Biochemical Engineering Journal 110 (2016) 134142

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Regular article

Application of magneto-responsive Oenococcus oeni for the malolactic

fermentation in wine
Peter Dusak a,b, , Mojca Bencina c,d , Martina Turk e , Dejan Bavcar f , Tatjana Kosmerl g ,
Marin Berovic h , Darko Makovec a,b
a
Department for Material Synthesis, Jozef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia
b
Jozef Stefan International Postgraduate School, Jamova 39, 1000 Ljubljana, Slovenia
c
Laboratory of Biotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia
d
EnFIST, The Centre of Excellence EN-FIST, 1000 Ljubljana, Slovenia
e
Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia
f
Agricultural Institute of Slovenia, Hacquetova ulica 17, 1000 Ljubljana, Slovenia
g
Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia
h
Department of Chemical, Biochemical and Environmental Engineering, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Vecna pot
113, 1000 Ljubljana, Slovenia

a r t i c l e i n f o a b s t r a c t

Article history: A new method for magnetic separation of the magnetized lactic acid bacteria (LAB) Oenococcus oeni
Received 9 September 2015 at a desired stage of the malolactic fermentation (MLF) in wine was developed. The method includes
Received in revised form the bonding of functionalized magnetic nanoparticles to the bacterial surface in the suspension, the
17 December 2015
application of the magneto-responsive bacteria (MRB) in the fermentation process and their magnetic
Accepted 27 February 2016
separation from the wine using high-gradient magnetic separation (HGMS). The controlled attachment
Available online 3 March 2016
of the superparamagnetic amino-functionalized silica-coated maghemite nanoparticles (aMNPs) to the
O. oeni was developed. The MRB were applied in the MLF and separated from the fermentation process
Keywords:
Superparamagnetic nanoparticles
at a certain stage using the HGMS. From the results it was found that the magnetization of the bacteria
Lactic acid bacteria using aMNPs attached to the cell surface had no inuence on the O. oeni metabolism. The O. oeni with the
High-gradient magnetic separation attached magnetic nanoparticles were efciently removed from the fermentation media using HGMS,
Malolactic fermentation which resulted in a complete stop of the fermentation process.
Controlled fermentation 2016 Elsevier B.V. All rights reserved.

1. Introduction enhances the organoleptic characteristic, and increases the micro-

The fermentation of grape juice involves two fermentation pro-
cesses: an alcoholic fermentation and a malolactic fermentation
(MLF). The MLF represents a secondary fermentation that proceeds
in parallel or after the alcohol fermentation [1]. It starts as soon
as the lactic acid bacteria (LAB) population reaches a concentra-
tion of 106 colony-forming units per millilitre (CFU/mL) [2] and
its duration is approximately 5 days to 3 weeks, depending on the
physico-chemical properties of the fermentation [1]. The MLF is
desirable for some wine types, because it decreases the acidity,
Corresponding author at: Department for Materials Synthesis, Jozef Stefan Insti-
tute, Jamova 39, SI-1000 Ljubljana, Slovenia.
E-mail addresses: peter.dusak@ijs.si (P. Dusak), mojca.bencina@ki.si
(M. Bencina), martina.turk@bf.uni-lj.si (M. Turk), dejan.bavcar@kis.si (D. Bavcar),
tatjana.kosmerl@bf.uni-lj.si (T. Kosmerl), marin.berovic@fkkt.uni-lj.si (M. Berovic),
darko.makovec@ijs.si (D. Makovec).
biological stability of the wine [3]. The MLF is performed by LAB,
which convert the l-malic acid to l-lactic acid and carbon dioxide
[4]. Oenococcus oeni (formerly known as Leuconostoc oenos) [5] is
the predominant bacterial species found in wines during MLF, and
is well adapted to the low pH, high SO2 , and ethanol concentrations
in wines [6]. Such harsh conditions result in the very slow growth
of microorganisms and their poor cell density in the wine. Under
certain conditions, the contributions made by the MLF improve the
wines quality, but the same contributions may be considered as
highly undesirable under a different set of circumstances, as found
in the warm viticultural regions. Uncontrolled MLF or spontaneous
MLF implies several risks, such as a potential for increasing the
volatile acidity, the consumption of residual sugars and the for-
mation of undesirable metabolites, such as biogenic amines, that
can affect human health and lead to low-quality wines [3,7]. If MLF
is not desired, the growth of the LAB in the wine must be sup-
pressed by removing or inactivating the bacteria that are present.
The removal of LAB can be achieved by using standard winemak-

http://dx.doi.org/10.1016/j.bej.2016.02.016
1369-703X/ 2016 Elsevier B.V. All rights reserved.
 P. Dusak et al. / Biochemical Engineering Journal 110 (2016) 134142
ing technology, such as ltration, centrifugation, and the control of
MLF by using addition of anti-microbial including sulphur dioxide
or lysozyme [4]. In contrast, if MLF is desired or needed, its control
is essential.
In recent years several technologies have been proposed to
control a wines MLF by using LAB, principally O. oeni [8]. These
alternative technologies usually involve the use of high densities of
bacterial cells, free or immobilized by adsorption onto, or encap-
sulated into, different matrices, such as calcium alginate [9,10],
pectate [11], -carrageenan [1214], polyacrylamide [12], cellu-
lose [8,15], and poly(vinyl alcohol) hydrogel [16]. As a support
for the immobilization of the bacterial cells used during the MLF,
chemically modied chitosan (CCB) beads have also been used [17].
However, the encapsulation method has mass-transfer limitations
for nutrients that lead to inactivation or even to cell death in the
centre interior. To remove the LAB during or post the MLF in wine,
a rapid, convenient, selective and efcient separation method is
needed. This can be achieved by employing magnetic separation of
LAB from wine.
In biotechnology, magnetic separation is a well-known tech-
nique [1822], where magnetic carriers are dispersed into a
reaction mixture containing specic targets. After binding the spe-
cic targets with magnetic carriers, the conjugates are separated by
using an external magnetic eld [2325]. For the magnetic separa-
tion of larger objects, such as cells and microorganisms, the small
magnetic nanoparticles can be attached to their surfaces. Even if a
relatively low surface concentration of the nanoparticles is attached
and the magnetization of the object is small, its magnetic moment
in the magnetic eld can be large enough for effective separa-
tion, because of its relatively large volume [26]. It is benecial if
the magnetic nanoparticles are small enough to be in the super-
paramagnetic state. Superparamagnetism is a phenomenon related
to ferri/ferromagnetic particles when their size is reduced below
a certain limit and thermal excitation induces rapid uctuations,
compared to the observation time, of the nanoparticles magnetic
moments. The superparamagnetic limit is at approximately 20 nm
for soft magnetic materials [27,28]. At this point, these superparam-
agnetic nanoparticles no longer exhibit any spontaneous magnetic
moments and, in contrast to larger ferromagnetic particles, they
do not agglomerate in suspensions due to magnetic dipole-dipole
interactions.
As the magnetic material for magnetic separation, simple mag-
netic iron-oxides like maghemite (-Fe2 O3 ) or magnetite (Fe3 O4 )
are normally used [2933], because of their low cost and rela-
tively simple synthesis. These iron-oxides have also been used
for magnetic separation in environmental engineering [34,35], in
chemical engineering [36,37], as well as in bioseparation processes
[3841]. Iron-oxide magnetic nanoparticles have also attracted a lot
of attention in biomedicine, in drug delivery or in the detection and
targeting of specic (bio) molecules or cells [25,4245]. Iron-oxide
nanoparticles are considered to be nontoxic and were approved by
the U.S. Food and Drug Administration (FDA) for in-vivo medical
applications [31].
Low-magnetisation particles are usually separated in a con-
tinuous process from suspensions using high magnetic gradients
(HGMS). In biotechnology, for protein purication [4649], or in cell
separation [5052], the HGMS techniques have become very use-
ful. The magnetic nanoparticles can be adsorbed onto the surface
of microorganisms, by attractive electrostatic interactions between
the microorganisms and the magnetic nanoparticles with an oppo-
site surface charge, or with chemical interactions, (biotin-avidin
interactions) [53] or by antigen-antibody recognition [54].
In this research the separation of LAB O. oeni at a desired time of
the MLF process was developed. The desired time could be any time
during or at the end of the MLF. Instead of entrapping or immo-
bilizing the bacterial cells onto different matrices, functionalized
135
superparamagnetic iron-oxide nanoparticles were electrostatically
adsorbed onto the surface of the bacteria in the suspension. Thus,
magnetized bacteria were removed from the fermentation media
using HGMS.
2. Materials and methods
2.1. Fermentation procedure
2.1.1. Microorganism and the preparation of the inoculum
The freeze-dried LAB strain of Oenococcus oeni (Uvaferm BETA,
MBR process) used in the experiments was provided by Lallemand
Inc. (EU) and stored in accordance with the manufacturers recom-
mendations. The freeze-dried bacteria were removed from 20 C
around 30 min prior to use. The reactivation of the freeze-dried
bacteria was conducted in accordance with suppliers recommen-
dations. The freeze-dried bacteria were rehydrated in 20 times their
weight of sterile de-ionized water at 20 C for a maximum of 15 min.
2.1.2. Media, fermentation conditions and preparation of the
samples for analysis
The MLFs and HGMS were carried out in a synthetic liquid
medium: 81 g of ethanol, 5.2 g of glycerol, 0.7 g of glucose, 0.9 g of
fructose, 0.5 g of citric acid, and 2 g of malic acid per litre of de-
ionized water, the pH was adjusted to 3.2. The MLFs were also
carried out in unltered, unsulphurized wine, (Chardonnay and
Pinot blanc blend) from the winery Ptujska klet vinarstvo d.o.o.
(Ptuj, Slovenia), after alcoholic fermentation with pH 3.07. The ini-
tial total residual sugar content in the wine before the MLF was
0.9 g/L, with 11% of ethanol (v/v), 3 g/L of l-malic acid, and <0.1 g/L
of l-lactic acid. A glass bioreactor (with a total volume of 0.5 L,
closed with a fermentation bung) was lled with a fermentation
substrate and inoculated with bacteria or magneto-responsive bac-
teria (MRB) at a concentration of 107 CFU/mL. The fermentation
was carried out for 21 days at 22 C without mixing. Samples were
taken at the beginning, after 7, 14, and 21 days. For the enzymatic
analysis the samples were ltered through a 0.2-m lter (regener-
ated cellulose, Chromal, USA). The experiments were carried out
in triplicates and the averages of the three runs were calculated.
2.2. Nanoparticles
2.2.1. Synthesis of nanoparticles
The superparamagnetic maghemite (-Fe2 O3 ) nanoparti-
cles coated with an approximately 4-nm-thick layer of silica
were synthesized as described in Ref. [55]. The silica-coated
maghemite nanoparticles were functionalized by grafting 3-
(2-aminoethylamino) propylmethyldimethoxysilane onto their
surfaces, as described elsewhere [56]. TEM analyses showed that
the globular aMNPs were of uniform size, equal to 24 4 nm
(11 3 nm maghemite nanoparticle core), corresponding to a cal-
culated specic surface area of 100 m2 /g (Fig. A.1 in Supplementary
data). The aMNPs were superparamagnetic with a saturation mag-
netization of 32 emu/g (for details see Fig. A.2 in Supplementary
data).
2.2.2. Adsorption of the superparamagnetic amino-functionalized
maghemite nanoparticles onto the O. oeni surface
The MRB were prepared by the electrostatic adsorption of posi-
tively charged aMNPs onto the O. oeni displaying a negative surface
charge. First, the prepared bacterial suspension of 2 mL was washed
with 6 mL of de-ionized water and either harvested by centrifu-
gation (6900 g, 2 min) or ultraltered (Solvent resistant stirred
cell, Millipore, USA; ultraltration membrane 30 kDa; 2 bar of N2
pressure) to remove any impurities, such as the salts from growth
136 P. Dusak et al. / Biochemical Engineering Journal 110 (2016) 134142
media, that might change the ionic strength of the suspension,
affecting the binding of aMNPs to the bacterial surface.
To adsorb the positively charged aMNPs onto the negatively
charged bacterial cell surface, the bacterial suspension at pH 4
was vigorously admixed into the suspension of aMNPs (1 mg/mL,
pH 4). To study the efciency of the attachment of the aMNPs
onto the bacterial cell surface in an aqueous medium, two differ-
ent concentrations of bacterial cells (B1 and B2) and two different
aMNPs/bacteria cells ratios (R1 and R2) were used (presented in
Table 1).
For the viability tests, the combination of a higher bacterial con-
centration (B1 = 5 109 cells/mL) and a higher aMNP/bacteria ratio
(R1 = 1:8745) was used.
2.3. Analyses
2.3.1. Analysis of the metabolites
The concentrations of the l-malic and l-lactic acids were deter-
mined with enzymatic test kits (specic for the determination of
the l-malic acid or the l-lactic acid) from Oenolab Diagnostics (Hen-
daye, France). The d-glucose together with the d-fructose were
measured with specic enzymatic test kits from the same producer
and all the analyses were performed with a BS-200 Auto-Analyser
(Mindray, China). The estimated relative error of the measurement
for each parameter is below 10%. The amount of citric acid was
determined with the HPLC method following the protocol proposed
by the International Organisation of Vine and Wine in Mthodes
internationales danalyse des vins [57].
2.3.2. Dissolved Fe analysis
The dissolved Fe was analysed using an elemental mass spec-
trometer with ionization in an inductively coupled plasma (Agilent
Technologies 7500ce ICP-MS, USA).
2.3.3. Zeta-potential
The zeta()-potentials of the aMNPs and the LAB strain of O. oeni
in their aqueous suspensions were measured using a Brookhaven
Instruments Corp., Zeta PALS, Holtsville, USA.
2.3.4. Electron microscopy
The MRB were characterized using transmission electron
microscopy (TEM; JEOL 2100) and scanning electron microscopy
(SEM; JEOL 7600F, USA). For the TEM and SEM analyses the bac-
terial suspension was diluted 10 times in 20% ethanol (v/v). For
the TEM analyses the diluted suspension was transferred and dried
on a copper-grid-supported transparent carbon foil, while for the
SEM analyses the diluted suspension was transferred to a graphite
specimen mount, dried and coated with a 3-nm platinum surface
coating (Gatan, Model 682 PECS, USA).
The TEM analyses showed that the O. oeni had an oval shape with
a long dimension of 1.5 m, and 0.5 m in the transverse direction.
Based on these values, the specic surface area of the O. oeni was
estimated to be 2 m2 .
2.3.5. Flow-cytometry analysis
The number of bacteria cells was determined by ow cytometry.
A CyFlow Space cytometer (Partec, Mnster, Germany) equipped
with a 50-mW blue laser emitting at 488 nm was used. A forward
scatter (FSC, for the cell size) and side scatter (SSC, for the cell granu-
larity) were used to dene the population of cells. For the detection
of the cells stained with propidium iodide (PI) dye and SYTO9 dye
we used optical lters of 675/25 nm (FL3) and 590/50 nm (FL2),
respectively. The setting region on the FSC/SSC was used to dis-
criminate the bacteria from the background. Gates were dened
in the histogram plots of green uorescence and red uorescence.
The samples were analysed at low rate settings of approximately
200 cells s1 , and at least 20,000 gated cells were analysed. The data
were analysed using FlowJo software (Tree Star, Ashland, OR, USA).
A high precision of better than 5% was guaranteed by the precise
counting and the mechanical volume measurement. The counting
reproducibility was better than 2% relative standard deviation [58].
Stock solutions of the dyes were prepared as follows.
Red-uorescent nucleic acid stain propidium iodide (PI) and
green-uorescent nucleic acid stain SYTO9 were used from the
LIVE/DEAD BacLightTM Bacterial Viability kit (Molecular Probes,
USA), as proposed by the manufacturer. The SYTO9 dye enters all
the cells, while the PI was only internalised in the dead cells.
All the stock solutions were stored at 20 C. Six L of the stock
solution was added to the 2 mL of culture containing approximately
106 cells and mixed thoroughly by pipetting. The prepared sample
was incubated at room temperature in the dark for 15 min and then
measured.
2.3.6. Enumeration of O. oeni on agar plates
The number of O. oeni cells was determined using the
plate-count method. One mL of suspension containing 50 mg of
freeze-dried cells was diluted in Milli-Q water and serial 10-fold
dilutions were spread plated on MRS agar (Biolife Italiana, Italia)
in duplicates. Inoculated plates were incubated at 30 C for 7 days
under anaerobic conditions. After incubation, the colonies were
counted on each plate and plates with 30 to 300CFUs/plate were
used to calculate the CFUs/ml.
2.3.7. Magnetic separation
The HGMS experiments were performed with a model L 1CN
Frantz laboratory canister separator (S. G. Frantz Co., Inc. Trenton,
USA). The HGMS system consisted of a nonmagnetic stainless-steel
column with a working space of 0.6 cm in width by 2.5 cm in depth
and 22.2 cm in length, for a volume of 35.3 cm3 lled with type-
430 ne-grade stainless-steel wool, also supplied by S. G. Frantz
Co., Inc. The column was packed with approximately 5 vol.% (15 g)
of matrix material, which is the maximum packing fraction that
could be obtained manually. For the magnetic separation, the can-
ister was placed vertically in the 1-cm gap between the two pole
pieces of the separator. A magnetic eld between the pole pieces,
which could be varied in strength, was generated with an attached
electromagnet. The direction of the magnetic eld was transverse
with respect to the direction of the ow through the column. The
maximum magnetic ux density generated between the two plates
was 1 T, as measured with a handheld gauss meter. The maximum
magnetic ux density was used in all the experiments.
The continuous magnetic separation experiments were per-
formed at room temperature by passing 250 mL of the suspension
(MRB in synthetic media or MRB in wine) from the reaction ves-
sel, through the HGMS column with the electromagnet on, into
the ltrate vessel. The suspension was rst roughly shaken and
then pumped steadily at 4.3 mL/min with a peristaltic pump (Wat-
son Marlow 400, United Kingdom) through the HGMS column. The
efciency of the HGMS was evaluated by ow-cytometry analysis.
3. Results and discussion
3.1. Adsorption of magnetic nanoparticles onto the bacteria
The adsorption of the superparamagnetic nanoparticles onto
the LAB strain was studied to prepare the MRB. These MRB were
prepared by the adsorption of positively charged aMNPs onto the
O. oeni in water. O. oeni display a negative surface charge, due
to its cell-wall composition consisting of several polymers and
macromolecules, which possess carboxyl, hydroxyl and phosphate
surface groups [59]. The adsorption of the nanoparticles onto the
 P. Dusak et al. / Biochemical Engineering Journal 110 (2016) 134142 137

Table 1
Different combinations of bacterial cell concentrations and aMNP/bacteria ratios.

Samplename Bacterial concentration [cells/mL] Bacterial suspension volume [mL] aMNP/bacterialnumber ratio aMNP (1 mg/mL) volume added [mL]

B1R1 5 109 1 1:8745 0.5

B2R1 5 107 1 1:8745 0.005
B1R2 5 109 1 1:3336 0.2
B2R2 5 107 1 1:3336 0.002

Fig. 1. -potential of O. oeni and aMNPs as a function of the pH value of their aqueous suspension.

LAB surface will therefore be stimulated if they display a positive
surface charge. Due to the terminal amino groups, the aMNPs dis-
play a strong positive -potential at pH 4. At this pH value, they were
adsorbed onto the negatively charged bacterial surface (Fig. 1).
The bacterial suspension, prepared from the freeze-dried
cells, might contain salts or some other components from the
growth medium. These water-soluble components might cause
the agglomeration of the aMNPs by adsorbing onto the nanopar-
ticles or by increasing the ionic strength of the suspension. Even
a relatively small amount of salt (e.g., less than 10 mM of KCl)
caused the agglomeration of aMNPs in the aqueous suspension
(data not shown). To remove the impurities, the bacterial suspen-
sion was ultraltered before the cells were added to the suspension
of aMNPs.
Fig. 2 shows a SEM image of the bacteria with the adsorbed
aMNPs. The aMNPs (1 mg/mL, pH 4) in Fig. 2a were adsorbed at a
higher aMNPs/bacteria-number ratio (R1 = 1:8745) onto the receiv-
ing bacteria (5 109 cells/mL, pH 4) in the suspension, which was
not puried by ultraltration. The bacteria were non-uniformly
covered with the aMNPs. The aMNPs were mainly attached to the
bacterial cells in the form of larger agglomerates, while vast areas
of the cells were uncovered. The agglomeration was most probably
induced by an increased ionic strength of the suspension caused by
dissolved impurities.
The coverage of the bacteria with aMNPs was clearly improved
when the bacterial suspension was puried by ultraltration
(Fig. 2b). The cells were covered more homogenously with the indi-
vidual aMNPs, although some smaller agglomerates of aMNPs were
also observed.
Previous studies dealing with the adsorption of magnetic
nanoparticles onto microorganisms surfaces show that apart from
the suspension pH [60], the suspensions concentrations [61,62]
and the number ratio R between the nanoparticles and the
microorganisms inuence the surface coverage of the microorgan-
isms [26,63]. TEM analyses showed that different aMNPs/bacterial
number ratios and different bacterial concentrations inuenced
the coverage of the ultraltered O. oeni with the aMNPs. The
aMNPs/bacterial-number ratios were chosen according to the
specic surface area of the bacterial cells, determined from micro-
scopic analyses of the dry cells. The coverage of O. oeni with
aMNPs was better in the case of a higher (R1 = 1:8745) (Fig. 3a
and b) compared to a lower (R2 = 1:3336) aMNPs/bacteria ratio
(Fig. 3c and d). The O. oeni were more homogenously covered
at the higher (B1 = 5 109 cells/mL) (Fig. 3a) than at the lower
(B2 = 5 107 cells/mL) (Fig. 3b) bacterial concentration at the same
aMNPs/bacteria ratio (R1).
It is evident that by decreasing the aMNPs/bacteria ratio fewer
aMNPs are attached to the bacterial cells (Fig. 3c and d). In the
case when the aMNPs are in excess (R1), it was assumed that
non-attached aMNPs act like stabilizers, preventing the aggre-
gation of the magnetically modied O. oeni. In contrast, at the
lower aMNPs/bacteria ratio (R2), the adsorbed nanoparticles make
bridges between the bacterial cells (Fig. 3c), similar to the situa-
tion observed with the chemically driven heteroaggregation of two
types of nanoparticles [64].
3.2. Inuence of magnetic nanoparticles on bacterial viability and
metabolism
The number of bacterial cells in the starting suspension was
determined using the ow-cytometry technique and the plate-
count method. The use of uorescent stains in combination with
ow cytometry allows the detection and discrimination of viable
culturable, viable nonculturable, and nonviable organisms [65]. The
concentration of 3 109 cells/mL was determined by ow cytome-
try in the starting suspension. The determined viability for O. oeni
without any attached magnetic nanoparticles in the bacterial sus-
pension was 98%.
In practice, bacterial viability is measured using the plate-count
technique. However, in the case of O. oeni, the plate-count tech-
nique requires a very long incubation time of about 10 days or more
[66]. To verify the number of bacterial cells obtained by ow cytom-
etry, the bacteria were grown on MRS agar plates. The number of
5 109 CFU/mL was determined by the plate counting, which is in
good agreement with the determined number for lyophilized or
dried bacteria in the International Oenological Codex [67].
Bonding of the nanoparticles on the cell wall can damage the
cell membrane of the microorganism [63,68]. The cell membrane
of O. oeni used in our experiments was reinforced by the MBR
process, developed by Lallemand to adapt the cells to the harsh con-
138 P. Dusak et al. / Biochemical Engineering Journal 110 (2016) 134142

Fig. 2. SEM images of aMNPs adsorbed on O. oeni: (a) the aMNPs were adsorbed onto the bacteria in the suspension not previously puried, (b) bacteria in the suspension
were puried by ultraltration prior to the aMNPs adsorption.

Fig. 3. Attachment of aMNPs on bacteria at different bacterial concentrations and aMNP/bacteria ratios: (a) B1R1, (b) B1R2, (c) B2R1 and (d) B2R2 (see Table 1).

ditions in MLF, such as high amount of ethanol, a low pH, etc. [69].
The preparation process of the MRB and the presence of magnetic
nanoparticles attached on the bacterial cells might have an inu-
ence on the viability of the O. oeni. However, the ow cytometry
results on the viability of the bacterial cells that were ultraltered
or the bacterial cells with attached aMNPs were the same as in
the case of the starting bacterial suspension. The percentage of live
bacteria in the suspension remained the same, i.e., 98%. The prepa-
ration process for the MRB and the attached magnetic nanoparticles
on the bacterial cells show no cytotoxic effect on the O. oeni.
A literature survey of the inuence of attached nanoparticles on
the bacterial metabolism in general shows that this topic has not
been well researched yet. The inuence of the attached nanoparti-
cles on the growth of bacteria was studied by other researchers in
order to assess the cytotoxic effect [7073]. Although it was shown
that the attached magnetic nanoparticles accelerate the metabolic
activity of the wine yeast by speeding up the fermentation process
kinetics [26], the attached magnetic nanoparticles on the surface of
the O. oeni did not have any inuence on its metabolism. The inu-
ence of the attached magnetic nanoparticles on the metabolism
of O. oeni was tested by performing the MLF in wine after alco-
holic fermentation. The organic acid concentrations obtained from
an enzymatic analysis of the wine, inoculated according to the
manufacturers recommendations, wine inoculated with puried
bacterial suspension (centrifuged or ultraltrated bacteria) and
wine inoculated with the MRB (centrifuged or ultraltrated bac-
teria with the aMNPs) (for details see Table A.1 in Supplementary
data) were compared. The comparison between the start and the
end pH values and the organic acid concentrations conrmed that
the MLF occurred in all experiments. The results also showed that
the purication methods, e.g., centrifugation or ultraltration, that
were used for the preparation of the MRB do not have any inuence
on the bacterial metabolism and the MLF process.
3.3. Control of the malolactic fermentation of wine using
magnetic separation of magneto-responsive bacteria
With the aim being to develop a method for the continuous
HGMS of the MRB from wine, they were rst separated from the
synthetic medium with a chemical composition similar to wine. The
 P. Dusak et al. / Biochemical Engineering Journal 110 (2016) 134142
Fig. 4. Graphs FSC vs. SSC before (a) and after (b) HGMS of MRB. The oval denes the region of MRB.
efciency of the HGMS was evaluated by determining the number
of remaining cells after the separation using ow-cytometry anal-
yses. No difference in the ow-cytometry analysis results, as the
background noise, was observed if the O. oeni cells were dispersed
in de-ionized water or in the synthetic medium. Fig. 4 shows the
presence of the MRB before (a) and after (b) the HGMS.
The tail-shaped part marked on the graph in Fig. 4a between
10 and 1000 FSC, represents the MRB before the HGMS. It is clear
that this part is missing after the HGMS (Fig. 4b). The efciency of
the HGMS was estimated to be 96%. The number of bacterial cells
per mL after the HGMS was 4 103 , which is less than the number
of cells needed for the start or continuation of the MLF [2].
After testing on the synthetic medium, the HGMS was carried
out on the wine sample after 14 days of MLF. However, the results
of the ow-cytometry analysis could not be quantied because
two populations of cells were present in the wine (see Fig. A.3 in
Supplementary data). Apart from the MRB, another population of
microorganisms was detected, which can be ascribed to the wine
yeasts [74]. Yeast cells might already be present in the wine, since it
was not ltered before the experiment, or they were added with the
O. oeni as bio-activators. The efciency of the HGMS was therefore
tested with a TEM analysis and by observations of the MLF process
after the HGMS.
The TEM analysis detected no bacteria in the wine samples after
the HGMS, whereas some larger yeast cells were observed (data not
shown). The sedimented cells from the magnetic separator were
also analysed using TEM and proved to be the MRB. Although the
cells multiplied during the fermentation process, the nanoparti-
cles remained on their surface (Fig. 5b). After drying the bacterial
suspension on a TEM specimen support, the vast majority of the
bacterial cells were deposited in the form of larger clusters con-
taining several cells. The surface concentration of the nanoparticles
on the bacteria after the MLF (Fig. 5b) is smaller than the initial
concentration (Fig. 5a). During the MLF the magneto-responsive
O. oeni cells multiply and their magnetization decreases because
of the increased number in the newly grown bacteria. Oenococ-
cus oeni is known to grow slowly in comparison to other bacteria
[75]. Their slow growth and chain-like structures, formed during
multiplication [75], are advantageous in the magnetic separation.
Although the cells of O. oeni multiplied during the fermentation
process, the magnetic nanoparticles remained on their surface in
a relatively high concentration (Fig. 5), and the newly grown bac-
teria without magnetic nanoparticles were always attached to the
original magnetic bacteria, thus enabling efcient separation using
the HGMS.
The MLF can be controlled by the removal of O. oeni at a desired
time. To test this hypothesis the MLF was started in the wine after
139
alcoholic fermentation and terminated after 7 days with the HGMS
of the magneto-responsive O. oeni. The enzymatic tests for organic
acids and residual sugars were performed on samples taken before
and after the magneto-responsive O. oeni were separated from
the wine, and also an additional 7 and 14 days after the separa-
tion. The results obtained for the magneto-responsive O. oeni were
compared with the results for the bacteria without the attached
magnetic nanoparticles (the control, labelled as pristine O. oeni)
(Fig. 6). Under conditions of limiting sugar availability or low pH,
the growth of the O. oeni is enhanced in the presence of organic acids
such as malate or citrate. Although malic acid is the most impor-
tant acid metabolized by O. oeni in wine, other organic acids are also
metabolized. Citric acid metabolism by O. oeni has been correlated
with the synthesis of acetic acid, diacetyl and acetoin [57]. In biore-
actors containing the pristine O. oeni, the starting concentrations of
l-malic acid and citric acid decreased in 7 days after the inoculation
of the wine. On the other hand, the concentration of l-lactic acid
and the pH value increased and the exhaust of CO2 was visually
observed. These results indicate that the MLF proceeded. The con-
centration of l-malic acid and citric acid continued to decrease and
the concentration of l-lactic acid and pH continued to increase in
the next 7 days. Oenococcus oeni cannot grow with l-malic acid as
a unique carbon source; therefore, these microorganisms need an
additional energy source, such as residual fermentable sugars, i.e.,
glucose and/or fructose, to allow the cell growth [3]. It is a hetero-
fermentative microorganism and converts glucose to D-lactic acid,
CO2 and acetic acid (or ethanol) [4]. The low pH of the wine inhibits
the sugar metabolism of O. oeni [4]. The concentration of residual
sugars did not change from the starting value (0.9 g/L) during the
MLF in our experiments. This result indicates that residual sugars
were not metabolized and the produced lactic acid was the conver-
sion of malic acid. The same results were obtained if the MLF was
performed with the MRB.
In the bioreactor containing the magneto-responsive O. oeni, the
starting pH value and organic acid concentrations changed in 7 days
after the inoculation of the wine (Fig. 6). After the HGMS the organic
acid concentrations and the pH value remained the same. There
was no further exhausting of CO2 observed in the bioreactors after
the HGMS. The results prove that the fermentation process stopped
completely. It is therefore reasonable to expect that the fermenta-
tion can be completely stopped at the desired stage of the process
with the separation of the MRB using HGMS.
After the HGMS of the magneto-responsive O. oeni, the HGMS
column containing trapped magneto-responsive O. oeni was back
ushed with de-ionized water in order to study the inuence of the
magnetic separation process on the bacterial viability and the pos-
sibility of their reuse in subsequent MLFs. To study their reuse, the
140 P. Dusak et al. / Biochemical Engineering Journal 110 (2016) 134142

Fig. 5. TEM images of MRB before (a) and after (b) MLF. Before MLF (a) the surface of O. oeni is densely covered with aMNPs. The chain-like structures of O. oeni form when
the bacteria multiply (b), which results in a decrease in the number of aMNPs on the bacterial surface.

Fig. 6. Changing of the pH value (a) and the content of organic acids (b-d) during the MLF of wine. The analyses were performed at the beginning of the MLF and after the
HGMS of bacteria with attached magnetic nanoparticles: (a) fermentation pH, (b) consumption of l-malic acid, (c) production of l-lactic acid and (d) consumption of citric
acid. The dashed lines serve as a guide to the eyes only.

recycled magneto-responsive O. oeni cells were inoculated into a
new bioreactor containing wine. The changes of organic acid con-
centrations and the pH value clearly indicate that the MLF occurred
(Fig. 6). During the HGMS the bacteria were exposed to the high
magnetic eld. The exposure of microorganisms to the magnetic
eld could have an inuence on their metabolism or viability [76].
The results proved that the separation process does not have a neg-
ative inuence on the magneto-responsive O. oeni in the HGMS
column. It is therefore reasonable to expect that the recycled MRB
could be used again in another MLF.
To simplify the process of magnetic separation, the mag-
netic nanoparticles could be added to the wine at a desired
time of the MLF to stop the process by using HGMS. To test
this hypothesis, the magnetic nanoparticles were added to the
bioreactor containing the O. oeni without adsorbed nanoparticles
7 days after inoculation, just before the HGMS. After the HGMS
of postmagneto-responsive O. oeni there was no change in the
pH value or in the organic acid concentrations (Fig. 6). To ensure
an efcient magnetic separation, a high concentration of magnetic
nanoparticles has to be added. In the experiment, the nanoparticles
were added in excess approximately 16,200 aMNPs were added
per cell. The excess of nanoparticles is needed because they adsorb
non-selectively, not only onto the surfaces of the O. oeni, but also
onto other particles present in the wine, e.g., yeast cells, cell debris,
polymerized phenolic compounds, etc. Moreover, the positively
charged nanoparticles added directly into the wine quickly adsorb
negatively charged molecules, which change their surface charge.
-potential measurements showed that the nanoparticles changed
their surface charge from positive to negative only a few minutes
after they were dispersed in the wine. The change in the nanoparti-
cles surface charge inuences the adsorption of the nanoparticles
onto the bacteria. It was found experimentally that negatively
 P. Dusak et al. / Biochemical Engineering Journal 110 (2016) 134142
charged nanoparticles also adsorb onto the microorganisms; how-
ever, to a much smaller extent, compared to the positively charged
nanoparticles [26]. In spite of the decrease of the nanoparticles -
potential after their addition to the wine, it seems that they were
still attached to the bacteria cells in a sufcient number to enable
effective magnetic separation.
The non-selective adsorption of the wine components onto the
magnetic nanoparticles during the magnetic separation of the O.
oeni, with the addition of the nanoparticles directly to the wine,
can change the wines composition, thus inuencing its quality.
It was assumed that the excess nanoparticles that were not
adsorbed onto the bacterial surface and other particles in the wine
would agglomerate in the wine due to the increased ionic strength
and due to the adsorption of different molecules. Due to their rel-
atively large size, the agglomerates can be effectively magnetically
separated.
Based on the obtained results in this research it can be concluded
that the MLF process can be controlled by the magnetic separation
of the MRB at a specic stage of the fermentation.
There are concerns about the presence of nanoparticles in the
wine after the separation. The wine after the separation was ana-
lysed using ICP-MS; however, an increase in the concentration of
the iron could not be detected. The concentration of Fe in the
wine was 1.48 mg/L. After the separation the concentration was
equal within the limits of the analytical uncertainty, which was
0.09 mg/L. It needs to be mentioned here that the concentration
of Fe in the wine would only increase by 2 mg/L if all the nanoparti-
cles added to the wine with the MRB were to dissolve. According to
the OIV and the national legislation of several countries, the allowed
concentration of Fe in white wine is 10 mg/L [77].
Generally, the magnetic separation of bacteria is important for
applications where the bacteria need to be removed from com-
plex environments and conventional purication methods, e.g.,
ltration, centrifugation, are not cost-effective [78] or in order to
control the fermentation process, e.g., in the fermentation of wine.
Several technologies have been proposed to control the MLF of
wines by using immobilized O. oeni [8]. However, the adsorption
or encapsulation of O. oeni on/into different matrices has mass-
transfer limitations for nutrients that might lead to cell death. Some
of these entrapment techniques are sensitive to the presence of
wine components, which may affect their mechanical stability. The
adsorbed magnetic nanoparticles do not have a signicant inu-
ence on the transfer limitations for nutrients. Furthermore, the MRB
can be efciently removed at a desired time or at the end of MLF
by applying an external magnetic eld. To minimize the expenses
during the production of wine the method for the preparation
of MRB should be an inexpensive process. Here, the electrostati-
cally driven adsorption of magnetic nanoparticles is the method of
choice, mainly for economic reasons.
4. Conclusions
Malolactic fermentation (MLF) plays a remarkable role in wine
technology. For the wines from colder areas that have a sharp
malic acid taste, the MLF conversion of malic acid to the smoother-
tasting lactic acid represents a technical solution to this problem.
The main difculty is one of process control. The present research
introduces a new method for the control of MLF. The method is
based on the electrostatic adsorption of functionalized superpara-
magnetic iron-oxide nanoparticles on the surface of the lactic acid
bacterium O. oeni, its application in MLF, and magnetic separation at
a desired stage of the process. The results of the enzymatic analysis
showed that magnetization with nanoparticles did not inuence
the metabolic activity of the used bacteria. The ow-cytometry
results before and after the magnetic separation showed that the
141
magneto-responsive bacteria were quickly and efciently removed
from the fermentation media.
The main advantage of it is that the magnetized O. oeni could
be quick and effectively removed from the process after the bio-
transformation of the malic to the lactic acid in this process.
Alternatively, pristine bacteria without the magnetic nanoparticles
can be used for the MLF and the nanoparticles can be added to the
wine after a certain time to stop the process using HGMS.
Acknowledgements
The support of the Ministry of Higher Education, Science
and Technology of the Republic of Slovenia within the National
Research Program P2-0089 is gratefully acknowledged. We
acknowledge the use of equipment in the Centre of Excellence on
Nanoscience and Nanotechnology-Nanocenter. Authors also thank
Dr. Darja Lisjak from the Jozef Stefan Institute for the SEM analy-
ses, Bojan Kobal from Ptujska klet for providing the wine samples,
Gordana Veber from Jurana d.o.o for providing the freeze-dried
bacteria, Dr. Vid Simon Selih from National Institute of Chemistry
Slovenia for the ICP-MS analysis and Prof. Dr. Rok Kostanjsek from
the Biotechnical Faculty, University of Ljubljana for fruitful discus-
sions.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bej.2016.02.016.
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