Electric field charge polarity triggers the organization and promotes the stability of electrosprayed probiotic cells

The encapsulation and organization of Bifidobacterium animalis subsp. lactis (BIFIDO) probiotic cells within maltodextrin microcapsules using electrospray processing was investigated. By choosing an appropriate polarity of the DC electric field, the surface charged probiotic cells were localized either in the core or towards the surface of the capsule, as visualized using confocal microscopy. Negatively charged probiotic cells encapsulated using a negative polarity on the electrospraying nozzle, were ‘organized ’ mostly in the core of the microcapsules. The organization of the cells affected the evaporation of the solvent (water) and subsequently the glass transition temperature (Tg) of the electrosprayed microcapsules. Moreover, the viability of the encapsulated cells was significantly improved for up to 2 weeks of storage at 25 ◦ C and 35% RH, when the cells were located at the core of the microcapsules, compared to the case where the probiotics were distributed towards the surface. Overall, this study presents a novel organization process that promotes the stability of the probiotic cells.


Introduction
Encapsulation provides a physical barrier to protect sensitive compounds and cells, including probiotics, when incorporated into various products, as well as through the gastrointestinal track (García- Moreno, Mendes, Jacobsen, & Chronakis, 2018).Electrohydrodynamic (EHD) processes (electrospinning and electrospray) have been considered appealing technologies for the encapsulation of probiotics (Mendes & Chronakis, 2021).EHD processes utilize high-voltage electrostatic fields to charge the surface of biopolymer solution droplets, which induce the ejection of a liquid jet through a spinneret.The EHD encapsulation processes can be performed utilizing aqueous dispersions of a broad range of encapsulation materials, at room temperature without heat, and without compromising the viability of the living cells (Jacobsen, Garcia-Moreno, Mendes, Mateiu, & Chronakis, 2018;Mendes & Chronakis, 2021).
Lopez-Rubio et al., reported that the encapsulation of Bifidobacterium animalis subsp.lactis by using an electrospinning coaxial setup, could increase the viability of the probiotics compared to non-encapsulated strains (López-Rubio, Sanchez, Sanz, & Lagaron, 2009).Moreno et al., also confirmed that the viability of BIFIDO probiotics encapsulated within the core-shell ETC electrosprayed capsules could be extended, presenting a loss in viable cells of no more than 3 log loss CFU/g after four weeks at 30 • C and 40%RH (Moreno, Dima, Chronakis, & Mendes, 2021).Moreover, similar results have been reported for several probiotic microorganisms, such as other members of genus Bifidobacterium (Bifidobacterium longum subsp.infantis CECT 4552) and Lactobacillus (Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus paracasei) (Mendes & Chronakis, 2021).
EHD encapsulation of probiotics has been done so far, in a conventional manner, by applying a positive polarity at the nozzle, and using a grounded collector.The polarities of the electric field utilized at EHD processing, are rarely alternated.However, a few studies have been reporting the effect of the polarity on the EHD processing.Kilic et al., assessed the electrospinning of polyvinyl(alcohol)/water using conventional and reversed polarities configuration.It was observed that the conventional setup had a noticeably higher nanofiber production efficiency than the setup with reversed polarities, due to a lack of Coulombic forces acting on the polymer jet.The morphology of the nanofibers (diameter and web layer pore size) was also affected by the polarity of the electric field (Kilic et al., 2008).
Tsaroom et al., investigated the electrospinning of poly(ethylene oxide) (PEO) nanofibers containing metal salts.The formation of coresheath nanofibers was observed, due to the repulsion of the metal atoms by the positive surface charge of the nozzle.Moreover, the PEOmetal salts electrospun nanofibers using positive polarity at the needle, exhibited a lower melting point (Tm) compared to the neat PEO nanofibers, indicating that the presence of the metal salts disrupted the crystallization of PEO (Tsaroom, Matyjaszewski, & Silverstein, 2011).Urbanek and co-workers also studied the effect of polarity of the electric field on the electrospinning of PLC-chitosan nanofibers, and how it affects the properties, the morphology and the efficiency of surface modification of the nanofibers.It was observed that the repulsive forces by the application of negative polarity at the nozzle caused the movement of the positively charged chitosan to the surface of the fibers and increased the PCL-chitosan interactions, resulting in fibers with lower PCL crystallinity, crystal size distribution as well as lower wettability (Urbanek, Sajkiewicz, & Pierini, 2017).
Furthermore, EHD processing commonly uses Direct Current (DC) external electric field.The effect of a DC external electric field on bacterial cells has been investigated, and various cell responses, depending on for the strength of the electric field, the treatment time and the suspending medium, have been observed.The morphological and physiological characteristics of the bacterial cells, such as the microorganism's phenotype, shape, size as well as surface charges, among others, differentiate the response of bacterial cells under the application of electric field (Esteban, 2014;Galván-D'Alessandro & Carciochi, 2018).
The majority of the bacterial cell walls have a negative charge at neutral pH (Boonaert & Rouxhet, 2000;Dickson & Koohmaraie, 1989).This is consistent with their known cell wall structure, as Gram positive bacteria have teichoic acids linked to either the peptidoglycan or to the underlying plasma membrane with phosphodiester bonds, thus conferring a negative charge, while Gram negative bacteria have an outer covering of phospholipids and lipopolysaccharides, imparting a strongly negative charge to the cell surface.Thus, the electrostatic surface charges of the bacteria cells (which is related to their zeta potential) dominate their movement under DC electric fields (Deflaun & Condee, 1997;Dickson & Koohmaraie, 1989;Luo, Wang, Zhang, & Qian, 2005).
In a previous study, Li et al. reported the manipulation and transportation of the inherently negatively-charged yeast, erythrocytes, and Escherichia coli bacteria within a microfluidic capillary system, due to the electrophoresis and electroosmosis processes induced by DC-current (Li & Harrison, 1997).Cabrera et al., utilized the electrophoresis and isoelectric focusing phenomena to concentrate the Gram-negative bacteria Erwinia herbicola in a multilayer polymeric device.It was observed that the bacteria would concentrate either at the positive electrode due to their negative surface charges, or at the isoelectric point by switching the polarity of the electrodes and simultaneously applying a perpendicular pressure-driven flow (Cabrera & Yager, 2001).Moreover, Armstrong et al., recorded the electro-migration of Bifidobacteria infantis, Lactobacillus acidophilus and Saccharomyces cerevisiae dispersed in PEO under the application of DC electric field, using a CCD imaging system (camera) coupled with Laser-induced fluorescence (LIF).The electrophoretic process of microbial migration was used in order to separate the two different bacteria and Baker's yeast based on their negatively charged surface, and subsequent movement towards the positively-charged anode.It was also observed, that the effect of PEO molecular weight and concentration, as well as the pH, manipulated further the microbial surface charges and their migration under the application of DC (Armstrong et al., 2002).
Other studies also support that DC electric fields induce electrokinetic transport of charged bacteria, towards the electrodes of opposite charge (Deflaun & Condee, 1997;Kłodzińska et al., 2010;Qin et al., 2015;Shan, Harms, & Wick, 2018;Shi, Müller, Harms, & Wick, 2008;Voldman, 2006).For instance, Shan et al., studied the electrokinetic forces acting on Pseudomonas putida KT2440, Rhodococcus opacus X9, Pseudomonas fluorescens LP6a and Sphingomonas species S3 in percolation columns packed with glass beads.The authors intended to quantify the effect of DC fields on bacteria with different surface charges, and the electrophoretic drag force that the bacteria experience.It was suggested, that the zeta potential ratio between the bacteria and the collector surface (glass beads) was a crucial parameter effecting the bacterial deposition and transport in porous media (Shan et al., 2018).
Maltodextrin is a D-glucose polymer resulted from hydrolysis of starch (Xiao, Xia, Zhao, Niu, & Zhao, 2022).It consists of linear amylose and branched amylopectin joined by a-1,4-linked glucose units and a-1, 6-glycosidic bonds branching points occurring every 25 to 30 glucose units.Unlike native starches, maltodextrins are very soluble in water and their physicochemical properties depend on the dextrose equivalent (DE) value, which is lower than 20 (Chronakis, 1998).Maltodextrins have been used as wall material for the encapsulation of sensitive compounds, including probiotics (Moreno et al., 2021;Samedi & Charles, 2019), mainly due to their high water solubility, low viscosity even at higher concentration, low relative cost, neutral taste, and aroma (Xiao et al., 2022).In addition, maltodextrins can be easily digested and absorbed in the intestinal tract (Chronakis, 1998), and are known for their prebiotic properties, which is linked to the growth of probiotics (Yeo & Liong, 2010).Maltodextrin can also reduce the oxygen permeability trough the wall matrix of the microcapsules (Shishir, Xie, Sun, Zheng, & Chen, 2018) which is beneficial in the encapsulation and protection of anaerobic probiotics.This property is caused by the fact that maltodextrin exhibits high water solubility and low viscosity even at a high concentration, which permits rapidly formation of a dense microcapsule surface (Anandharamakrishnan & Ishwarya, 2015).Inter-chain bonding interactions increases as the moisture content reduces, which favors increased barrier properties toward oxygen and water (Vilstrup, 2001).
Although the electrospray encapsulation of probiotics has been explored by several studies, none investigated the effect of the electric fields charge polarity on the organization and stability of electrosprayed encapsulated probiotics.Therefore, the main goal of this study was to investigate and develop a novel method of encapsulation of probiotics by electrospray, that allows to control the location and organization of the probiotics inside the electrosprayed maltodextrin microcapsules.The effect of probiotics surface properties and the polarity of the applied electric field were tested using Bifidobacterium animalis subsp.lactis (BIFIDO) probiotic cells.Correlations between electric field charge polarity and BIFIDO organization within the capsules, glass transition temperature and stability were also assessed.

Materials
The probiotic cells Bifidobacterium animalis subsp.lactis (BIFIDO) of concentration 3 × 10 11 cells/g were provided by Chr.Hansen A/S (Hørsholm, Denmark).The strain was subjected to freeze pelletization and was stored at − 80 • C until further use.KH 2 PO 4 (VWR International, Leuven, Belgium) was utilized as suspending medium for the evaluation of the probiotic cells' surface properties and hexadecane (Thermo Fisher Scientific, Waltham, MA, USA) for carrying out the hydrophobicity measurements.Maltodextrin of Dextrose Equivalent 12 (Glucidex IT12, Roquette, Lestrem, France) was used for the encapsulation of BIFIDO.Sodium chloride (NaCl) (Merck, Kenilworth, NJ, USA) and tryptone (Oxoid, Hampshire, UK) were used for the preparation of isotonic solutions.Fluorescent dye Thiazole Orange, dye content ~90% (Sigma-Aldrich, St. Louis, Missouri, USA) was employed for the staining of the BIFIDO.De Man Rogosa Sharpe (MRS) agar (Merck, Kenilworth, NJ, USA), L-cysteine hydrochloride monohydrate (Merck, Kenilworth, NJ, USA), and anaerobic atmosphere generation sachets (AnaeroGen, Oxoid, Hampshire, UK) were utilized for the viability evaluation tests of BIFIDO.
BIFIDO probiotic cells at a concentration of approximately 3 × 10 8 cells/mL were suspended in 10 mM KH 2 PO 4 to obtain an optical density (OD 600 ) ~ 1.0.The pH of solutions was adjusted to 1, 2, 4, 6 and 8 with 1 M HCl, or 1 M NaOH.The electrophoretic mobility was determined using a Nano ZS Zetasizer (Malvern Instruments Ltd, Worcestershire, UK).A volume of 1 mL of sample was injected into a folded capillary cell with gold plated beryllium/copper electrodes (DTS1070 cell, Malvern Panalytical Ltd, Malvern, UK).The measurements were carried out at 25 • C and each sample was analyzed in quintuplicate.The electrophoretic mobilities were converted to zeta potential using the Helmholtz-Schmoluchowski equation: Where U p is the electrophoretic mobility, V p is the electrophoretic velocity, E is the electric field strength, ζ is the zeta potential, ε is the permittivity, and η is the viscosity of the medium (Li & Shi, 2013).

Probiotic cells' surface hydrophobic/hydrophilic character
The hydrophobic/hydrophilic character of the surface of BIFIDO cells was evaluated by the microbial adhesion to hexadecane (MATH) assay, as it has been previously described by Deepika et al. (Deepika, Green, Frazier, & Charalampopoulos, 2009).The probiotic cells were suspended in 10 mM KH 2 PO 4 of different pH (1, 2, 4, 6, 8) until an optical density (OD 600 ) ~ 0.8 was obtained.Two milliliters of the probiotic cell suspension were mixed with equal volume of hexadecane, vortexed for 1 min and were allowed to stand for 20 min to ensure complete phase separation.The aqueous phase was carefully removed after equilibration and the OD 600 was measured.
The percentage of hydrophobicity was calculated utilizing the equation: where A 0 is the initial absorbance of the probiotic cell suspension and A 1 is the absorbance after phase separation (OD 600 ).Each measurement was performed in triplicate.

Electrospraying process
Maltodextrin was dispersed in deionized Milli Q water (Millipore Corporation, USA) in a concentration of 75% w/v and stirred until obtaining a homogeneous solution.The frozen probiotics' cell concentrate was thawed at room temperature (20 ± 1 • C) and BIFIDO cells (~10 10 cells/mL) were added to the maltodextrin solution and gently stirred until they were fully dispersed.The dispersions had approximately pH = 5 -6, unless otherwise stated.The pH of the solutions was adjusted to 1, 4 and 8 with 1 M HCl, or 1 M NaOH.
The electrospray setup included a high voltage supply (ES50P-10W, Gamma High Voltage Research, Inc., USA) to provide a voltage of 15 -40 kV, and a syringe pump (New Era Pump Systems, Inc., Farmingdale, NY, USA) to feed the maltodextrin-BIFIDO and BIFIDO dispersions at a flow rate of 0.03 mL/min.The electrosprayed maltodextrin-BIFIDO microcapsules and BIFIDO cells were collected horizontally on a steel collector, placed at a distance of 10 cm from the end of the needle.The electrospray setup was placed inside a chamber with nitrogen flow, and the temperature and relative humidity were set to 20 ± 1 • C and 20 ± 3% respectively.
When the polarity of the electrode connected at the needle tip of the syringe was positive or negative, the sample is referred as electrosprayed under positive or negative polarity, respectively.Moreover, the charge applied on the needle tip is denoted first, followed by the charge applied on the collector.For example, when − 15kV was applied on the nozzle tip and +5 kV on the collector, then it is referred as (− 15 kV)(+5 kV), and the absolute value of the electric potential is presented as |20 kV|.
The probiotic cells were stained with the fluorescent dye Thiazole Orange, dye content~90% and washed with NaCl 0.85% w/v prior to encapsulation.The dye was in powder format; therefore, it was dissolved in dimethyl sulfoxide (DMSO) at concentration 42 μmol/L.All images were acquired at an excitation wavelength of 488 nm and emission bandpass filter between 505 and 550 nm.Z-stack series of images were acquired at different focal planes and were reconstructed in a 2D image.The profile of fluorescence intensity across the diameter of the capsules was also plotted and included in the CLSM micrographs using the software Zeiss Zen 2009 (Carl Zeiss MicroImaging GmbH, Jena, Germany).Circles were drawn around the capsules to approximate the edge of the capsules and to facilitate imaging.
The Fourier transform infrared spectroscopy (FT-IR) spectra of the samples were recorded using a Nicolet iS50 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) in attenuated total reflection (ATR) mode over the range of 400-4000 cm − 1 .All spectra were recorded in transmission mode, with a scanning resolution of 4 cm − 1 and 32 scans, and at room temperature (25 • C).Spectral analysis was carried out using the Omnic software (Driver version: 9.12.928,Thermo Fisher Scientific, Waltham, MA, USA).

Differential scanning calorimetry
Modulated Differential Scanning Calorimetry (MDSC) experiments were performed to determine the glass transition temperature (Tg) of the electrosprayed microcapsules.The thermograms of the samples were obtained using the DSC 250 (TA Instruments, New Castle, Delaware, USA), equipped with Refrigerated Cooling System 90 (TA Instruments, New Castle, Delaware, USA).The instrument was calibrated in terms of heat flow and temperature using distilled water (melting point (m.p.) = 0 • C; DHm = 334 J/g) and indium (m.p. = 156.5 • C; DHm = 28.5 J/g).Nitrogen was used as a carrier gas at a flow rate of 50 mL/min.
Approximately 5 mg of the electrosprayed microcapsules (immediately collected after processing) were placed in pre-weighted standard DSC aluminum pans (Tzero aluminum Hermetic pans, TA Instruments, USA) and were hermetically sealed.An empty hermetically sealed aluminum pan was used as reference.The pans were first equilibrated for 5 min at 10 • C, after which a heating ramp followed with 3 • C/min to 120 • C. The protocol was adapted from Elnagaar et al., in order to evaluate the effect of the electrospray processing on the thermal properties of the capsules (Elnaggar, El-Massik, Abdallah, & Ebian, 2010).The settings of the run were set at least 30 • C below and above the expected Tg of the electrosprayed microcapsules, as suggested in literature, based on the Tg of maltodextrin DE12 (Drake et al., 2018;Siemons, Politiek, Boom, van der Sman, & Schutyser, 2020).
The obtained DSC thermograms were analyzed using Trios software interfaced with the DSC, and the Tg was determined from the midpoint temperature of the detected step change.All measurements were done in triplicate and the Tg values were averaged.

Stability of microencapsulated BIFIDO during storage
The stability of the microencapsulated BIFIDO was evaluated according to a previously described protocol (Moreno et al., 2021).Microcapsules containing BIFIDO electrosprayed with negative (− 15 kV) (+5 kV) and positive (+15 kV)(-5 kV) polarities and non-encapsulated Bifidobacterium animalis as a reference sample, were stored in an incubating cabinet (Model KB 8400F, A/S Ninolab, Solrød Strand, Denmark) at a relative humidity of 38 ± 2% RH at 30 • C. The viability of the encapsulated and non-encapsulated BIFIDO cells as a function of time (0-14 days) was determined by Colony Forming Unit (CFU) analysis.In particular, the electrosprayed microcapsules were dispersed in 0.85% w/v sodium chloride (NaCl) and 0.1% w/v peptone (tryptone) solution, homogenized by vortexing and then 0.1 mL of appropriate decimal dilutions was spread-plated in de Man Rogosa Sharpe (MRS) agar plates supplemented with 0.05% w/v L-cysteine hydrochloride monohydrate.The MRS agar plates were incubated at 37 • C under anaerobic conditions for 3 days and the cell viability was calculated as the average of 4 plates.
The Encapsulation Efficiency (EE) of the microcapsules was determined by defining the number of viable cells inside the microcapsules (N) divided by the number of viable cells in the initial solution (N 0 ) as expressed in (3):

Statistical analysis
The results are expressed as mean value ± Standard Deviation (SD).A single factor ANOVA, using Excel software (Microsoft corporation), was employed to analyze the differences in the samples that were each time compared.Statistically significant differences among the samples' results were considered for p-values below (p < 0.05).Samples with the same letter indicate no statistically significant differences between samples.

Probiotic cells' surface electrical charge and cell surface hydrophobic/hydrophilic character
The zeta potential of BIFIDO in monopotassium phosphate buffer as function of pH is shown in Fig. 1. Cell surface electronegativity decreased with decreasing pH, showing negative zeta potential values above pH 2 and slightly positive values at pH 1. Similar zeta potential profiles have been previously reported for other Bifidobacterium strains (Bifidobacterium bifidum and Bifidobacterium pseudolongum) (Gómez Zavaglia, Kociubinski, Pérez, Disalvo, & De Antoni, 2002).The negative surface charge of the cells is due to the surface composition of the cells, which is dominated by anionic domains, including strong acids (phosphate based (lipo-)teichoic acids) and weak acids (acidic polysaccharides and surface proteins) (Deepika et al., 2009).The gradual protonation of the aforementioned chemical groups could justify the cells' decreasing electronegativity with decreasing pH (Pelletier et al., 1997).
The hydrophobicity of the BIFIDO cell surface was evaluated by studying the adhesion of BIFIDO to hexadecane (Fig. 2).At pH values 1 to 6, BIFIDO showed a similar affinity to hexadecane ranging from 73.1 to 88.4%, suggesting a hydrophobic cell surface character.At pH 8, BIFIDO showed a moderate hydrophobic character, with a percentage of affinity to the apolar hexadecane below 40%.
Covalently bound proteins, surface layer proteins and fatty acids that are present at the surface of BIFIDO cells (Dianawati & Shah, 2011;Shakirova, Auzina, Zikmanis, Gavare, & Grube, 2010, 2013), are known   P. Dima et al. to promote the adherence to hydrophobic solvents and contribute to elevated cell hydrophobicity.

Electrospray of non-encapsulated probiotic cells: effect of electric field charge polarity
The FT-IR spectra of (non-encapsulated) electrosprayed BIFIDO (under the influence of different electric field polarity), as well as BIFIDO not treated with an external electric field, are presented in Fig. 3.The band at ~3280 cm − 1 represents the stretching of the hydroxyl-group bond (-OH) and is associated with the water related bonds of the samples.The band appeared more intense for the non-treated BIFIDO, while the lowest hydroxyl-group stretching intensity was noted for BIFIDO electrosprayed with negative polarity (− 20 kV) (Fig. 3).Increased -OH band intensity of spin-coated BB-12 biofilms in water moisture environment has been previously reported (Bozkurt et al., 2019).
Differences at the intensity of bands in the fatty-acid region between 3000 and 2800 cm − 1 were apparent between the differently treated samples, being more intense in the spectrum of the electrosprayed samples (Fig. 3).The bands at 2961, 2930 and 2876 cm − 1 are assigned to the asymmetric CH 3 , CH 2 stretching and the symmetric CH 3 stretching of the nonpolar site of phospholipid bilayer, respectively (Dianawati, Mishra, & Shaha, 2012;Santos, Gerbino, Tymczyszyn, & Gomez-Zavaglia, 2015;Shakirova et al., 2010).Higher transmittance intensity was observed for BIFIDO electrosprayed using negative polarity in comparison with the positive polarity electric field.The presence of water is known to hinder the spectroscopy signal intensity of these groups (Dianawati et al., 2012).Therefore, the peak intensity of the -CH 3 and -CH 2 groups is related with the amount of water present in the samples and decreases in the following order: negative polarity > positive polarity > no-electric fields, where those peaks cannot be seen due to the highest water content (Fig. 3).
The protein amide bands for biological samples are typically found at frequencies ~3200 cm − 1 (Amide A), 1660-1500 cm − 1 (Amide I and Amide II), and 1310-1240 cm − 1 (Amide III) (Gautier et al., 2013;Krimm & Bandekar, 1986).For the non-electrosprayed BIFIDO, the carbonyl stretching vibration of the peptide bonds from secondary amides (at ~1640 cm − 1 , Amide I) was noticed to be more pronounced, in comparison to the N-H bending and C-N stretching vibrational modes of Amide II peak (at ~1550 cm − 1 ).Nevertheless, for the electrosprayed BIFIDO, a less pronounced peak for Amide I in comparison to Amide II was observed, and this peak appeared to also be the least intense for the BIFIDO electrosprayed using negative polarity (Fig. 3).Bozkurt et al., studied the influence of relative humidity on the surface structure of spin-coated BB-12 biofilms, concluding that the increased Amide I absorption is indicative of an increased contact of BB-12 with water molecules (Bozkurt et al., 2019).Therefore, the decreased Amide I peak intensity of sample electrosprayed at negative polarity is suggestive for more dried samples.
Furthermore, differences at the intensity of bands at the polar site of the phospholipid bilayers of the cells (1070 cm − 1 and 1040 cm − 1 ) due to the valence of C-O-C group vibrations were also observed (Fig. 3).The least pronounced peaks of the non-electrosprayed BIFIDO (No electric field) is an indication of extended contact of the cells with water molecules, while the more prominent peaks of the samples electrosprayed at negative polarity suggests that less water was present in BIFIDO compared to the sample electrosprayed using positive (Bozkurt et al., 2019).

Effect of electric field charge polarity and voltage on the cell organization within the electrosprayed microcapsules
The effect of negative and positive polarity of the electric field on the distribution of probiotics cells within the electrosprayed maltodextrin -BIFIDO microcapsules at pH 6, was studied by confocal laser scanning microscopy (CLSM) (Fig. 4).It was observed that the microcapsules were polydispersed and had a diameter range between 30 and 100 μm.For imaging purposes and to facilitate the visualization of the distribution of the encapsulated BIFIDO, the electrosprayed microcapsules with higher diameter are presented.
Negative polarity at the needle triggered an organization of the probiotic cells at the core of the microcapsules.This is due to the electrostatic repulsion between the negatively charged BIFIDO cells (at pH 6), and the negatively charged electric field at the needle walls, as well as at the Taylor cone inner surfaces.Analogous spontaneous organization of BIFIDO cells at the core of the electrosprayed microcapsules was observed utilizing different voltages with negative polarity at the needle (Fig. 4).On the contrary, when a positive polarity was applied at the needle, the cells were distributed towards the surface of the electrosprayed microcapsules (Fig. 4).A suggested graphic model representing the cell organization within the microcapsules influenced by the applied polarity, is depicted in Fig. 5.
The CLSM images of electrosprayed maltodextrin-BIFIDO dispersions at different pH values are presented in Fig. 6.Herein, negatively surface charged BIFIDO cells at pH = 4-8 were organized at the core of the microcapsules when negative polarity electric field was applied at the needle.Cells at pH 1, with slightly positive surface charges, were distributed closer to the surface of the microcapsules when positive polarity was applied.Thus, the encapsulation of the BIFIDO cells and their location and organization within the electrosprayed microcapsules can be controlled by the electrostatic forces between the charged cells and the DC electric field.Moreover, it is to note that as the probiotics exhibited similar hydrophobic character at pH 1 to 6, as discussed previously.Therefore the organization of the cells within the electrosprayed microcapsules was mainly attributed to their surface charge, rather than due to the cell's surface hydrophobic/hydrophilic character.

Glass transition temperature of electrosprayed microcapsules
The effect of voltage and polarity of the electric field, on the glass transition temperature (Tg) of the maltodextrin -BIFIDO electrosprayed microcapsules was also assessed (Fig. 7).The Tg is related to the moisture content of the electrosprayed microcapsules (Tg increases with decreasing the moisture content), and to the subsequent stability and viability of the encapsulated cells (Drake et al., 2018;Haque & Roos, 2004;Terpou et al., 2019).Removal of sufficient water and formation of non-crystalline glassy solid matrix is required in order to achieve stability during storage of the cells in the dried state (Drake et al., 2018).
The glass transition temperature (Tg) of the electrosprayed maltodextrin microcapsules without probiotic cells remained nearly constant with increasing electrospray voltage, both for positive and negative polarity.Slightly higher Tg values of maltodextrin microcapsules were observed using the positive polarity electric field.On the contrary, the electrosprayed maltodextrin -BIFIDO microcapsules at pH 6 (Fig. 7) presented a maximum Tg value at |20 kV| ((− 15 kV)(+5 kV)), and a subsequent decrease of the Tg with increasing electrospraying voltage.Moreover, the maltodextrin -BIFIDO microcapsules processed using a negative polarity were characterized by much higher Tg values than the microcapsules produced using a positive polarity.Particularly, the maltodextrin -BIFIDO microcapsules produced using a negative polarity of (− 15 kV)(+5 kV) were characterized by a Tg of 68.21 ± 1.66 • C, which is a Tg value significantly higher than the Tg(s) of the electrosprayed microcapsules processed at other voltages under negative polarity.When a positive polarity (+15 kV)(-5 kV) was used, the Tg was about 10 • C lower than the microcapsules produced using the same voltage of negative polarity (− 15 kV)(+5 kV).As described previously, at pH 6, the negative surface charged probiotic cells electrosprayed using a negative polarity electric field were repelled and 'organized' in the core of the microcapsules, while the solvent accumulated near to the highly charged microcapsule surface.Thus, an enhanced evaporation of the solvent during electrospray is expected when a negative polarity electric field is applied, comparatively to the positive one.This enhanced evaporation leads to a lower moisture content for the negative polarity electrosprayed maltodextrin -BIFIDO microcapsules, and subsequent higher Tg values in comparison to the corresponding microcapsules processed using positive polarity.The effect of polarity on the water content and Tg is in agreement with FT-IR analysis on BIFIDO cells.Thus, the different electric field voltages and polarities affect the thermal properties (glass transition) of the electrosprayed microcapsules due to the presence of the surface charged probiotics.

Stability and cell viability of microencapsulated probiotic cells
As shown in Fig. 8, the probiotics encapsulated using negative polarity, presented a significantly higher viability (~7.3 times higher, 0.06 log loss), compared to the encapsulated probiotics using positive polarity (0.44 log loss), even after 3 days of storage at 25 • C and 35% RH.Similar trend in viability (about 2.4 and 1.5 times higher) was monitored even after 7 and 14 days, respectively.Non encapsulated BIFIDO had 29.5, 9, and 5.5 times lower viability than cell encapsulated using negative polarity after 3, 7, and 14 days of storage, respectively.Moreover, it is to note, that the encapsulation efficiency of the microcapsules was similar for both polarities (92.9 ± 2.1% and 92.4 ± 1.2% for positive and negative polarities, respectively).
Thus, the encapsulated maltodextrin -BIFIDO electrosprayed using negative polarity, showed higher viability up to about 14 days of storage in comparison to the cells electrosprayed using positive polarity.This arises from the organization of the cells in the core of the electrosprayed microcapsules using negative polarity, and the lower moisture content (and water activity) of these microcapsules, which certainly provide better stability and viability conditions for the encapsulated cells.Hence, the stability and the viability of the encapsulated probiotics can be effectively controlled utilizing the polarity of the external electric field (Dima, Stubbe, Mendes, & Chronakis, 2022).

Conclusions
In the present study, a novel method allowing the control of the location and organization of the encapsulated probiotics within electrosprayed microcapsules, is presented.The charge polarity of the needle of a DC electric field, directed the negatively surface charged Bifidobacterium animalis subsp.lactis (BIFIDO) probiotic cells, either to the core or towards the surface of the maltodextrin microcapsules, as documented by confocal microscopy images.
Charge polarity applied to the electrospray needle controlled not only the organization of the probiotic cells within the liquid jet, but as well the mass transfer, the solvent evaporation and the physicochemical properties (e.g., glass transition) of the microcapsules.The probiotics encapsulated using negative polarity, presented significantly higher glass transition values (nearly 10 • C) than the microcapsules produced using a positive polarity, and much greater viability (~7.3 times higher), compared to the encapsulated probiotics using positive polarity, even after 3 days of storage at RH of 30% and 25 • C.This novel method enhanced the viability and stability of probiotic cells encapsulated by electrospray.The viability of the encapsulated cells was significantly enhanced when the cells were located at the core of the microcapsules, compared to the case where the probiotics were distributed towards the surface of the microcapsules.Further work would be valuable to evaluate the selective localization, by electrospraying, of various strains of probiotic cells.In addition, the effect of charged density biopolymers (i.e., polyelectrolytes) or salts, that can change the charge density of the solution could be assessed, which could further optimize the organization and distribution of the cells inside electrosprayed microcapsules.

Fig. 3 .
Fig. 3. FT-IR spectra of BIFIDO probiotic cells with and without the influence of an external electric field.

Fig. 4 .
Fig. 4. CLSM micrographs of BIFIDO cells' (pH = 6) distribution inside microcapsules electrosprayed with different applied voltage and polarities.The circles represent the edge of the microcapsules, and the graphs signify the BIFIDO fluorescence intensity profile across the diameter of the microcapsules.

Fig. 5 .
Fig. 5. Proposed model of the negatively charged cells' organization within the electrosprayed microcapsules, using negative polarity (a) and positive polarity (b) at the electrospraying nozzle.

Fig. 6 .
Fig. 6.CLSM micrographs of BIFIDO cells at different pH values and their distribution inside microcapsules electrosprayed using both positive and negative polarities at applied voltage of |20 kV|.The circles represent the edge of the microcapsules, and the graphs signify the BIFIDO fluorescence intensity profile across the diameter of the microcapsules.