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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.
26
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
27
28
Fig. 1. Values of O. oeni E5003 area under the curve (AUC) in the 27 different culture media (
media is described in Table 1.
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.
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
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.
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.
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
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% (;).
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