-acidication, which results from the transformation
of L-malic acid into L-lactic acid by lactic acid bacteria (LAB), mainly
by Oenococcus oeni (Lonvaud-Funel, 1999; Versari, Parpinello, &
Cattaneo, 1999; Wibowo, Eschenbruch, Davis, Fleet, & Lee, 1985).
MLF induces an increase in pH, contributes to microbiological stability, and changes wine taste (Davis, Wibowo, Fleet, & Lee, 1988;
Kunkee, 1991; Maicas, Gil, Pardo, & Ferrer, 1999). MLF can be produced by two strategies: spontaneously or by inoculation with
starter cultures. Traditionally, spontaneous and indigenous LAB
carried out a natural MLF process. But this MLF method can take
weeks or months and is uncontrolled (Zhang & Lovitt, 2006). The
induction of MLF by using a starter culture ensures faster fermentation, reduces potential spoilage by other LAB, and allows the
improvement of wine quality when using selected bacterial strains
~ a, Patchett, Liu, & Pilone, 2001; Pozo-Bayo
n et al.,
(Mira de Ordun

Abbreviations: AUC, area under the curve; LAB, lactic acid bacteria; MLF,
malolactic fermentation; OPM, Oenococcus production medium.
* Corresponding author. Tel.: 34 963 544 518.
E-mail address: sergi.ferrer@uv.es (S. Ferrer).
http://dx.doi.org/10.1016/j.lwt.2015.05.020
0023-6438/ 2015 Elsevier Ltd. All rights reserved.

2005). Nevertheless, successful inoculation of the starter into


wine depends not only on the suitable strain of O. oeni, but on the
preparation and use of the cultures. For the production of a suitably
large biomass, selected strains of O. oeni are grown under conditions
that permit rapid growth and result in a high cell yields, but these
conditions are very different to those present in wine. Consequently, when the starter culture is inoculated directly into wine, it
loses much viability (Henick-Kling, 1988). O. oeni is usually grown in
the laboratory in complex culture media such as MRS (De Man,
~ iga, Ferrer, & Pardo, 1994), or
Rogosa, & Sharpe, 1960), MLO (Zn
AGB (Dicks & van Vuuren, 1990). Most of them frequently contain
grape juice (Davis, Wibowo, Eschenbruch, Lee, & Fleet, 1985) or
apple juice (Champagne, Gardner, & Lafond, 1989). We prefer to use
tomato juice instead, as it is a source of pantothenic acid, commonly
used as a growth factor for wine bacteria (Amachi, Imamoto, &
Yoshizumi, 1971; Richter, Vlad, & Unden, 2001; Terrade & Mira de
~ a, 2009). The culture media are generally supplemented
Ordun
with other nutrients like yeast extract, peptone, and Tween 80, to
increase biomass production (Champagne, Gardner, & Doyon, 1989;
Guerrini, Bastianini, Granchi, & Vincenzini, 2002; Krieger, Hammes,
& Henick-Kling, 1990; Kunkee, 1974; Pilone & Kunkee, 1970). The
growth of O. oeni has been also investigated in single sugars and
their mixtures. The best growth was obtained with sugar mixtures
(glucoseefructose) rather than growth on a single sugar (Maicas,

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C. Berbegal et al. / LWT - Food Science and Technology 64 (2015) 25e31

lez-Cabo, Ferrer, & Pardo, 1999;


Ferrer, & Pardo, 2002; Maicas, Gonza
Zhang & Lovitt, 2005a). Other components like manganese or yeast
mannoproteins and nutrient requirements have been studied
nig,
(Stamer, Albury, & Pederson, 1964; Theobald, Pfeiffer, & Ko
2005). Results revealed that the essential bacterial nutrients were
strain-specic and O. oeni strains showed a large number of auxotrophies (Diez, Guadalupe, Ayestar
an, & Ruiz-Larrea, 2010; Terrade
~ a, 2009; Terrade, Noel, Couillaud, & De Mira
& Mira de Ordun
~ a, 2009; Theobald et al., 2005). Hayman and Monk (1982)
Ordun
evaluated the effect of adding wine to a medium for the production of O. oeni biomass, and found that a content of 40e80% wine in
the medium improved LAB survival and malolactic activity. Nevertheless, further studies are necessary to nd a medium for biomass
production with a cheap easy recipe that allows any O. oeni strain to
grow and adapt before being inoculated into wine.
The objectives of this work were to develop a liquid O. oeni
production medium (OPM) that would permit high levels of
biomass production, but also an adequate pre-adaptation to wine
conditions, and evaluate cell viability and malolactic activity of the
MLF starter culture in wine. Preservation conditions for the liquid
starter culture were also studied.
2. Material and methods
2.1. Microorganisms
O. oeni strains E5003, E5067, E5259, and E5245 were taken from
the Enolab culture collection of the University of Valencia (Spain).
All strains had been previously isolated from spontaneous mid-MLF
in Tempranillo wines from the Ribera del Duero region (Spain).
2.2. Growth in biomass production media
~ iga, Pardo, &
O. oeni strains were pre-grown in MLO broth (Zn
Ferrer, 1993) to early stationary phase. Cells were then centrifuged (rotational speed of 8000 min1, 10 min), washed and
transferred to the 27 culture media (Table 1) at a nal concentration
of CFU 1  106 mL1. The base of all these media contained: yeast
extract (5 g/L), commercial tomato juice with no preservatives
(23 mL/L), Tween 80 (0.5 mL/L), L-malic acid (3 g/L) and concentrate
reconstituted white wine (glucose 0.13 g/L, fructose 0.14 g/L, L-malic
acid 0.22 g/L, free SO2 1 mg/L, ethanol ASv 0%) from Agrovin S.A.
(Spain) (400 mL/L). This basal medium was supplemented with 4X
concentrated white grape must (glucose 360 g/L, fructose 360 g/L, Lmalic acid 5.3 g/L, free SO2 1.4 mg/L) from Agrovin S.A. (Spain),
which was diluted 4, 6 and 8 times (576.5 mL/L) and the pH was
adjusted to 3.8, 4 and 4.5. Media were sterilized by autoclave at
115  C, 30 min. After sterilization, ethanol degree was adjusted at
ASv 4%, ASv 6% and ASv 8%. All growth studies were carried
out in duplicate, in 10 mL screw cap tubes and incubated at 28  C,
7 d. Growth was monitored by measuring optical density at 600 nm
(OD600) with a spectrophotometer (CECIL, CE 373). The bacterial
growth curve was transformed into a value of area under the curve
(AUC) (Gagnon & Peterson, 1998).
2.3. Malolactic fermentation in red wine
The cultures were inoculated into red wine at a nal concentration of CFU 1  106 mL1, and MLF was monitorized for 15 d.
All fermentations were carried out in duplicate. The red wine was
made at the laboratory using a red Tempranillo grape must, inoculated with S. cerevisiae Viniferm B4 (Agrovin S.A.) at a nal concentration of CFU 2  106 mL1, and was incubated at 28  C, 10 d,
until the AF was nished. When the sugar concentration was lower
than 1 g/L, the ethanol was adjusted to ASv 11%, ASv 12%,

Table 1
Combination of diluted 4X grape must (1/4, 1/6 or 1/8), ethanol content (ASv 4%,
ASv 6% or ASv 8%) and pH level (3.8, 4 or 4.5) resulted in 27 different media for
the O. oeni biomass production.
Medium

Diluted must

Ethanol %

pH

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27

1/4
1/4
1/4
1/4
1/4
1/4
1/4
1/4
1/4
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/8
1/8
1/8
1/8
1/8
1/8
1/8
1/8
1/8

4
4
4
6
6
6
8
8
8
4
4
4
6
6
6
8
8
8
4
4
4
6
6
6
8
8
8

3.8
4
4.5
3.8
4
4.5
3.8
4
4.5
3.8
4
4.5
3.8
4
4.5
3.8
4
4.5
3.8
4
4.5
3.8
4
4.5
3.8
4
4.5

ASv 13% and ASv 14%, 3.0 g/L of malic were added, and the pH
was adjusted to 3.5. Then, it was sterilized by ltering through
0.22 mm pore lter and stored at 15  C until use.
2.4. Viable cell counts
Cell viability in red wine was studied by plate counting. The
volume of 0.1 mL of decimal serial dilutions in sterile saline solution
~ iga et al., 1993)
were spread in duplicate on MLO agar plates (Zn
and were incubated at 28  C for 7 d and then the colonies were
counted.
2.5. Culture preservation
Freeze-drying was performed after growing the bacteria until
the end of the exponential phase in 50 mL of OPM medium. Cells
were recovered by centrifugation at rotational speed of 6000 min1,
15 min in a Heraeus Multifuge 1 S-R centrifuge. Then, cells were
washed twice with glutamic acid 0.98%, recovered with the same
above centrifugation conditions, and resuspended in 2 mL of 0.98%
glutamic acid. The bacterial solution was distributed in aliquots of
400 mL per tube. Tubes were frozen at 20  C, 1 h. Freeze-dying was
performed at 60  C for 18 h under vacuum (Virtis Sentry). Tubes
were vacuum sealed and stored at 4  C under dark. Refrigeration at
4  C and freezing at 20  C were performed after growing O. oeni
until the exponential phase in OPM medium and later transferring
aliquots to the respective temperature. Bacterial cultures were
preserved and checked for viability up to 250 d.
2.6. Analytical methods
Glucose, fructose, malic acid and ethanol were quantied by
high-pressure liquid chromatography (HPLC) (Agilent series 1200)
with an isocratic pump (Agilent G1310A) following the procedure
described by Frayne (1986) with minor modications. The mobile
phase consisted of a solution of 0.75 mL of 85% H3PO4 per litre of

C. Berbegal et al. / LWT - Food Science and Technology 64 (2015) 25e31

deionized water with a ow rate of 0.7 mL/min. An Agilent G1322A


degasser was employed. Samples (5 mL) were injected automatically (Agilent G1367B). The components were separated using an
Aminex HPX-87H precolumn (Bio-Rad) coupled with two ion
exclusion columns of 300 mm by 7.8 mm Aminex HPX-87H (BioRad) thermostatically controlled at 65  C (Agilent G1316A). Compounds were detected by a G1314B variable-wavelength detector
(Agilent) set to 210 nm and a refractive index detector (Agilent
G1362A) in series. Elution time was 45 min. External calibration
was performed. All samples were centrifuged (rotational speed of
8000 min1, 10 min) and ltered through a membrane lter with a
mean pore size of 0.22 mm before use. Quantication was performed by measuring peak height compared to external standards.
2.7. Scale-up of biomass production
The scale up started with a culture of CFU 1  106 mL1 of
O. oeni in 50 mL of liquid OPM medium incubated statically at 28  C.
When the bacterial population reached the maximum biomass
yield, the 50 mL were inoculated into 0.5 L of OPM sterile medium,
and were incubated at 28  C until the bacteria population reached
the maximum biomass yield. Then, the O. oeni culture was inoculated into the 10 L fermenter with 8 L of OPM sterile medium and
was incubated stirred at room temperature. The 0.5 L and the 10 L
fermenter were built using Pyrex bottles with a screw cap GL 45
with three ports GL 14 thread (Duran). In the 0.5 L fermenter, one of
these ports was closed with a screw cap GL 14, the second one was
connected to a set for pressure compensation (Duran) to permit
fermentation gases to vent out, and the third one was connected
with a screw Cap GL 14 for hose connection (Duran) with a joint
silicone diameter 0.6 mm and a silicone tube of 3 mm  6 mm. This
silicone tube was joined to one of the ports in the PVDF Luer lock 3
way valve (Cole Palmer) of the 10 L fermenter. In the 10 L fermenter,
one of these three ports was connected to a pressure compensation
set (Duran) to enable fermentation gases to vent out, and the other
two were connected with a screw cap GL 14 for hose connection
(Duran) to two silicone tubes. One of these tubes was used to
inoculate the starter from the 0.5 L fermenter, and the other one
was used to collect samples with a 50 mL Luer lock syringe. The
industrial biomass production process was carried out inoculating
the 10 L of the O. oeni culture into a fermenter with 80 L of OPM
sterile medium (Bioprocess technology Bio-pro 100 L). The inoculated culture was incubated at 28  C until the bacteria population
reached CFU 1  109 mL1. A microbiological analysis of the three
scale-up steps was carried out to certify the correct sterilization of
the culture medium. After the sterilization of 50 mL and 0.5 L
fermenter of OPM medium by autoclave, 115  C 30 min, and the 10 L
and 100 L fermenters, 115  C 45 min, 0.1 mL sample from each
~ iga
fermenter were plated on MRS (De Man et al., 1960), MLO (Zn
et al., 1993) and YPD (Landete, Ferrer, & Pardo, 2007) media. The
plates were incubated at 28  C for 7 d.
2.8. Statistical analysis
Statistical signicance of differences between averages of
duplicate measurements were evaluated by performing one-way
ANOVA at a condence level of p 0.05 and Tukey's Studentized
Range Test.
3. Results and discussion
3.1. Growth in biomass production media
O. oeni strain E5003 was selected to carry out the assays because
it presented interesting technological features as malolactic starter

27

(Berbegal, 2014). The culture medium designed to grow bacteria


contained mainly grape must and wine in order to adapt cells to the
harsh conditions of wine. White wine and must were used because
the sterilization of red must and wine caused precipitates, and also
turbidity made it difcult to monitor bacterial growth properly.
Commercial wine concentrate and grape must were used to standardize the culture medium.
When the white concentrate must was diluted 4 times (media
1e9), the growth was lower than when the must was diluted 6
(media 10e18) or 8 (media 19e27) times (Fig. 1). Between must
diluted 6 or 8 times differences were smaller, although there was
slightly better growth with the must diluted 6 times. The best
growth was obtained with ASv 4% (1e3, 10e12, 19e21), and the
worst results were found with ASv 8% (7e9, 16e18, 25e27). In all
cases, growth with a pH of 4.5 resulted in the highest value of AUC
followed by growth with a pH of 4, and the lowest value was obtained with a pH of 3.8. It was observed that at lower pH, more time
was needed to reach to the same OD600 nm. The best results were
found, as expected, when the must was diluted 6 times with the
lowest ethanol content (ASv 4%) and with the highest pH (4.5)
(media 12), and the worst growth was observed with the must
diluted 4 times, the highest ethanol content (ASv 8%) and 3.8 pH
(medium 7) (Fig. 1).
Results were analyzed using an ANOVA test. This analysis
revealed a signicant effect (p < 0.05) for must, ethanol, pH and for
the interactions musteethanol and mustepH. Through the Tukey
post hoc test, the media with the lowest AUC were rejected (1, 2, 4,
5, 7, 8, 16, 17, and 25). All these media obtained an AUC below 2, and
their pH levels were 3.8 or 4. The cultures from the 18 remaining
media were selected and their malolactic activity in wine was
studied. HPLC analysis showed that the must concentrate contained
around 360 g/L of glucose and 360 g/L of fructose. When must
concentrate was diluted 4 times bacterial growth was lower than
when the must was diluted 1/6 or 1/8 probably because of the
osmotic pressure.
Composition of OPM medium was based on previous studies of
growth, metabolism and biomass production of O. oeni.
Champagne, Gardner, and Lafond (1989) studied the production of
O. oeni biomass in apple juice media and grape juice media. Their
results showed that there were variations between strains in their
ability to grow, and the optimum pH was also strain dependent,
varying from 4.5 to 4.8. Yeast extract supplementation was the most
benecial for growth. In our study, 5 g/L of yeast extract was added
to the media and a range of pH was studied but always below 4.8
because this value was too high, compared to the wine pH. Also,
malic acid was added to stimulate LAB growth and MLF activity
(Terrade et al., 2009). The studies of Zhang and Lovitt (2005a) and
lez-Cabo, et al. (1999)
Maicas et al. (2002) and Maicas, Gonza
showed that better growth of O. oeni was obtained in the presence of sugar mixtures (glucose:fructose, 1:1) in the culture medium compared to glucose alone. The optimal growth of O. oeni was
at pH 4.5, with the maximum biomass yield and specic growth
rate. In the present work, the highest biomass also was obtained
when the bacterium was grown at pH 4.5 and the medium contained a sugar mixture of glucose:fructose (1:1).
3.2. Malolactic activity in red wine
All the cultures from media with a value of AUC above 2 (Fig. 1)
were inoculated in red wine with ASv 12% and pH 3.5. Some
differences were found in malolactic activity, and 5 cultures (10, 13,
19, 22, and 26) consumed all the malic acid in 6 d (Fig. 2). These
differences in malolactic activity depended mainly on the pH of the
medium where the bacteria were grown before. The cultures that
were grown in a medium with a pH of 4.5 resulted in a slower

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C. Berbegal et al. / LWT - Food Science and Technology 64 (2015) 25e31

Fig. 1. Values of O. oeni E5003 area under the curve (AUC) in the 27 different culture media (
media is described in Table 1.

must diluted 1/4,

must diluted 1/6,

must diluted 1/8). The code number of the

Fig. 2. Malic acid consumption (%) in red wine after 6 d of incubation of the O. oeni strain E5003 previously grown in the different culture media ( must diluted 1/4,
1/6, must diluted 1/8). The code number of the media is described in Table 1.

consumption of malic acid in red wine. The best results of activity in


wine were found when O. oeni strain E5003 was previously grown
in a medium with a pH of 3.8. This can be correlated to the fact that
this pH was more similar to the wine pH (3.5), so the cells were
more adapted to it. Out of these 5 media, 4 of them (10, 13, 19 and
22) had been grown in a pH of 3.8, proving the importance of the
pH as a growth factor in the culture media for MLF in wine later on.
The culture with the highest biomass in the culture medium was
selected for further studies. Medium 10 had the highest value of
area under the curve (2.44 AUC) (Fig. 1) so this medium was chosen
to be the OPM. That was the medium with the must diluted 6 times,
with an ASv 4% and a pH of 3.8 (Table 2). HPLC analysis of this
medium showed around 40 g/L of glucose, 40 g/L of fructose, and
3.5 g/L of malic acid. The OPM medium contains must and wine and
pH is 3.8, so growth conditions are similar to wine conditions, cells
are adapted to wine and do not need successive transfers before
inoculation. The starter culture kept high cell viability and malolactic activity when it was inoculated in wine.

Table 2
Composition of OPM medium.
Constituent/conditions

Concentration

Yeast extract
Commercial tomato juice (with no preservatives)
Tween 80
L-Malic acid
White wine
White grape must
Ethanol
pH
Growth temperature

5 g/L
23 mL/L
0.5 mL/L
3 g/L
400 mL/L
536.5 mL/L
4%
3.8
28  C

must diluted

3.3. Scale-up of biomass production


Microbiological analysis of the 4 scaled-up steps was performed
to certify the correct sterilization of the OPM culture medium. The
results showed that sterilization of all media was adequate, with no
growth appearing in any control media (CFU < 1  102 mL1 in all
plate media).
Scale up started with a culture of CFU 1  106 mL1 O. oeni
E5003 in 50 mL of OPM medium. The bacterial population
reached CFU 1  109 mL1 after 6 d incubation. Then, the 50 mL
were inoculated into 0.5 L of OPM sterile medium (Fig. 3a). The
inoculated fermenter was incubated at 28  C and the bacterial
population reached CFU 1  109 mL1 after 4 d of incubation
(Fig. 3b). The O. oeni culture was inoculated into the 10 L
fermenter with 8 L of OPM sterile medium and became a nal
concentration of CFU 1.3  109 mL1 after 6 d of incubation
(Fig. 3c). The 8 L of O. oeni culture were inoculated into the 80 L of
OPM medium, and incubated at 28  C to a nal concentration of
CFU 1  109 mL1. The culture reached this cell concentration in
6 d (Fig. 3d). The total time of the production process to obtain
80 L of O. oeni liquid starter culture, from the stock culture with a
nal population of CFU 1  109 mL1, were 22 d. Many authors
have studied the medium composition required to achieve
maximum biomass production and to improve the malolactic
activity of O. oeni in wine (Champagne, Gardner, & Doyon, 1989;
Champagne, Gardner, & Lafond, 1989; Henick-Kling, 1988;
Krieger, Hammes, & Henick-Kling, 1992; Zhang & Lovitt, 2005b). A
high biomass is needed to assure MLF by the inoculated bacteria,
but a high biomass yield usually means that these cells are not
well adapted to harsh wine conditions. On the contrary, good
adaptation and acclimation of bacteria to wine involves poor

C. Berbegal et al. / LWT - Food Science and Technology 64 (2015) 25e31

29

Fig. 3. Growth of O. oeni strain E5003 (CFU mL1) in OPM medium in the different fermenters during the scale-up: a) 50 mL, b) 0.5 L, c) 8 L, d) 80 L.

growth and a low biomass yield. Besides, nowadays cells are


regularly collected and freeze-dried after growth, in order to
preserve them and permit convenient use in cellars. These concentration and preservation processes can involve loss of viability,
or imply a change in the adaptation abilities of the cells to the
wine. The production of a liquid starter overcomes these two last
drawbacks, and it has been demonstrated that it is possible to
produce well-adapted bacteria with a good yield. A liquid active
malolactic starter can be obtained in a short time, and this process
is easily scalable.
The whole process was tested with other O. oeni strains from
Enolab collection with technological features (E5067, E5259, and
E5245), to be used as malolactic starters. In all 3 cases, a nal
population between CFU 8.9  108 and 1.3  109 mL-1 was achieved in the 80 L fermenter. Total time of the production process to
obtain the O. oeni liquid starters, from the stock culture was also
22 d for every strain. Therefore, the medium designed could be
effectively used to produce biomass of different O. oeni strains.

Preservation by refrigeration at 4  C was better than freezedrying for a short period of time, up to 50 d, but it was worse
than freezing at 20  C. Therefore, for preservation from 2 to 4
months, refrigeration was the best option because cell viability was
CFU 2.5  107 mL1 after 124 d. Storage at 4  C of the liquid starter
is feasible for most industrial productions and uses. It provides a
good viability for periods up to 3 or 4 months, and it is cheaper and
easier to store and distribute than freeze-dried or frozen cultures.
Many authors have studied the effect of different preservation
methods, mainly freeze-drying, of O. oeni and its inuence on MLF
and survival in wine (G-Alegria et al., 2004; Huaa, WenYing, Huaa,
Zhong Chao, & AiLian, 2009; Maicas, Pardo, & Ferrer, 2000; Zhao &
Zhang, 2005, 2009). Maicas et al. (2000) studied the effects of
freezing at 20  C in glycerol, by volume, of 40% and freeze-drying
of O. oeni upon induction of MLF in red wine. Their results in freezedrying experiments were similar to those described for storage
at 20  C, but the ability to induce MLF in wine depended on the
medium where the cells were previously grown before being stored
frozen or freeze-dried. These results are in agreement with ours.

3.4. Malolactic starter culture preservation


Maintenance of the liquid starter culture O. oeni strain E5003
produced in OPM medium was studied using freezing at 20  C
and refrigeration at 4  C. These methods were compared to
freeze-drying, the most common technique for preserving MLF
starter culture. Culture preservation after freeze-drying showed
that cell viability was stabilized and maintained for 8 months. At
this point, this method presented the best viability results, so this
preservation method would be the most adequate for long-term
storage (Fig. 4). Alternatively, freezing at 20  C presented a
very low drop in cell viability throughout the rst month. This
technique would be the most adequate for culture preservation
for 30 d, as during this time the freezing method presents the best
results, preserving the bacterial population over 51.31%
(CFU 7  108 mL1). After that period LAB viability decreased
drastically (Fig. 4).

Fig. 4. Cell viability of O. oeni strain E5003 (CFU mL1) preserved by refrigeration at
4  C (C), freezing at 20  C (-) and freeze-drying (:) during 250 d.

30

C. Berbegal et al. / LWT - Food Science and Technology 64 (2015) 25e31

Fig. 5. Viable cells (CFU mL1) and malic acid consumption (%) of O. oeni strain E5003 previously grown in OPM medium in red wine with ASv 11% (C), ASv 12% (-),
ASv 13% (:) and ASv 14% (;).

3.5. Malolactic activity and cell viability in red wine


Fig. 5 shows the malolactic activity and cell viability of the
starter culture produced in the 100 L fermenter when it was
inoculated in red wine with ASv 11%, ASv 12%, ASv 13% and
ASv 14% in a nal concentration of CFU 1  106 mL1. When
wine had an ASv 11% or ASv 12%, the number of viable cells
increased promptly and depleted all malic acid (3 g/L) in 7 d. With
an ASv 13%, the O. oeni culture grew slower but malic acid was
consumed in 14 d. During the time of the assay, MLF was not
accomplished for wines with ASv 14%, however, the viability of
the bacteria remained stable during this period, which allowed the
bacteria to grow later and perform MLF correctly (data not shown).
If a particular winery has difcult or unusual wines, with high
ethanol content, low pH, etc., grape must and wine from the OPM
medium can be replaced by grape must and wine from the winery.
Thus, the O. oeni strain that is going to be inoculated in the winery
will be more adapted to their specic wine conditions.
4. Conclusions
A liquid culture medium has been designed (OPM) to grow
O. oeni and obtain high levels of biomass, and also adequate
adaptation to the wine conditions. Composition of the biomass
production medium permits to reach a population of O. oeni of
CFU 1  109 mL1 in 6 d. The process to obtain industrial levels of
O. oeni biomass has been scaled up using 80 L of the OPM medium.
The bacterial population reaches CFU 1  109 mL1 in 22 d from
the laboratory stock. For a long storage period of the O. oeni strain
E5003 starter culture, freeze-drying is the best option; nevertheless, for up to 3 months of preservation, storage at 4  C is the best
choice, and for a 1 month storage period, freezing at 20  C is the
best option. This new culture medium also allowed the bacteria to
adapt to the wine conditions, consuming all the wine malic acid in
7 d with an inoculum of CFU 1  106 mL1.
Acknowledgements
This work was supported by the project CENIT-2008 1002, V
segles Universitat-Empresa grant of the University of Valencia, and
Agrovin S.A. English text revised by the English language reviewer
Beverly Johnson.
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