Biochemical Engineering Journal 110 (2016) 134–142
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Application of magneto-responsive Oenococcus oeni for the malolactic fermentation in wine Peter Duˇsak a,b,∗ , Mojca Benˇcina c,d , Martina Turk e , Dejan Bavˇcar f , Tatjana Koˇsmerl g , Marin Beroviˇc h , Darko Makovec a,b a
Department for Material Synthesis, Joˇzef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia Joˇzef 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, Veˇcna 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, Veˇcna pot 113, 1000 Ljubljana, Slovenia b
a r t i c l e
i n f o
Article history: Received 9 September 2015 Received in revised form 17 December 2015 Accepted 27 February 2016 Available online 3 March 2016 Keywords: Superparamagnetic nanoparticles Lactic acid bacteria High-gradient magnetic separation Malolactic fermentation Controlled fermentation
a b s t r a c t A new method for magnetic separation of the magnetized lactic acid bacteria (LAB) Oenococcus oeni at a desired stage of the malolactic fermentation (MLF) in wine was developed. The method includes the bonding of functionalized magnetic nanoparticles to the bacterial surface in the suspension, the application of the “magneto-responsive” bacteria (MRB) in the fermentation process and their magnetic separation from the wine using high-gradient magnetic separation (HGMS). The controlled attachment 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 at a certain stage using the HGMS. From the results it was found that the magnetization of the bacteria using aMNPs attached to the cell surface had no inﬂuence on the O. oeni metabolism. The O. oeni with the attached magnetic nanoparticles were efﬁciently removed from the fermentation media using HGMS, which resulted in a complete stop of the fermentation process. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The fermentation of grape juice involves two fermentation processes: an alcoholic fermentation and a malolactic fermentation (MLF). The MLF represents a secondary fermentation that proceeds in parallel or after the alcohol fermentation . It starts as soon as the lactic acid bacteria (LAB) population reaches a concentration of 106 colony-forming units per millilitre (CFU/mL)  and its duration is approximately 5 days to 3 weeks, depending on the physico-chemical properties of the fermentation . The MLF is desirable for some wine types, because it decreases the acidity,
∗ Corresponding author at: Department for Materials Synthesis, Joˇzef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia. E-mail addresses: [email protected]
(P. Duˇsak), [email protected]
(M. Benˇcina), [email protected]
(M. Turk), [email protected]
(D. Bavˇcar), [email protected]
(T. Koˇsmerl), [email protected]
(M. Beroviˇc), [email protected]
(D. Makovec). http://dx.doi.org/10.1016/j.bej.2016.02.016 1369-703X/© 2016 Elsevier B.V. All rights reserved.
enhances the organoleptic characteristic, and increases the microbiological stability of the wine . The MLF is performed by LAB, which convert the l-malic acid to l-lactic acid and carbon dioxide . Oenococcus oeni (formerly known as Leuconostoc oenos)  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 . 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 wine’s 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 formation 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 suppressed by removing or inactivating the bacteria that are present. The removal of LAB can be achieved by using standard winemak-
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ing technology, such as ﬁltration, centrifugation, and the control of MLF by using addition of anti-microbial including sulphur dioxide or lysozyme . In contrast, if MLF is desired or needed, its control is essential. In recent years several technologies have been proposed to control a wine’s MLF by using LAB, principally O. oeni . These alternative technologies usually involve the use of high densities of bacterial cells, free or immobilized by adsorption onto, or encapsulated into, different matrices, such as calcium alginate [9,10], pectate , -carrageenan [12–14], polyacrylamide , cellulose [8,15], and poly(vinyl alcohol) hydrogel . As a support for the immobilization of the bacterial cells used during the MLF, chemically modiﬁed chitosan (CCB) beads have also been used . 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 efﬁcient separation method is needed. This can be achieved by employing magnetic separation of LAB from wine. In biotechnology, magnetic separation is a well-known technique [18–22], where magnetic carriers are dispersed into a reaction mixture containing speciﬁc targets. After binding the speciﬁc targets with magnetic carriers, the conjugates are separated by using an external magnetic ﬁeld [23–25]. For the magnetic separation 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 ﬁeld can be large enough for effective separation, because of its relatively large volume . It is beneﬁcial if the magnetic nanoparticles are small enough to be in the superparamagnetic state. Superparamagnetism is a phenomenon related to ferri/ferromagnetic particles when their size is reduced below a certain limit and thermal excitation induces rapid ﬂuctuations, 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 superparamagnetic 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 magnetic iron-oxides like maghemite (␥-Fe2 O3 ) or magnetite (Fe3 O4 ) are normally used [29–33], because of their low cost and relatively 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 [38–41]. Iron-oxide magnetic nanoparticles have also attracted a lot of attention in biomedicine, in drug delivery or in the detection and targeting of speciﬁc (bio) molecules or cells [25,42–45]. 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 . Low-magnetisation particles are usually separated in a continuous process from suspensions using high magnetic gradients (HGMS). In biotechnology, for protein puriﬁcation [46–49], or in cell separation [50–52], the HGMS techniques have become very useful. The magnetic nanoparticles can be adsorbed onto the surface of microorganisms, by attractive electrostatic interactions between the microorganisms and the magnetic nanoparticles with an opposite surface charge, or with chemical interactions, (biotin-avidin interactions)  or by antigen-antibody recognition . 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 immobilizing the bacterial cells onto different matrices, functionalized
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 manufacturer’s recommendations. 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 supplier’s recommendations. 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 deionized water, the pH was adjusted to 3.2. The MLFs were also carried out in unﬁltered, 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 initial 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 ﬁlled with a fermentation substrate and inoculated with bacteria or magneto-responsive bacteria (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 ﬁltered through a 0.2-m ﬁlter (regenerated cellulose, Chromaﬁl, 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 ) nanoparticles coated with an approximately 4-nm-thick layer of silica were synthesized as described in Ref. . The silica-coated maghemite nanoparticles were functionalized by grafting 3(2-aminoethylamino) propylmethyldimethoxysilane onto their surfaces, as described elsewhere . 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 calculated speciﬁc surface area of 100 m2 /g (Fig. A.1 in Supplementary data). The aMNPs were superparamagnetic with a saturation magnetization 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 positively 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 centrifugation (6900 × g, 2 min) or ultraﬁltered (Solvent resistant stirred cell, Millipore, USA; ultraﬁltration membrane 30 kDa; 2 bar of N2 pressure) to remove any impurities, such as the salts from growth
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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 efﬁciency of the attachment of the aMNPs onto the bacterial cell surface in an aqueous medium, two different 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 concentration (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 determined with enzymatic test kits (speciﬁc for the determination of the l-malic acid or the l-lactic acid) from Oenolab Diagnostics (Hendaye, France). The d-glucose together with the d-fructose were measured with speciﬁc 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 Méthodes internationales d’analyse des vins . 2.3.2. Dissolved Fe analysis The dissolved Fe was analysed using an elemental mass spectrometer 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 bacterial 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 speciﬁc 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 ﬂow cytometry. A CyFlow Space cytometer (Partec, Münster, 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 granularity) were used to deﬁne the population of cells. For the detection of the cells stained with propidium iodide (PI) dye and SYTO9 dye we used optical ﬁlters of 675/25 nm (FL3) and 590/50 nm (FL2), respectively. The setting region on the FSC/SSC was used to discriminate the bacteria from the background. Gates were deﬁned in the histogram plots of green ﬂuorescence and red ﬂuorescence. The samples were analysed at low rate settings of approximately
200 cells s−1 , 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 . Stock solutions of the dyes were prepared as follows. Red-ﬂuorescent nucleic acid stain propidium iodide (PI) and green-ﬂuorescent 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 ﬁlled with type430 ﬁne-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 canister was placed vertically in the 1-cm gap between the two pole pieces of the separator. A magnetic ﬁeld between the pole pieces, which could be varied in strength, was generated with an attached electromagnet. The direction of the magnetic ﬁeld was transverse with respect to the direction of the ﬂow through the column. The maximum magnetic ﬂux density generated between the two plates was 1 T, as measured with a handheld gauss meter. The maximum magnetic ﬂux density was used in all the experiments. The continuous magnetic separation experiments were performed at room temperature by passing 250 mL of the suspension (MRB in synthetic media or MRB in wine) from the reaction vessel, through the HGMS column with the electromagnet on, into the ﬁltrate vessel. The suspension was ﬁrst roughly shaken and then pumped steadily at 4.3 mL/min with a peristaltic pump (Watson Marlow 400, United Kingdom) through the HGMS column. The efﬁciency of the HGMS was evaluated by ﬂow-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 . The adsorption of the nanoparticles onto the
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Table 1 Different combinations of bacterial cell concentrations and aMNP/bacteria ratios. Samplename
Bacterial concentration [cells/mL]
Bacterial suspension volume [mL]
aMNP (1 mg/mL) volume added [mL]
B1R1 B2R1 B1R2 B2R2
5 × 109 5 × 107 5 × 109 5 × 107
1 1 1 1
1:8745 1:8745 1:3336 1:3336
0.5 0.005 0.2 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 display 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 nanoparticles 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 suspension was ultraﬁltered 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 receiving bacteria (5 × 109 cells/mL, pH 4) in the suspension, which was not puriﬁed by ultraﬁltration. 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 puriﬁed by ultraﬁltration (Fig. 2b). The cells were covered more homogenously with the individual 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 , the suspensions’ concentrations [61,62] and the number ratio R between the nanoparticles and the microorganisms inﬂuence the surface coverage of the microorganisms [26,63]. TEM analyses showed that different aMNPs/bacterial number ratios and different bacterial concentrations inﬂuenced the coverage of the ultraﬁltered O. oeni with the aMNPs. The aMNPs/bacterial-number ratios were chosen according to the speciﬁc surface area of the bacterial cells, determined from microscopic 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 aggregation of the magnetically modiﬁed 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 situation observed with the chemically driven heteroaggregation of two types of nanoparticles . 3.2. Inﬂuence of magnetic nanoparticles on bacterial viability and metabolism The number of bacterial cells in the starting suspension was determined using the ﬂow-cytometry technique and the platecount method. The use of ﬂuorescent stains in combination with ﬂow cytometry allows the detection and discrimination of viable culturable, viable nonculturable, and nonviable organisms . The concentration of 3 × 109 cells/mL was determined by ﬂow cytometry in the starting suspension. The determined viability for O. oeni without any attached magnetic nanoparticles in the bacterial suspension was 98%. In practice, bacterial viability is measured using the plate-count technique. However, in the case of O. oeni, the plate-count technique requires a very long incubation time of about 10 days or more . To verify the number of bacterial cells obtained by ﬂow cytometry, 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 . 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-
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Fig. 2. SEM images of aMNPs adsorbed on O. oeni: (a) the aMNPs were adsorbed onto the bacteria in the suspension not previously puriﬁed, (b) bacteria in the suspension were puriﬁed by ultraﬁltration 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. . The preparation process of the MRB and the presence of magnetic nanoparticles attached on the bacterial cells might have an inﬂuence on the viability of the O. oeni. However, the ﬂow cytometry results on the viability of the bacterial cells that were ultraﬁltered 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 preparation 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 inﬂuence of attached nanoparticles on the bacterial metabolism in general shows that this topic has not been well researched yet. The inﬂuence of the attached nanoparticles on the growth of bacteria was studied by other researchers in order to assess the cytotoxic effect [70–73]. Although it was shown that the attached magnetic nanoparticles accelerate the metabolic activity of the wine yeast by speeding up the fermentation process kinetics , the attached magnetic nanoparticles on the surface of the O. oeni did not have any inﬂuence on its metabolism. The inﬂuence of the attached magnetic nanoparticles on the metabolism
of O. oeni was tested by performing the MLF in wine after alcoholic fermentation. The organic acid concentrations obtained from an enzymatic analysis of the wine, inoculated according to the manufacturer’s recommendations, wine inoculated with puriﬁed bacterial suspension (centrifuged or ultraﬁltrated bacteria) and wine inoculated with the MRB (centrifuged or ultraﬁltrated bacteria 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 conﬁrmed that the MLF occurred in all experiments. The results also showed that the puriﬁcation methods, e.g., centrifugation or ultraﬁltration, that were used for the preparation of the MRB do not have any inﬂuence 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 ﬁrst separated from the synthetic medium with a chemical composition similar to wine. The
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Fig. 4. Graphs FSC vs. SSC before (a) and after (b) HGMS of MRB. The oval deﬁnes the region of MRB.
efﬁciency of the HGMS was evaluated by determining the number of remaining cells after the separation using ﬂow-cytometry analyses. No difference in the ﬂow-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 efﬁciency 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 . 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 ﬂow-cytometry analysis could not be quantiﬁed 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 . Yeast cells might already be present in the wine, since it was not ﬁltered before the experiment, or they were added with the O. oeni as bio-activators. The efﬁciency 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 nanoparticles 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 containing 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. Oenococcus oeni is known to grow slowly in comparison to other bacteria . Their slow growth and chain-like structures, formed during multiplication , 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 bacteria without magnetic nanoparticles were always attached to the original magnetic bacteria, thus enabling efﬁcient 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
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 separation. 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 important 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 . In bioreactors 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 concentration 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 . It is a heterofermentative microorganism and converts glucose to D-lactic acid, CO2 and acetic acid (or ethanol) . The low pH of the wine inhibits the sugar metabolism of O. oeni . 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 conversion 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 fermentation 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 ﬂushed with de-ionized water in order to study the inﬂuence of the magnetic separation process on the bacterial viability and the possibility of their reuse in subsequent MLFs. To study their reuse, the
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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 concentrations and the pH value clearly indicate that the MLF occurred (Fig. 6). During the HGMS the bacteria were exposed to the high magnetic ﬁeld. The exposure of microorganisms to the magnetic ﬁeld could have an inﬂuence on their metabolism or viability . The results proved that the separation process does not have a negative inﬂuence 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 magnetic 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 efﬁcient 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 nanoparticles’ surface charge inﬂuences the adsorption of the nanoparticles onto the bacteria. It was found experimentally that negatively
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charged nanoparticles also adsorb onto the microorganisms; however, to a much smaller extent, compared to the positively charged nanoparticles . 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 sufﬁcient 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 wine’s composition, thus inﬂuencing 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 relatively 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 speciﬁc stage of the fermentation. There are concerns about the presence of nanoparticles in the wine after the separation. The wine after the separation was analysed 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 nanoparticles 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 . Generally, the magnetic separation of bacteria is important for applications where the bacteria need to be removed from complex environments and conventional puriﬁcation methods, e.g., ﬁltration, centrifugation, are not cost-effective  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 . However, the adsorption or encapsulation of O. oeni on/into different matrices has masstransfer 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 signiﬁcant inﬂuence on the transfer limitations for nutrients. Furthermore, the MRB can be efﬁciently removed at a desired time or at the end of MLF by applying an external magnetic ﬁeld. To minimize the expenses during the production of wine the method for the preparation of MRB should be an inexpensive process. Here, the electrostatically 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 smoothertasting lactic acid represents a technical solution to this problem. The main difﬁculty 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 superparamagnetic 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 inﬂuence the metabolic activity of the used bacteria. The ﬂow-cytometry results before and after the magnetic separation showed that the
magneto-responsive bacteria were quickly and efﬁciently 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 biotransformation 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 Joˇzef Stefan Institute for the SEM analyses, Bojan Kobal from Ptujska klet for providing the wine samples, Gordana Veber from Jurana d.o.o for providing the freeze-dried ˇ bacteria, Dr. Vid Simon Selih from National Institute of Chemistry Slovenia for the ICP-MS analysis and Prof. Dr. Rok Kostanjˇsek from the Biotechnical Faculty, University of Ljubljana for fruitful discussions. 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. References  H. König, G. Unden, J. Fröhlich, Biology of Microorganisms on Grapes in Must and in Wine, Springer, Berlin, 2009.  A. Lonvaud-Funel, Lactic acid bacteria in the quality improvement and depreciation of wine, Antonie Van Leeuwenhoek 76 (1999) 317–331.  A. Versari, G.P. Parpinello, M. Cattaneo, Leuconostoc oenos and malolactic fermentation in wine: a review, J. Ind. Microbiol. Biotech. 23 (1999) 447–455.  P. Ribéreau-Gayon, D. Dubourdieu, B. Donèche, A. Lonvaud, Handbook of Enology, the Microbiology of Wine and Viniﬁcations, Wiley, Chichester, 2006.  L.M.T. Dicks, F. Dellaglio, M.D. Collins, Proposal to reclassify Leuconostoc oenos as Oenococcus oeni [corrig] gen-Nov, comb-Nov, Int. J. Syst. Bacteriol. 45 (1995) 395–397.  D. Wibowo, R. Eschenbruch, C.R. Davis, G.H. Fleet, T.H. Lee, Occurrence and growth of lactic acid bacteria in wine: a review, Am. J. Enol. Vitic. 36 (1985) 302–313.  I. Lopez, R. Lopez, P. Santamaria, C. Torres, F. Ruiz-Larrea, Performance of malolactic fermentation by inoculation of selected Lactobacillus plantarum and Oenococcus oeni strains isolated from Rioja red wines, Vitis 47 (2008) 123–129.  S. Maicas, The use of alternative technologies to develop malolactic fermentation in wine, Appl. Microbiol. Biotechnol. 56 (2001) 35–39.  V.A. Nedovic, A. Durieux, L. Van Nedervelde, P. Rosseels, J. Vandegans, A.M. Plaisant, J.P. Simon, Continuous cider fermentation with co-immobilized yeast and Leuconostoc oenos cells, Enzyme Microb. Technol. 26 (2000) 834–839.  M. Herrero, A. Laca, L.A. Garcia, M. Diaz, Controlled malolactic fermentation in cider using Oenococcus oeni immobilized in alginate beads and comparison with free cell fermentation, Enzyme Microb. Technol. 28 (2001) 35–41.  M.R. Kosseva, J.F. Kennedy, Encapsulated lactic acid bacteria for control of malolactic fermentation in wine, Artif. Cells Blood Substit. Immobil. Biotechnol. 32 (2004) 55–65.  A. Crapisi, P. Spettoli, M.P. Nuti, A. Zamorani, Comparative traits of Lactobacillus-brevis, lact fructivorans and Leuconostoc-oenos immobilized cells for the control of malo-lactic fermentation in wine, J. Appl. Bacteriol. 63 (1987) 513–521.  J.D. Mccord, D.D.Y. Ryu, Development of malolactic fermentation process using immobilized whole cells and enzymes, Am. J. Enol. Vitic. 36 (1985) 214–218.  P. Spettoli, M.P. Nuti, A. Crapisi, A. Zamorani, Technological improvement of malolactic fermentation in wine by immobilized microbial-cells in a continuous-ﬂow reactor, Ann. N. Y. Acad. Sci. 501 (1987) 386–389.  N. Agouridis, N. Kopsahelis, S. Plessas, A.A. Koutinas, M. Kanellaki, Oenococcus oeni cells immobilized on deligniﬁed cellulosic material for malolactic fermentation of wine, Bioresour. Technol. 99 (2008) 9017–9020.  A. Durieux, X. Nicolay, J.P. Simon, Continuous malolactic fermentation by Oenococcus oeni entrapped in LentiKats, Biotechnol. Lett. 22 (2000) 1679–1684.
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