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Biochemical Engineering Journal 110 (2016) 134142

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Biochemical Engineering Journal

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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
Department for Material Synthesis, Jozef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia
Jozef Stefan International Postgraduate School, Jamova 39, 1000 Ljubljana, Slovenia
Laboratory of Biotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia
EnFIST, The Centre of Excellence EN-FIST, 1000 Ljubljana, Slovenia
Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia
Agricultural Institute of Slovenia, Hacquetova ulica 17, 1000 Ljubljana, Slovenia
Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia
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
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-

biological stability of the wine [3]. The MLF is performed by LAB,
The fermentation of grape juice involves two fermentation pro- which convert the l-malic acid to l-lactic acid and carbon dioxide
cesses: an alcoholic fermentation and a malolactic fermentation [4]. Oenococcus oeni (formerly known as Leuconostoc oenos) [5] is
(MLF). The MLF represents a secondary fermentation that proceeds the predominant bacterial species found in wines during MLF, and
in parallel or after the alcohol fermentation [1]. It starts as soon is well adapted to the low pH, high SO2 , and ethanol concentrations
as the lactic acid bacteria (LAB) population reaches a concentra- in wines [6]. Such harsh conditions result in the very slow growth
tion of 106 colony-forming units per millilitre (CFU/mL) [2] and of microorganisms and their poor cell density in the wine. Under
its duration is approximately 5 days to 3 weeks, depending on the certain conditions, the contributions made by the MLF improve the
physico-chemical properties of the fermentation [1]. The MLF is wines quality, but the same contributions may be considered as
desirable for some wine types, because it decreases the acidity, 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-
Corresponding author at: Department for Materials Synthesis, Jozef Stefan Insti- mation of undesirable metabolites, such as biogenic amines, that
tute, Jamova 39, SI-1000 Ljubljana, Slovenia. can affect human health and lead to low-quality wines [3,7]. If MLF
E-mail addresses: (P. Dusak),
(M. Bencina), (M. Turk), (D. Bavcar),
is not desired, the growth of the LAB in the wine must be sup- (T. Kosmerl), (M. Berovic), pressed by removing or inactivating the bacteria that are present. (D. Makovec). The removal of LAB can be achieved by using standard winemak-
1369-703X/ 2016 Elsevier B.V. All rights reserved.
P. Dusak et al. / Biochemical Engineering Journal 110 (2016) 134142 135

ing technology, such as ltration, centrifugation, and the control of superparamagnetic iron-oxide nanoparticles were electrostatically
MLF by using addition of anti-microbial including sulphur dioxide adsorbed onto the surface of the bacteria in the suspension. Thus,
or lysozyme [4]. In contrast, if MLF is desired or needed, its control magnetized bacteria were removed from the fermentation media
is essential. using HGMS.
In recent years several technologies have been proposed to
control a wines MLF by using LAB, principally O. oeni [8]. These 2. Materials and methods
alternative technologies usually involve the use of high densities of
bacterial cells, free or immobilized by adsorption onto, or encap- 2.1. Fermentation procedure
sulated into, different matrices, such as calcium alginate [9,10],
pectate [11], -carrageenan [1214], polyacrylamide [12], cellu- 2.1.1. Microorganism and the preparation of the inoculum
lose [8,15], and poly(vinyl alcohol) hydrogel [16]. As a support The freeze-dried LAB strain of Oenococcus oeni (Uvaferm BETA,
for the immobilization of the bacterial cells used during the MLF, MBR process) used in the experiments was provided by Lallemand
chemically modied chitosan (CCB) beads have also been used [17]. Inc. (EU) and stored in accordance with the manufacturers recom-
However, the encapsulation method has mass-transfer limitations mendations. The freeze-dried bacteria were removed from 20 C
for nutrients that lead to inactivation or even to cell death in the around 30 min prior to use. The reactivation of the freeze-dried
centre interior. To remove the LAB during or post the MLF in wine, bacteria was conducted in accordance with suppliers recommen-
a rapid, convenient, selective and efcient separation method is dations. The freeze-dried bacteria were rehydrated in 20 times their
needed. This can be achieved by employing magnetic separation of weight of sterile de-ionized water at 20 C for a maximum of 15 min.
LAB from wine.
In biotechnology, magnetic separation is a well-known tech-
2.1.2. Media, fermentation conditions and preparation of the
nique [1822], where magnetic carriers are dispersed into a
samples for analysis
reaction mixture containing specic targets. After binding the spe-
The MLFs and HGMS were carried out in a synthetic liquid
cic targets with magnetic carriers, the conjugates are separated by
medium: 81 g of ethanol, 5.2 g of glycerol, 0.7 g of glucose, 0.9 g of
using an external magnetic eld [2325]. For the magnetic separa-
fructose, 0.5 g of citric acid, and 2 g of malic acid per litre of de-
tion of larger objects, such as cells and microorganisms, the small
ionized water, the pH was adjusted to 3.2. The MLFs were also
magnetic nanoparticles can be attached to their surfaces. Even if a
carried out in unltered, unsulphurized wine, (Chardonnay and
relatively low surface concentration of the nanoparticles is attached
Pinot blanc blend) from the winery Ptujska klet vinarstvo d.o.o.
and the magnetization of the object is small, its magnetic moment
(Ptuj, Slovenia), after alcoholic fermentation with pH 3.07. The ini-
in the magnetic eld can be large enough for effective separa-
tial total residual sugar content in the wine before the MLF was
tion, because of its relatively large volume [26]. It is benecial if
0.9 g/L, with 11% of ethanol (v/v), 3 g/L of l-malic acid, and <0.1 g/L
the magnetic nanoparticles are small enough to be in the super-
of l-lactic acid. A glass bioreactor (with a total volume of 0.5 L,
paramagnetic state. Superparamagnetism is a phenomenon related
closed with a fermentation bung) was lled with a fermentation
to ferri/ferromagnetic particles when their size is reduced below
substrate and inoculated with bacteria or magneto-responsive bac-
a certain limit and thermal excitation induces rapid uctuations,
teria (MRB) at a concentration of 107 CFU/mL. The fermentation
compared to the observation time, of the nanoparticles magnetic
was carried out for 21 days at 22 C without mixing. Samples were
moments. The superparamagnetic limit is at approximately 20 nm
taken at the beginning, after 7, 14, and 21 days. For the enzymatic
for soft magnetic materials [27,28]. At this point, these superparam-
analysis the samples were ltered through a 0.2-m lter (regener-
agnetic nanoparticles no longer exhibit any spontaneous magnetic
ated cellulose, Chromal, USA). The experiments were carried out
moments and, in contrast to larger ferromagnetic particles, they
in triplicates and the averages of the three runs were calculated.
do not agglomerate in suspensions due to magnetic dipole-dipole
As the magnetic material for magnetic separation, simple mag- 2.2. Nanoparticles
netic iron-oxides like maghemite (-Fe2 O3 ) or magnetite (Fe3 O4 )
are normally used [2933], because of their low cost and rela- 2.2.1. Synthesis of nanoparticles
tively simple synthesis. These iron-oxides have also been used The superparamagnetic maghemite (-Fe2 O3 ) nanoparti-
for magnetic separation in environmental engineering [34,35], in cles coated with an approximately 4-nm-thick layer of silica
chemical engineering [36,37], as well as in bioseparation processes were synthesized as described in Ref. [55]. The silica-coated
[3841]. Iron-oxide magnetic nanoparticles have also attracted a lot maghemite nanoparticles were functionalized by grafting 3-
of attention in biomedicine, in drug delivery or in the detection and (2-aminoethylamino) propylmethyldimethoxysilane onto their
targeting of specic (bio) molecules or cells [25,4245]. Iron-oxide surfaces, as described elsewhere [56]. TEM analyses showed that
nanoparticles are considered to be nontoxic and were approved by the globular aMNPs were of uniform size, equal to 24 4 nm
the U.S. Food and Drug Administration (FDA) for in-vivo medical (11 3 nm maghemite nanoparticle core), corresponding to a cal-
applications [31]. culated specic surface area of 100 m2 /g (Fig. A.1 in Supplementary
Low-magnetisation particles are usually separated in a con- data). The aMNPs were superparamagnetic with a saturation mag-
tinuous process from suspensions using high magnetic gradients netization of 32 emu/g (for details see Fig. A.2 in Supplementary
(HGMS). In biotechnology, for protein purication [4649], or in cell data).
separation [5052], the HGMS techniques have become very use-
ful. The magnetic nanoparticles can be adsorbed onto the surface 2.2.2. Adsorption of the superparamagnetic amino-functionalized
of microorganisms, by attractive electrostatic interactions between maghemite nanoparticles onto the O. oeni surface
the microorganisms and the magnetic nanoparticles with an oppo- The MRB were prepared by the electrostatic adsorption of posi-
site surface charge, or with chemical interactions, (biotin-avidin tively charged aMNPs onto the O. oeni displaying a negative surface
interactions) [53] or by antigen-antibody recognition [54]. charge. First, the prepared bacterial suspension of 2 mL was washed
In this research the separation of LAB O. oeni at a desired time of with 6 mL of de-ionized water and either harvested by centrifu-
the MLF process was developed. The desired time could be any time gation (6900 g, 2 min) or ultraltered (Solvent resistant stirred
during or at the end of the MLF. Instead of entrapping or immo- cell, Millipore, USA; ultraltration membrane 30 kDa; 2 bar of N2
bilizing the bacterial cells onto different matrices, functionalized 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, 200 cells s1 , and at least 20,000 gated cells were analysed. The data
affecting the binding of aMNPs to the bacterial surface. were analysed using FlowJo software (Tree Star, Ashland, OR, USA).
To adsorb the positively charged aMNPs onto the negatively A high precision of better than 5% was guaranteed by the precise
charged bacterial cell surface, the bacterial suspension at pH 4 counting and the mechanical volume measurement. The counting
was vigorously admixed into the suspension of aMNPs (1 mg/mL, reproducibility was better than 2% relative standard deviation [58].
pH 4). To study the efciency of the attachment of the aMNPs Stock solutions of the dyes were prepared as follows.
onto the bacterial cell surface in an aqueous medium, two differ- Red-uorescent nucleic acid stain propidium iodide (PI) and
ent concentrations of bacterial cells (B1 and B2) and two different green-uorescent nucleic acid stain SYTO9 were used from the
aMNPs/bacteria cells ratios (R1 and R2) were used (presented in LIVE/DEAD BacLightTM Bacterial Viability kit (Molecular Probes,
Table 1). USA), as proposed by the manufacturer. The SYTO9 dye enters all
For the viability tests, the combination of a higher bacterial con- the cells, while the PI was only internalised in the dead cells.
centration (B1 = 5 109 cells/mL) and a higher aMNP/bacteria ratio All the stock solutions were stored at 20 C. Six L of the stock
(R1 = 1:8745) was used. solution was added to the 2 mL of culture containing approximately
106 cells and mixed thoroughly by pipetting. The prepared sample
2.3. Analyses was incubated at room temperature in the dark for 15 min and then
2.3.1. Analysis of the metabolites
The concentrations of the l-malic and l-lactic acids were deter- 2.3.6. Enumeration of O. oeni on agar plates
mined with enzymatic test kits (specic for the determination of The number of O. oeni cells was determined using the
the l-malic acid or the l-lactic acid) from Oenolab Diagnostics (Hen- plate-count method. One mL of suspension containing 50 mg of
daye, France). The d-glucose together with the d-fructose were freeze-dried cells was diluted in Milli-Q water and serial 10-fold
measured with specic enzymatic test kits from the same producer dilutions were spread plated on MRS agar (Biolife Italiana, Italia)
and all the analyses were performed with a BS-200 Auto-Analyser in duplicates. Inoculated plates were incubated at 30 C for 7 days
(Mindray, China). The estimated relative error of the measurement under anaerobic conditions. After incubation, the colonies were
for each parameter is below 10%. The amount of citric acid was counted on each plate and plates with 30 to 300CFUs/plate were
determined with the HPLC method following the protocol proposed used to calculate the CFUs/ml.
by the International Organisation of Vine and Wine in Mthodes
internationales danalyse des vins [57].
2.3.7. Magnetic separation
2.3.2. Dissolved Fe analysis The HGMS experiments were performed with a model L 1CN
The dissolved Fe was analysed using an elemental mass spec- Frantz laboratory canister separator (S. G. Frantz Co., Inc. Trenton,
trometer with ionization in an inductively coupled plasma (Agilent USA). The HGMS system consisted of a nonmagnetic stainless-steel
Technologies 7500ce ICP-MS, USA). 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-
2.3.3. Zeta-potential 430 ne-grade stainless-steel wool, also supplied by S. G. Frantz
The zeta()-potentials of the aMNPs and the LAB strain of O. oeni Co., Inc. The column was packed with approximately 5 vol.% (15 g)
in their aqueous suspensions were measured using a Brookhaven of matrix material, which is the maximum packing fraction that
Instruments Corp., Zeta PALS, Holtsville, USA. could be obtained manually. For the magnetic separation, the can-
ister was placed vertically in the 1-cm gap between the two pole
2.3.4. Electron microscopy pieces of the separator. A magnetic eld between the pole pieces,
The MRB were characterized using transmission electron which could be varied in strength, was generated with an attached
microscopy (TEM; JEOL 2100) and scanning electron microscopy electromagnet. The direction of the magnetic eld was transverse
(SEM; JEOL 7600F, USA). For the TEM and SEM analyses the bac- with respect to the direction of the ow through the column. The
terial suspension was diluted 10 times in 20% ethanol (v/v). For maximum magnetic ux density generated between the two plates
the TEM analyses the diluted suspension was transferred and dried was 1 T, as measured with a handheld gauss meter. The maximum
on a copper-grid-supported transparent carbon foil, while for the magnetic ux density was used in all the experiments.
SEM analyses the diluted suspension was transferred to a graphite The continuous magnetic separation experiments were per-
specimen mount, dried and coated with a 3-nm platinum surface formed at room temperature by passing 250 mL of the suspension
coating (Gatan, Model 682 PECS, USA). (MRB in synthetic media or MRB in wine) from the reaction ves-
The TEM analyses showed that the O. oeni had an oval shape with sel, through the HGMS column with the electromagnet on, into
a long dimension of 1.5 m, and 0.5 m in the transverse direction. the ltrate vessel. The suspension was rst roughly shaken and
Based on these values, the specic surface area of the O. oeni was then pumped steadily at 4.3 mL/min with a peristaltic pump (Wat-
estimated to be 2 m2 . son Marlow 400, United Kingdom) through the HGMS column. The
efciency of the HGMS was evaluated by ow-cytometry analysis.
2.3.5. Flow-cytometry analysis
The number of bacteria cells was determined by ow cytometry. 3. Results and discussion
A CyFlow Space cytometer (Partec, Mnster, Germany) equipped
with a 50-mW blue laser emitting at 488 nm was used. A forward 3.1. Adsorption of magnetic nanoparticles onto the bacteria
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 The adsorption of the superparamagnetic nanoparticles onto
of the cells stained with propidium iodide (PI) dye and SYTO9 dye the LAB strain was studied to prepare the MRB. These MRB were
we used optical lters of 675/25 nm (FL3) and 590/50 nm (FL2), prepared by the adsorption of positively charged aMNPs onto the
respectively. The setting region on the FSC/SSC was used to dis- O. oeni in water. O. oeni display a negative surface charge, due
criminate the bacteria from the background. Gates were dened to its cell-wall composition consisting of several polymers and
in the histogram plots of green uorescence and red uorescence. macromolecules, which possess carboxyl, hydroxyl and phosphate
The samples were analysed at low rate settings of approximately 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 aMNPs was better in the case of a higher (R1 = 1:8745) (Fig. 3a
surface charge. Due to the terminal amino groups, the aMNPs dis- and b) compared to a lower (R2 = 1:3336) aMNPs/bacteria ratio
play a strong positive -potential at pH 4. At this pH value, they were (Fig. 3c and d). The O. oeni were more homogenously covered
adsorbed onto the negatively charged bacterial surface (Fig. 1). at the higher (B1 = 5 109 cells/mL) (Fig. 3a) than at the lower
The bacterial suspension, prepared from the freeze-dried (B2 = 5 107 cells/mL) (Fig. 3b) bacterial concentration at the same
cells, might contain salts or some other components from the aMNPs/bacteria ratio (R1).
growth medium. These water-soluble components might cause It is evident that by decreasing the aMNPs/bacteria ratio fewer
the agglomeration of the aMNPs by adsorbing onto the nanopar- aMNPs are attached to the bacterial cells (Fig. 3c and d). In the
ticles or by increasing the ionic strength of the suspension. Even case when the aMNPs are in excess (R1), it was assumed that
a relatively small amount of salt (e.g., less than 10 mM of KCl) non-attached aMNPs act like stabilizers, preventing the aggre-
caused the agglomeration of aMNPs in the aqueous suspension gation of the magnetically modied O. oeni. In contrast, at the
(data not shown). To remove the impurities, the bacterial suspen- lower aMNPs/bacteria ratio (R2), the adsorbed nanoparticles make
sion was ultraltered before the cells were added to the suspension bridges between the bacterial cells (Fig. 3c), similar to the situa-
of aMNPs. tion observed with the chemically driven heteroaggregation of two
Fig. 2 shows a SEM image of the bacteria with the adsorbed types of nanoparticles [64].
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- 3.2. Inuence of magnetic nanoparticles on bacterial viability and
ing bacteria (5 109 cells/mL, pH 4) in the suspension, which was metabolism
not puried by ultraltration. The bacteria were non-uniformly
covered with the aMNPs. The aMNPs were mainly attached to the The number of bacterial cells in the starting suspension was
bacterial cells in the form of larger agglomerates, while vast areas determined using the ow-cytometry technique and the plate-
of the cells were uncovered. The agglomeration was most probably count method. The use of uorescent stains in combination with
induced by an increased ionic strength of the suspension caused by ow cytometry allows the detection and discrimination of viable
dissolved impurities. culturable, viable nonculturable, and nonviable organisms [65]. The
The coverage of the bacteria with aMNPs was clearly improved concentration of 3 109 cells/mL was determined by ow cytome-
when the bacterial suspension was puried by ultraltration try in the starting suspension. The determined viability for O. oeni
(Fig. 2b). The cells were covered more homogenously with the indi- without any attached magnetic nanoparticles in the bacterial sus-
vidual aMNPs, although some smaller agglomerates of aMNPs were pension was 98%.
also observed. In practice, bacterial viability is measured using the plate-count
Previous studies dealing with the adsorption of magnetic technique. However, in the case of O. oeni, the plate-count tech-
nanoparticles onto microorganisms surfaces show that apart from nique requires a very long incubation time of about 10 days or more
the suspension pH [60], the suspensions concentrations [61,62] [66]. To verify the number of bacterial cells obtained by ow cytom-
and the number ratio R between the nanoparticles and the etry, the bacteria were grown on MRS agar plates. The number of
microorganisms inuence the surface coverage of the microorgan- 5 109 CFU/mL was determined by the plate counting, which is in
isms [26,63]. TEM analyses showed that different aMNPs/bacterial good agreement with the determined number for lyophilized or
number ratios and different bacterial concentrations inuenced dried bacteria in the International Oenological Codex [67].
the coverage of the ultraltered O. oeni with the aMNPs. The Bonding of the nanoparticles on the cell wall can damage the
aMNPs/bacterial-number ratios were chosen according to the cell membrane of the microorganism [63,68]. The cell membrane
specic surface area of the bacterial cells, determined from micro- of O. oeni used in our experiments was reinforced by the MBR
scopic analyses of the dry cells. The coverage of O. oeni with 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]. of O. oeni was tested by performing the MLF in wine after alco-
The preparation process of the MRB and the presence of magnetic holic fermentation. The organic acid concentrations obtained from
nanoparticles attached on the bacterial cells might have an inu- an enzymatic analysis of the wine, inoculated according to the
ence on the viability of the O. oeni. However, the ow cytometry manufacturers recommendations, wine inoculated with puried
results on the viability of the bacterial cells that were ultraltered bacterial suspension (centrifuged or ultraltrated bacteria) and
or the bacterial cells with attached aMNPs were the same as in wine inoculated with the MRB (centrifuged or ultraltrated bac-
the case of the starting bacterial suspension. The percentage of live teria with the aMNPs) (for details see Table A.1 in Supplementary
bacteria in the suspension remained the same, i.e., 98%. The prepa- data) were compared. The comparison between the start and the
ration process for the MRB and the attached magnetic nanoparticles end pH values and the organic acid concentrations conrmed that
on the bacterial cells show no cytotoxic effect on the O. oeni. the MLF occurred in all experiments. The results also showed that
A literature survey of the inuence of attached nanoparticles on the purication methods, e.g., centrifugation or ultraltration, that
the bacterial metabolism in general shows that this topic has not were used for the preparation of the MRB do not have any inuence
been well researched yet. The inuence of the attached nanoparti- on the bacterial metabolism and the MLF process.
cles on the growth of bacteria was studied by other researchers in
order to assess the cytotoxic effect [7073]. Although it was shown 3.3. Control of the malolactic fermentation of wine using
that the attached magnetic nanoparticles accelerate the metabolic magnetic separation of magneto-responsive bacteria
activity of the wine yeast by speeding up the fermentation process
kinetics [26], the attached magnetic nanoparticles on the surface of With the aim being to develop a method for the continuous
the O. oeni did not have any inuence on its metabolism. The inu- HGMS of the MRB from wine, they were rst separated from the
ence of the attached magnetic nanoparticles on the metabolism synthetic medium with a chemical composition similar to wine. The
P. Dusak et al. / Biochemical Engineering Journal 110 (2016) 134142 139

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 alcoholic fermentation and terminated after 7 days with the HGMS
of remaining cells after the separation using ow-cytometry anal- of the magneto-responsive O. oeni. The enzymatic tests for organic
yses. No difference in the ow-cytometry analysis results, as the acids and residual sugars were performed on samples taken before
background noise, was observed if the O. oeni cells were dispersed and after the magneto-responsive O. oeni were separated from
in de-ionized water or in the synthetic medium. Fig. 4 shows the the wine, and also an additional 7 and 14 days after the separa-
presence of the MRB before (a) and after (b) the HGMS. tion. The results obtained for the magneto-responsive O. oeni were
The tail-shaped part marked on the graph in Fig. 4a between compared with the results for the bacteria without the attached
10 and 1000 FSC, represents the MRB before the HGMS. It is clear magnetic nanoparticles (the control, labelled as pristine O. oeni)
that this part is missing after the HGMS (Fig. 4b). The efciency of (Fig. 6). Under conditions of limiting sugar availability or low pH,
the HGMS was estimated to be 96%. The number of bacterial cells the growth of the O. oeni is enhanced in the presence of organic acids
per mL after the HGMS was 4 103 , which is less than the number such as malate or citrate. Although malic acid is the most impor-
of cells needed for the start or continuation of the MLF [2]. tant acid metabolized by O. oeni in wine, other organic acids are also
After testing on the synthetic medium, the HGMS was carried metabolized. Citric acid metabolism by O. oeni has been correlated
out on the wine sample after 14 days of MLF. However, the results with the synthesis of acetic acid, diacetyl and acetoin [57]. In biore-
of the ow-cytometry analysis could not be quantied because actors containing the pristine O. oeni, the starting concentrations of
two populations of cells were present in the wine (see Fig. A.3 in l-malic acid and citric acid decreased in 7 days after the inoculation
Supplementary data). Apart from the MRB, another population of of the wine. On the other hand, the concentration of l-lactic acid
microorganisms was detected, which can be ascribed to the wine and the pH value increased and the exhaust of CO2 was visually
yeasts [74]. Yeast cells might already be present in the wine, since it observed. These results indicate that the MLF proceeded. The con-
was not ltered before the experiment, or they were added with the centration of l-malic acid and citric acid continued to decrease and
O. oeni as bio-activators. The efciency of the HGMS was therefore the concentration of l-lactic acid and pH continued to increase in
tested with a TEM analysis and by observations of the MLF process the next 7 days. Oenococcus oeni cannot grow with l-malic acid as
after the HGMS. a unique carbon source; therefore, these microorganisms need an
The TEM analysis detected no bacteria in the wine samples after additional energy source, such as residual fermentable sugars, i.e.,
the HGMS, whereas some larger yeast cells were observed (data not glucose and/or fructose, to allow the cell growth [3]. It is a hetero-
shown). The sedimented cells from the magnetic separator were fermentative microorganism and converts glucose to D-lactic acid,
also analysed using TEM and proved to be the MRB. Although the CO2 and acetic acid (or ethanol) [4]. The low pH of the wine inhibits
cells multiplied during the fermentation process, the nanoparti- the sugar metabolism of O. oeni [4]. The concentration of residual
cles remained on their surface (Fig. 5b). After drying the bacterial sugars did not change from the starting value (0.9 g/L) during the
suspension on a TEM specimen support, the vast majority of the MLF in our experiments. This result indicates that residual sugars
bacterial cells were deposited in the form of larger clusters con- were not metabolized and the produced lactic acid was the conver-
taining several cells. The surface concentration of the nanoparticles sion of malic acid. The same results were obtained if the MLF was
on the bacteria after the MLF (Fig. 5b) is smaller than the initial performed with the MRB.
concentration (Fig. 5a). During the MLF the magneto-responsive In the bioreactor containing the magneto-responsive O. oeni, the
O. oeni cells multiply and their magnetization decreases because starting pH value and organic acid concentrations changed in 7 days
of the increased number in the newly grown bacteria. Oenococ- after the inoculation of the wine (Fig. 6). After the HGMS the organic
cus oeni is known to grow slowly in comparison to other bacteria acid concentrations and the pH value remained the same. There
[75]. Their slow growth and chain-like structures, formed during was no further exhausting of CO2 observed in the bioreactors after
multiplication [75], are advantageous in the magnetic separation. the HGMS. The results prove that the fermentation process stopped
Although the cells of O. oeni multiplied during the fermentation completely. It is therefore reasonable to expect that the fermenta-
process, the magnetic nanoparticles remained on their surface in tion can be completely stopped at the desired stage of the process
a relatively high concentration (Fig. 5), and the newly grown bac- with the separation of the MRB using HGMS.
teria without magnetic nanoparticles were always attached to the After the HGMS of the magneto-responsive O. oeni, the HGMS
original magnetic bacteria, thus enabling efcient separation using column containing trapped magneto-responsive O. oeni was back
the HGMS. ushed with de-ionized water in order to study the inuence of the
The MLF can be controlled by the removal of O. oeni at a desired magnetic separation process on the bacterial viability and the pos-
time. To test this hypothesis the MLF was started in the wine after 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 of postmagneto-responsive O. oeni there was no change in the
new bioreactor containing wine. The changes of organic acid con- pH value or in the organic acid concentrations (Fig. 6). To ensure
centrations and the pH value clearly indicate that the MLF occurred an efcient magnetic separation, a high concentration of magnetic
(Fig. 6). During the HGMS the bacteria were exposed to the high nanoparticles has to be added. In the experiment, the nanoparticles
magnetic eld. The exposure of microorganisms to the magnetic were added in excess approximately 16,200 aMNPs were added
eld could have an inuence on their metabolism or viability [76]. per cell. The excess of nanoparticles is needed because they adsorb
The results proved that the separation process does not have a neg- non-selectively, not only onto the surfaces of the O. oeni, but also
ative inuence on the magneto-responsive O. oeni in the HGMS onto other particles present in the wine, e.g., yeast cells, cell debris,
column. It is therefore reasonable to expect that the recycled MRB polymerized phenolic compounds, etc. Moreover, the positively
could be used again in another MLF. charged nanoparticles added directly into the wine quickly adsorb
To simplify the process of magnetic separation, the mag- negatively charged molecules, which change their surface charge.
netic nanoparticles could be added to the wine at a desired -potential measurements showed that the nanoparticles changed
time of the MLF to stop the process by using HGMS. To test their surface charge from positive to negative only a few minutes
this hypothesis, the magnetic nanoparticles were added to the after they were dispersed in the wine. The change in the nanoparti-
bioreactor containing the O. oeni without adsorbed nanoparticles cles surface charge inuences the adsorption of the nanoparticles
7 days after inoculation, just before the HGMS. After the HGMS onto the bacteria. It was found experimentally that negatively
P. Dusak et al. / Biochemical Engineering Journal 110 (2016) 134142 141

charged nanoparticles also adsorb onto the microorganisms; how- magneto-responsive bacteria were quickly and efciently removed
ever, to a much smaller extent, compared to the positively charged from the fermentation media.
nanoparticles [26]. In spite of the decrease of the nanoparticles - The main advantage of it is that the magnetized O. oeni could
potential after their addition to the wine, it seems that they were be quick and effectively removed from the process after the bio-
still attached to the bacteria cells in a sufcient number to enable transformation of the malic to the lactic acid in this process.
effective magnetic separation. Alternatively, pristine bacteria without the magnetic nanoparticles
The non-selective adsorption of the wine components onto the can be used for the MLF and the nanoparticles can be added to the
magnetic nanoparticles during the magnetic separation of the O. wine after a certain time to stop the process using HGMS.
oeni, with the addition of the nanoparticles directly to the wine,
can change the wines composition, thus inuencing its quality. Acknowledgements
It was assumed that the excess nanoparticles that were not
adsorbed onto the bacterial surface and other particles in the wine The support of the Ministry of Higher Education, Science
would agglomerate in the wine due to the increased ionic strength and Technology of the Republic of Slovenia within the National
and due to the adsorption of different molecules. Due to their rel- Research Program P2-0089 is gratefully acknowledged. We
atively large size, the agglomerates can be effectively magnetically acknowledge the use of equipment in the Centre of Excellence on
separated. Nanoscience and Nanotechnology-Nanocenter. Authors also thank
Based on the obtained results in this research it can be concluded Dr. Darja Lisjak from the Jozef Stefan Institute for the SEM analy-
that the MLF process can be controlled by the magnetic separation ses, Bojan Kobal from Ptujska klet for providing the wine samples,
of the MRB at a specic stage of the fermentation. Gordana Veber from Jurana d.o.o for providing the freeze-dried
There are concerns about the presence of nanoparticles in the
bacteria, Dr. Vid Simon Selih from National Institute of Chemistry
wine after the separation. The wine after the separation was ana- Slovenia for the ICP-MS analysis and Prof. Dr. Rok Kostanjsek from
lysed using ICP-MS; however, an increase in the concentration of the Biotechnical Faculty, University of Ljubljana for fruitful discus-
the iron could not be detected. The concentration of Fe in the sions.
wine was 1.48 mg/L. After the separation the concentration was
equal within the limits of the analytical uncertainty, which was
Appendix A. Supplementary data
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-
Supplementary data associated with this article can be found, in
cles added to the wine with the MRB were to dissolve. According to
the online version, at
the OIV and the national legislation of several countries, the allowed
concentration of Fe in white wine is 10 mg/L [77].
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