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Biosynthesis of dextran by Weissella confusa and its In vitro functional characteristics
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Biosynthesis of dextran by Weissella confusa and its In vitro functional characteristics Irina Rosca a , Anca Roxana Petrovici a,∗ , Dragos Peptanariu a , Alina Nicolescu a , Gianina Dodi b , Mihaela Avadanei a , Iuliu Cristian Ivanov c , Andra Cristina Bostanaru d , Mihai Mares d , Diana Ciolacu a,∗ a
“Petru Poni” Institute of Macromolecular Chemistry, 700487, Iasi, Romania SCIENT - Research Center for Instrumental Analysis, 060114, Bucharest, Romania c Regional Oncology Institute, 700483, Iasi, Romania d “Ion Ionescu de la Brad” University, 700490, Iasi, Romania b
a r t i c l e
i n f o
Article history: Received 21 July 2017 Received in revised form 25 September 2017 Accepted 9 October 2017 Available online xxx Keywords: Exopolysaccharide UHT milk NMR GPC 16S rDNA Cytotoxicity
a b s t r a c t The aim of this study was to monitor the influence of the fermentation conditions on the exopolysaccharides (EPS) biosynthesis. For this, different culture media compositions were tested on an isolated lactic acid bacteria (LAB) strain, identified by 16S rDNA sequence as being Weissella confusa. It was proved that this bacterial strain culture in MRS medium supplemented with 80 g/L sucrose and dissolved in UHT milk produced up to 25.2 g/L of freeze-dried EPS, in static conditions, after 48 h of fermentative process. Using FTIR and NMR analysis, it was demonstrated that the obtained EPS is a dextran. The thermal analysis revealed a dextran structure with high purity while GPC analysis depicted more fractions, which is normal for a biological obtained polymer. A concentration up to 3 mg/mL of dextran proved to have no cytotoxic effect on normal human dermal fibroblasts (NHDF). Moreover, at this concentration, dextran breaks up to 70% of the biofilms formed by the Candida albicans SC5314 strain, and has no antimicrobial activity against standard bacterial strains. Due to their characteristics, these EPS are suitable as hydrophilic matrix for controlled release of drugs in pharmaceutical industry. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Exopolysaccharides (EPS) are extracellular biomacromolecules, who have received special attention in the last decade due to numerous applications, such as in the pharmaceutical, medical and food industries. These biopolymer are generally recognized as safe (GRAS) for human health and are ideal candidate for food industry, being used as gelling, emulsifying, stabilizing and thickening agents [1]. EPS produced by lactic acid bacteria (LAB) have immune-modulatory, antitumor, anti-inflammatory and immune-stimulator effects, and act as oxidizing agents. Their structural properties determine the biological activity and technological applications [2], therefore more knowledge about EPS isolation techniques, chemical composition and structure are of interest for the potential applications.
∗ Corresponding authors at: “Petru Poni” Institute of Macromolecular Chemistry, Centre of Advanced Research in Bionanoconjugates and Biopolymers Department, 41A Grigore Ghica-Voda Alley, 700487, Iasi, Romania. E-mail addresses: [email protected] (A.R. Petrovici), [email protected] (D. Ciolacu).
Dextran is an EPS biosynthesized by several types of lactic acid bacteria, such as Leuconostoc mesenteroides, Lactobacillus brevis, Streptococcus mutants and Weissella confusa. Depending on the strain and the composition of the culture medium, it can be obtained with a low or high molecular weight (10–150 kDa) [3]. This biopolymer is a very complex glycan composed of units of ␣-d-glucose with ␣-(1 → 6) linear bonds and different percentages of ␣-(1 → 4) ␣-(1 → 3) and ␣-(1 → 2) branches. The branching degree depends on the nature of dextransucrase biosynthesized by the microbial strain. This enzyme hydrolyses the glycosidic bond in sucrose, releasing glucose which is further use in the biosynthesis of dextran and fructose. Both of them are involved in different metabolic processes [4]. EPS molecules are associated with one another, but can also interact with other molecules situated in their proximity, such as proteins, lipids, inorganic ions or other macromolecules found on the cell membrane surface [5]. The study of dextran obtained from the fermentation of Weissella spp. strains, especially Weissella confusa (W. confusa), has recently entered into the attention of the scientific community. It was only in 2012 when the gene encoding (LBAE K39) of W. confusa biosynthesizes dextransucrase, which has a size of 180 kDa, was completely sequenced, thus becoming available for various appli-
https://doi.org/10.1016/j.ijbiomac.2017.10.048 0141-8130/© 2017 Elsevier B.V. All rights reserved.
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cations [6]. W. confusa is known to biosynthesis high amounts of non-digestible oligosaccharides and mainly dextran as extracellular polysaccharides [7–9]. These polymers are receiving increased attention because of both potential application as probiotics and wide range of industrial uses, especially for bakeries [10–12] and for the production of cereal-based fermented functional beverages [13]. Moreover, dextran has different applications in pharmaceutical and light industries [14]. It is used in medicine as antithrombotic agents, reducing blood viscosity and increasing its volume [15]. Dextran-based nanoparticles have applications in targeted drug therapy, where dextran is used as coating, or it can be functionalized with other compounds in order to obtain specific properties [16]. Dextran is also used as coating for protection against oxidation of metal nanoparticles [17]. The aim of this study was to find the optimum conditions for obtaining high EPS amounts by fermentative methods. For this purpose, the compositions of the culture medium were varied, and the obtained polymers were extracted, purified and characterized. The used bacterial strain was isolated from commercial yoghurt and identified by molecular biology techniques as Weissella confusa. In order to select the potential medical applications of the biopolymer we conducted a series of biological assays: determination of cytotoxicity on fibroblasts, antibacterial testing and the antifungal susceptibility test against one of the most known pathogen nowadays, Candida albicans. 2. Materials and methods 2.1. Microorganisms The lactic acid bacteria strain coded PP29 was isolated from Romanian commercial yoghurt in the laboratories of Centre of Advanced Research in Bionanoconjugates and Biopolymers (IntelCentru) of the “Petru Poni” Institute of Macromolecular Chemistry, Iasi, and kept at −80 ◦ C in Man Rogosa Sharpe medium (MRS) supplemented with 20% glycerol. 2.2. Molecular identification of the bacterial strain The PP29 LAB strain was identifying by 16S rRNA gene sequence analysis. The bacterial DNA was extracted from 24 h cultures grown on MRS agar plates at 30 ◦ C. DNA purification was made in duplicate with the Genomic DNA Purification Kit (Thermo Scientific) and the elution was made in 100 L nuclease free water. The spectrophotometric quantification was made in NanoDrop. A 10 ng/L dilution was made for both samples and 5 L were used for the PCR reactions. The primer pair: 27F 5 AGAGTTTGATCMTGGCTCAG 3 and 1492R 5 TACGGYTACCTTGTTACGACTT 3 were used for 16 S rDNA gene amplification of [18]. The PCR reactions were performed in ® a total volume of 25 L (12.5 L GoTaq Master Mix, 2.5 L forward primer 10 mM, 2.5 L reverse primer 10 mM, 2.5 L nuclease free water and 5 L DNA). The reaction mixture was first incubated for 10 min at 95 ◦ C and then cycled for 35 times 30 s at 95 ◦ C followed by one cycle of 4 min at 60 ◦ C. The electrophoresis migration of the products was conducted in 2% agarose gel electrophoresis in order to verify the reaction and the possible contaminations. The PCR products were purified with Wizard SV Gel and PCR CleanUp Kit (Promega) and a new electrophoresis migration was made with the final products in order to verify the purification and to quantify the quantity to be sequenced. Sequencing reactions were prepared using primers 27F/1492R. DNA sequencing was carried out by using GenomeLabTM Dye Terminator Cycle Sequencing with Quick Start Kit (Beckman Coulter). The sequencing products were purified with glycogen, sodium acetate and Na2 -EDTA, as indi-
cated by the sequencing kit and were migrated in the sequencer GenomeLabTM GeXP Genetic Analysis System with the migration program LFR-a. The sequences were interpreted, exported in Chromas Lite program (version 2.01) and examined with deposited sequences by using nucleotide BLAST program (NCBI, http://www. ncbi. nlm.nih.gov) and BLAST search tools [19]. 2.3. Fermentations conditions The strain isolation and purification was made in Petri dishes using MRS agar supplemented with 1% CaCO3 , incubated at 30 ◦ C for 48 h [20]. For experimental fermentations were used three culture media denoted MDI, MDII and MDIII. The culture medium compositions were the following: MDI: MRS (55.3 g/L), fructose (40 g/L), glucose (40 g/L), dissolve in distillate water; MDII: MRS (55.3 g/L) and sucrose (80 g/L) dissolved in distillate water; MDIII: MRS (55.3 g/L) and sucrose (80 g/L) dissolved in UHT milk (with the following nutritional information/100 mL: energy value − 44 kcal, proteins − 3 g, lipids − 1.5 g (saturated fatty acids − 0.9 g), sugar − 4.5 g, calcium ions − 120 mg). All the fermentations were made in static (S) and dynamic conditions (D). The culture medium was sterilized at 110 ◦ C for 30 min and inoculated with 30% of fresh inoculum (24 h) with A600nm of 0.5 [21]. The samples were incubated at 33 ◦ C for 48 h without pH correction during fermentation, under static and dynamic conditions (at 100 rpm in an orbital incubator) [22]. Before performing the EPS extraction and purification, the culture was heated at 100 ◦ C for 15 min in order to inactivate the enzymatic equipment capable of degrading the biopolymer [23]. 2.4. EPS isolation and purification The cells and proteins were removed by precipitation with 20% trichloroacetic acid (TCA) followed by centrifugation at 10,000 rpm for 10 min at 4 ◦ C. The EPS were separated by precipitation with three volumes of cold ethanol for 24 h at 4 ◦ C [22]. The EPS were collected by centrifugation at 12,000 rpm for 15 min at 4 ◦ C, washed with ethanol three times, resuspended in double distilled water (DDW) and subjected to dialysis through a membrane with a porosity of 14,000 Da against DDW for three days at room temperature. For analysis, the EPS samples were coded as it follows: I-PP-29 − the PP29 strain fermented in MDI in dynamic conditions, I-PP-29-S − the PP29 strain fermented in MDI in static conditions, II-PP-29 − the PP29 strain fermented in MDII in dynamic conditions, II-PP-29S − the PP29 strain fermented in MDII in static conditions, III-PP-29 − the PP29 strain fermented in MDIII in dynamic conditions, III-PP29-S − the PP29 strain fermented in MDIII in static conditions and subjected to freeze drying process. The amount of the polymer was expressed in g of dry biopolymer per liter culture medium [24]. 2.5. Gel permeation chromatography analysis To estimate the distribution of the EPS molar masses, gel permeation chromatography (GPC) was used. Measurements (weight average of molecular weight number (Mw), the average molecular number (Mn) and the polydispersity index (PDI)) were recorded on a Polymer Laboratories System (PL-GPC 120, Varian) equipped with refractive index detector and three PL-aquagel packed columns filled with beads of porous gel composed of vinyl copolymers (cross-linked) with polymeric hydroxyl functionality (8 m particle size and 20, 40 and 60 Å pore type), connected in series and placed in the column oven at 30 ◦ C. The samples concentration was 0.8 mg/mL in H2 O (filtered through a cellulose filter with 0.45 m pore size) and 0.02 M NaNO3 solution was used as mobile phase with a flow rate of 1.0 mL/min. The calibration curve was made
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Fig. 1. The infrared spectra of EPS samples (from top to bottom).
on P-82 standard pullulan (Shodex Denko) with Mw 0.6 × 104 , 1 × 104 , 2.17 × 104 , 4.88 × 104 and 11.3 × 104 , 104 × 21, 36, 6 × 104 , 80.5 × 104 g/mole in H2 O, and 100 L injection volume was used. Data recording and processing were made with Cirrus GPC online and offline software. 2.6. Fourier-transform infrared spectroscopy (FTIR) The FTIR spectra were recorded in transmission on a Bruker Vertex 70 spectrometer (Bruker Optics, Germany) at a resolution of 2 cm−1 , on KBr pellets and for the data processing OPUS 6.5 software (Bruker Optics, Germany) was used. The interest regions were baseline-corrected by using the interactive concave rubber band method. In the curve fitting procedure, the position and the estimated number of the sub-bands were determined by using the second derivative spectrum. The mixed Gauss-Lorentz functions were used, where the peak position was maintained fixed and the intensity, shape and width were considered as variable parameters. 2.7. Nuclear magnetic resonance studies For the NMR analysis, the EPS sample was dissolved in deuterated water with TSP as internal standard. The spectra have been recorded at room temperature (approx. 24 ◦ C). Chemical shifts are reported in ppm and referred to TSP (ref. 1 H 0.00 ppm and 13 C 0.00 ppm). The NMR spectra had been recorded on a Bruker Avance III 400 MHz Spectrometer, equipped with a 5 mm inverse detection z-gradient probe, operating at 400.1 and 100.6 MHz for 1 H and 13 C nuclei. Total correlation spectroscopy (TOCSY) and heteronuclear single quantum coherence (HSQC) experiments have been recorded using standard pulse sequences in the version with z-gradients, as delivered by Bruker with TopSpin 2.1 PL6 spectrometer control and processing software. 2.8. Thermal analysis measurements − thermogravimetry (TGA) and differential scanning calorimetry (DSC) The thermogravimetric (TG) analysis of EPS samples were performed on STA 449F1 Jupiter NETZSCH equipment. DSC measurements were performed on a Maia F3 200 DSC device (Netzsch, Germany) using 10 mg of freeze-dried sample. Measurements were carried out in the 30–700 ◦ C temperature range, applying a heating rate of 10 ◦ C min−1 . Nitrogen purge gas was used as inert atmosphere at a flow rate of 50 mL/min. Samples were heated in open
Fig. 2. The infrared spectra of I-PP-29 (blue trace) and III-PP-29 (red trace). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Al2 O3 crucibles. The device was calibrated for temperature and sensitivity with indium, according to standard procedure.
2.9. Biological tests 2.9.1. Cytotoxicity assay To assess the cytotoxicity, the CellTiter 96sAQueous One Solution Cell Proliferation Assay, MTS (Promega) was performed on normal human dermal fibroblasts, NHDF (PromoCell), by following the protocol recommended by the manufacturer [25]. The cells were grown in DMEM: F12 medium (Lonza) supplemented with 10% fetal bovine serum (Gibco), 1 mM sodium pyruvate (Lonza) and 1% penicillin–streptomycin–amphotericin B mixture (10 K/10 K/25 mg in 100 mL, Lonza). The same medium was used to prepare serial dilutions of the EPS to be tested. To ensure cell adhesion, NHDF were seeded at 5 × 103 cells per well in 96-well tissue culture plates and incubated for 24 h at 37 ◦ C under a humidified atmosphere with 5% CO2 . The medium was then replaced with 100 mL per well of the previously prepared EPS dilutions and the plates were further incubated for 20 h. The MTS reagent was then added to each well and, after final 4 h incubation, the absorbance at 490 nm was recorded with an EnVisions plate reader (PerkinElmer). The experiment included 6 replicates and was repeated 3 times.
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Fig. 3. NMR spectra of III-PP-29 EPS sample produced by W. confusa, recorded in D2 O. (A) 1 H NMR spectra for the dextran produced by fermentation in comparison with 1 H NMR spectra of a commercial dextran, (B) 1 H, 1 H TOCSY spectrum, (C) 13 C NMR spectra of III-PP-29 sample, (D) 1 H, 13 C HSQC spectra of III-PP-29 sample.
2.9.2. The antifungal activity The in vitro susceptibility testing was performed following the EUCAST EDef 7.2 guidelines using Candida albicans SC5314 strain, which is known to form abundant biofilms with a complex structure. The stock solutions were prepared using water as a solvent. The active principle concentration range of the tested antifungals was 0.0156–3 mg L−1 . The minimum inhibitory concentrations (MICs) were determined using spectrophotometric technique on an iMark Microplate Reader (Bio-Rad Laboratories, USA) at 405 nm, after 24 h of incubation at 35 ◦ C. 2.9.3. Antimicrobial activity The antimicrobial activity was considered on three different reference strains, i.e. Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6583 and Pseudomonas aeruginosa ATCC27853. The antimicrobial activity was measured by the agar disk diffusion method which supposes the addition of the EPS on the culture medium pre-inoculated with the microbial suspension (0.5 McFarland standards optical turbidity in sterile saline solution, yielding a suspension containing approximately 1 × 108 CFU mL−1 for all three microorganisms), and measuring of the clear zone caused by growth inhibition around the film disks after 24 h of incubation. 3. Results and discussions 3.1. The influence of the fermentative conditions on the EPS biosynthesis yield The resulted sequences from PP29 LAB strain DNA sequencing showed 98.4% identities with those available in the nucleotide BLAST data base and led to the identification of the Weissella confusa bacterial species. The fermentations of the W. confusa strain in three different culture medium compositions, in static and dynamic conditions, lead
Table 1 The EPS amount extracted from the culture medium. Fermentations conditions
I-PP-29, g/L
II-PP-29, g/L
III-PP-29, g/L
Static fermentations Dynamic fermentations
– 2.8
– 5.18
25.2 17.4
to different amounts of EPS biosynthesized after 48 h of fermentative processes (Table 1). Our observations confirmed the literature data according to which the amount and the properties of the EPS depend entirely on the composition of the culture medium and on the fermentation conditions [26]. Dextran production by W. confusa is affected profoundly by nitrogen and carbon sources, and by inorganic salts present in the culture medium [27]. MDI and MDII in static conditions had inhibitory effect on EPS biosynthesis due to the low oxygenation of the culture media. In the case of dynamic fermentations, after 48 h we obtained small amounts of EPS. For MDI, the MRS carbon source was supplemented with fructose and glucose, which are the components of sucrose, but there were obtained only 2.8 g/L EPS. This denoted that the LAB enzymatic system does not recognized the components as sucrose but as single sugars. For the MDII, the MRS carbon source was supplemented with sucrose, and after 48 h of fermentation there were obtained almost double amounts of EPS (5.18 g/L EPS), which means that the proper sugar was sucrose. The MDIII culture medium was very productive, especially in static conditions there were obtained 25.2 g/L EPS. This amount was obtained due to the presence in the culture medium of milk, which supplements the MRS nitrogen source, and also due to the calcium ions which enhances the viscosity of dextran [28]. 3.2. Gel permeation chromatography analysis (GPC) In order to characterize the EPS, gel permeation chromatography was used to obtain information on the molecular weight
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I. Rosca et al. / International Journal of Biological Macromolecules xxx (2017) xxx–xxx Table 2 Average molecular number (Mn), average molecular weight (Mw), and polydispersity index (PDI) of the EPS biosynthesis. Sample
fraction
Mn, Da
Mw, Da
PDI
I-PP-29
1 2 1 2 1 2 3
2.4 * 105 4.5 * 104 7.4 * 105 8.7 * 104 9.9 * 105 1.4 * 105 1.3 * 104
4.5 * 105 6.6 * 104 8.7 * 105 1.2 * 105 1.2 * 106 2.5 * 105 1.6 * 104
1.86 1.46 1.17 1.40 1.24 1.78 1.25
II-PP-29 III-PP-29-S
expressed in fractions of different weight. Molecular weight is a very important variable that provides additional information on the EPS physical properties, their molecule being dependent on the ionic strength and therefore on their concentration in solution. The weight average of the numerical molecular weight (Mw) and the average molecular number (Mn) were estimated by comparison with standard pullulan calibration curve. Because the chromatographic method allows selecting molecules by size, an important parameter is the average molecular weight that can reasonably approximate the molecular weight of the polymer fragments separated at the spine level. The obtained values for the EPS compounds extracted are presented in Table 2. Analyzing the results, it can be concluded that the numeric molecular mass is influenced by the fermentation process, a phenomenon that determines the proportional increase of the molecular weight of the parent compound. For the EPS extracted from MDI culture media it was obtained a biopolymer with Mn lower than the biopolymer extracted from MDII culture media supplemented with sucrose. This means that the sugar type is very important for the obtained EPS with different Mn. In the MDIII and static conditions (III-PP-29-S) there were obtained three fractions with high molecular weight and an uniform PDI. Also, by supplementing the MRS nitrogen source, it was increased the amount and the molecular weight of the biosynthesized polymer comparatively with the fermentation made in MDII. 3.3. Fourier-transform infrared spectroscopy (FTIR) The FTIR spectra of samples I-PP-29 and II-PP-29, on one hand, and III-PP-29 and III-PP-29-S, on the other hand, were very similar with each other (Fig. 1). Within the spectra of samples I-PP-29 and II-PP-29, the overlapped absorption bands of hydroxyl stretching and NH amine stretching were noticed at 3394 cm−1 . The amide I and amide II bands (1668 cm−1 and 1545 cm−1 ) indicate the presence of proteic residues. The region between 1490 and 1308 cm−1 covers signals from the bending vibrations of CH, CH2 and OH groups. The broad band with the maximum around 1058 cm−1 is characteristics to glucopyranose fragments, being a superposition of vibrations given by the C O C stretching and C OH stretching and bending. Identification of glycosidic linkages, of the bonded and free hydroxyl groups in various positions was made with the help of the second derivative spectra. The sub-band at 1054 cm−1 , specific to ␣-d-glucoses, is mainly given by (C2-O2H) [29,30]. A sub-band observed at 1022 cm−1 can be assigned to the (C6-O6H) vibrations in H-bonded primary alcoholic groups. An intense peak at 1080 cm−1 arises from the overlapped (C6-O6H), ␦(C4-C5) and ␦ (C1-H) vibrations [30,31]. The medium-to-intense sub-band peaking at 1129 cm−1 is confidently assigned to (C4-O4H) stretching vibration [29]. The (C O C) in glycosidic linkages of ␣-d-glucopyranoses is observed at 1152 cm−1 and is further confirmed by the weaker subband at 832 cm−1 ((C O)), which indicates the ␣-(1→3) anomeric configuration [32,33]. The sub-band at 844 cm−1 is specific to ␣-
5
(1→6) linkages (Heyn, 1974). Therefore, the spectra of I-PP-29 and II-PP-29 are very close to that of dextran, but with traces of proteins and other polysaccharides. Instead, the FTIR spectra of III-PP-29 and III-PP-29-S do not show any signals from protein residues. The “sugar” band has a fine structure, where the vibrations corresponding to (C6 O6H· · ·) (1015 cm−1 ), (C3 O3H· · ·) (1040 cm−1 ), (C6-O6H) + ␦(C4-C5) (1080 cm−1 ), or as (C O C) and (C O) in glycosidic linkages (1155 and 840 cm−1 ) are clearly observed (Fig. 2). Comparison with a sample of pure dextran from commercial source (M = 40.000) led to the almost perfect matching of spectra. 3.4. NMR spectroscopy analysis In order to confirm the presence of dextran in the analysed sample and to obtain more structural details there were recorded several one- and two-dimensional NMR experiments. The 1 H NMR spectrum of III-PP-29-S (Fig. 3A) was recorded with suppression of the water signal, to reveal the less intense signals from the region 4.5-5.5 ppm. The most intense signals were assigned to the glucose units as it follows: ␦ = 3.53 (1H, t, J = 9.4 Hz, H-4,), 3.59 (1H, dd, J = 9.7, 3.1 Hz, H-2), 3.73 (1H, t, J = 9.3 Hz, H-3), 3.77 (1H, d, J = 9.8 Hz, H-6a or H-6b), 3.93 (1H, d, J = 9.1 Hz, H-5), 4.00 (1H, d, J = 7.1 Hz, H-6a or H-6b), 4.99 (1H, d, J = 2.9 Hz, H-1). The signal centered at 4.99 ppm has been previously assigned [34] to anomeric protons of ␣-(1 → 6)-linked glucose units, in the main chain. In the TOCSY spectrum presented in Fig. 3B it can be noticed the couplings of the anomeric proton from 4.99 ppm with H-2, H-3, H-4 and H-5 protons. These correlations confirmed that the investigated signals belong to the same spin system. In the anomeric spectral region, three less intense signals were observed: a doublet centered at 4.67 ppm (J = 7.8 Hz), another doublet centered at 5.23 ppm (J = 3.2 Hz) and the third doublet centered at 5.34 ppm (J = 3.7 Hz). The first two doublets belong to free glucose remained from the culture medium, 4.67 ppm − anomeric proton from beta glucose and 5.23 ppm − anomeric proton from alpha glucose. The third doublet, from 5.34 ppm, has been previously assigned [34] to anomeric protons of ␣-(1 → 3)-linked glucose units, as branches of the main chain. The rest of the signals for the branch glucose units could not be assigned because of their low intensity and overlap with the signals from the main chain units. Based on the two anomeric signals and the ratio of their integrals, the percentage of glycosidic linkages was establish as being 96.2% for ␣-(1 → 6) and 3.8% for ␣-(1 → 3). Information about carbon chemical shifts was obtained from the 1D 13 C NMR and 2D 1 H, 13 C HSQC spectra (Fig. 3C and D). Based on the 1 H–13 C correlations from 1 H, 13 C HSQC spectrum, there were assigned the six intense signals as it follows: 68.5 (C-6), 72.5 (C-4), 73.1 (C-5), 74.4 (C-2), 76.4 (C-3), 100.7 (C-1). All these signals belong to ␣-(1 → 6)-linked glucose units, in the main chain. From the same HSQC spectrum it was assigned the C-1 from the branch ␣-(1 → 3)-linked glucose units at 102.6 ppm. The obtained NMR data indicated the obtaining of an exopolysaccharide. Based on the reported data from literature [34] it was deduced that the obtained exopolysaccharide is dextran, results supported by the FTIR analysis as well. 3.5. Thermal analysis measurements − thermogravimetry (TGA) and differential scanning calorimetry (DSC) By analyzing the thermal degradation curves, it can notice that all four samples displayed comparable thermal degradation tendency in two stages (Fig. 4, Table 3). Since there is a direct relationship between carboxyl group content and the material hydrophobicity, the initial material moisture content is given by the increased carboxyl group quantity. Regard-
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Fig. 4. TG and DTG curves of the studied samples.
Table 3 Thermal characteristics of the studied samples. Sample
I-PP-29 ◦
Stage I Stage II Wrez , %
II-PP-29 ◦
◦
◦
III-PP-29 ◦
◦
◦
III-PP-29-S ◦
◦
Ti , C
Tmax , C
Tf , C
w , %
Ti , C
Tmax , C
Tf , C
w , %
Ti , C
Tmax , C
Tf , C
w , %
Ti , ◦ C
Tmax , ◦ C
Tf , ◦ C
w , %
57 260 40.56
71 304
95 332
6.3 53.14
57 265 37.33
76 302
88 331
7.08 55.59
48 269 27.19
78 307
108 322
4.77 68.04
55 267 21.37
80 305
114 323
8.1 70.53
Ti − initial thermal degradation temperature; Tmax − temperature corresponding to the maximum rate of decomposition for each stage, evaluated from the peaks of the DTG curves; Tf − final thermal degradation temperature; Wm − weight loss rate corresponding to the Tmax values; Wrez − percentage of residue remained at the end of thermal degradation (700 ◦ C).
3.6. Biological tests
Fig. 5. Second heating curves of the studied samples.
ing to this, the samples structures suffer physical and chemical moisture loss during the first thermal degradation stage, between 71 and 80 ◦ C with a mass loss from 4.7-8.1% (Table 3) [35]. The second and principal degradation step is from 302 to 307 ◦ C recording an important mass loss of 53.17–70.53% [36], corresponding to random polymer backbone cleavages and depolymerisation, with remaining of inorganic material [37]. Fig. 5 depicts the second heating scans of the studied samples. All samples showed a glass transition temperature domain (Tg) between 217 and 223 ◦ C. This aspect is in accordance with the data reported by Scandola [38] reporting a Tg value of 223 ◦ C for natural dextran. The producing of a more amorphous (i.e. enhanced segmental chain mobility) dextran could explain the lower Tg domain values, down to 217 ◦ C.
3.6.1. Cytotoxicity assay By testing the biocompatibility it was proved that III-PP-29-S sample, dextran-based, does not have cytotoxic effect on fibroblasts up to a maximum concentration of 3 mg/mL, when cell viability drops below 70% (Fig. 6). Other results have been reported for EPS based on glucose and mannose, biosynthesized by Lactobacillus plantarum, where the fibroblast cell viability decreased to 70%, at a concentration of 100 mg/mL EPS [39]. These differences between results may be related to the different type of lactic acid bacteria strain and on the different type of EPS resulted from fermentation. For example, EPS based on galactose and glucose at a concentration of 5–50 g/L did not have cytotoxic effect on the normal intestinal cells [40]. EPS based on galactose and glucose, at a dosage of 5–50 g/mL showed no cytotoxic effect on normal intestine 407 cells [40]. 3.6.2. The antifungal activity Antifungal susceptibility testing using the species Candida albicans showed that III-PP-29-S can break by up to 70% of the biofilms formed by the pathogenic fungus to a maximum concentration of 3 mg/mL (Fig. 7), results that have not been reported yet in the literature. 3.6.3. EPS antimicrobial activity EPS based on dextran showed no antimicrobial activity against the reference strains. However, EPS based on small amounts of sulphate (1.43%), uronic acids (2.98%), proteins (4.08%), galactose, glucose and mannose, in a concentration of 4 mg/L, inhibited 65.82% of S. aureus biofilm formation, 43.58% P. aeruginosa and 33.41% of E. coli [41]. In the same time, EPS based on glucose had antibacterial activity against E. coli ATCC8739 and S. aureus ATCC6538 [42]. How-
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Fig. 6. Relative cell viability at 48 h. The relative cell viability of NHDF cells was determined by MTS assay after 48 h of treatment with EPS. Data is represented as means ± SD (n = 3).
Fig. 7. EPS antifungal assay.
ever, EPS produced by this particular strain have no antimicrobial effect against the reference pathogens. 4. Conclusions FTIR and NMR analyses confirm the dextran structure of the EPS extracted from experiment III-PP-29-S. According to TGA, the polymer has a very stable structure with high purity and its amorphous properties were demonstrated by DSC analysis. Because the EPS proved to have antifungal properties, the next step will be to load together with this compound an antifungal drug in order to test its capacity to destroy the biofilm formation and the yeast cells of Candida albicans, which is nowadays an important health issue. Statement of conflict of interest The authors declare no competing financial interest. Acknowledgements This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCS − UEFISCDI, project number PN-II-RU-TE-2014-4-0558. PhD I. Ros¸ca and MD PhD D. Peptanariu are grateful also for the financial support from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 667387 WIDESPREAD 2-2014 SupraChem Lab. References [1] P.B. Devi, D. Kavitake, P.H. Shetty, Physico-chemical characterization of galactan exopolysaccharide produced by Weissella confusa KR780676, Int. J. Biol. Macromol. 93 (2016) 822–828. [2] D. Kavitake, P.B. Devi, S.P. Singh, P.H. Shetty, Characterization of a novel galactan produced by Weissella confusa KR780676 from an acidic fermented food, Int. J. Biol. Macromol. 86 (2016) 681–689.
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Biosynthesis of dextran by Weissella confusa and its In vitro functional characteristics Irina Rosca a , Anca Roxana Petrovici a,∗ , Dragos Peptanariu a , Alina Nicolescu a , Gianina Dodi b , Mihaela Avadanei a , Iuliu Cristian Ivanov c , Andra Cristina Bostanaru d , Mihai Mares d , Diana Ciolacu a,∗ a
“Petru Poni” Institute of Macromolecular Chemistry, 700487, Iasi, Romania SCIENT - Research Center for Instrumental Analysis, 060114, Bucharest, Romania c Regional Oncology Institute, 700483, Iasi, Romania d “Ion Ionescu de la Brad” University, 700490, Iasi, Romania b
a r t i c l e
i n f o
Article history: Received 21 July 2017 Received in revised form 25 September 2017 Accepted 9 October 2017 Available online xxx Keywords: Exopolysaccharide UHT milk NMR GPC 16S rDNA Cytotoxicity
a b s t r a c t The aim of this study was to monitor the influence of the fermentation conditions on the exopolysaccharides (EPS) biosynthesis. For this, different culture media compositions were tested on an isolated lactic acid bacteria (LAB) strain, identified by 16S rDNA sequence as being Weissella confusa. It was proved that this bacterial strain culture in MRS medium supplemented with 80 g/L sucrose and dissolved in UHT milk produced up to 25.2 g/L of freeze-dried EPS, in static conditions, after 48 h of fermentative process. Using FTIR and NMR analysis, it was demonstrated that the obtained EPS is a dextran. The thermal analysis revealed a dextran structure with high purity while GPC analysis depicted more fractions, which is normal for a biological obtained polymer. A concentration up to 3 mg/mL of dextran proved to have no cytotoxic effect on normal human dermal fibroblasts (NHDF). Moreover, at this concentration, dextran breaks up to 70% of the biofilms formed by the Candida albicans SC5314 strain, and has no antimicrobial activity against standard bacterial strains. Due to their characteristics, these EPS are suitable as hydrophilic matrix for controlled release of drugs in pharmaceutical industry. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Exopolysaccharides (EPS) are extracellular biomacromolecules, who have received special attention in the last decade due to numerous applications, such as in the pharmaceutical, medical and food industries. These biopolymer are generally recognized as safe (GRAS) for human health and are ideal candidate for food industry, being used as gelling, emulsifying, stabilizing and thickening agents [1]. EPS produced by lactic acid bacteria (LAB) have immune-modulatory, antitumor, anti-inflammatory and immune-stimulator effects, and act as oxidizing agents. Their structural properties determine the biological activity and technological applications [2], therefore more knowledge about EPS isolation techniques, chemical composition and structure are of interest for the potential applications.
∗ Corresponding authors at: “Petru Poni” Institute of Macromolecular Chemistry, Centre of Advanced Research in Bionanoconjugates and Biopolymers Department, 41A Grigore Ghica-Voda Alley, 700487, Iasi, Romania. E-mail addresses: [email protected] (A.R. Petrovici), [email protected] (D. Ciolacu).
Dextran is an EPS biosynthesized by several types of lactic acid bacteria, such as Leuconostoc mesenteroides, Lactobacillus brevis, Streptococcus mutants and Weissella confusa. Depending on the strain and the composition of the culture medium, it can be obtained with a low or high molecular weight (10–150 kDa) [3]. This biopolymer is a very complex glycan composed of units of ␣-d-glucose with ␣-(1 → 6) linear bonds and different percentages of ␣-(1 → 4) ␣-(1 → 3) and ␣-(1 → 2) branches. The branching degree depends on the nature of dextransucrase biosynthesized by the microbial strain. This enzyme hydrolyses the glycosidic bond in sucrose, releasing glucose which is further use in the biosynthesis of dextran and fructose. Both of them are involved in different metabolic processes [4]. EPS molecules are associated with one another, but can also interact with other molecules situated in their proximity, such as proteins, lipids, inorganic ions or other macromolecules found on the cell membrane surface [5]. The study of dextran obtained from the fermentation of Weissella spp. strains, especially Weissella confusa (W. confusa), has recently entered into the attention of the scientific community. It was only in 2012 when the gene encoding (LBAE K39) of W. confusa biosynthesizes dextransucrase, which has a size of 180 kDa, was completely sequenced, thus becoming available for various appli-
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cations [6]. W. confusa is known to biosynthesis high amounts of non-digestible oligosaccharides and mainly dextran as extracellular polysaccharides [7–9]. These polymers are receiving increased attention because of both potential application as probiotics and wide range of industrial uses, especially for bakeries [10–12] and for the production of cereal-based fermented functional beverages [13]. Moreover, dextran has different applications in pharmaceutical and light industries [14]. It is used in medicine as antithrombotic agents, reducing blood viscosity and increasing its volume [15]. Dextran-based nanoparticles have applications in targeted drug therapy, where dextran is used as coating, or it can be functionalized with other compounds in order to obtain specific properties [16]. Dextran is also used as coating for protection against oxidation of metal nanoparticles [17]. The aim of this study was to find the optimum conditions for obtaining high EPS amounts by fermentative methods. For this purpose, the compositions of the culture medium were varied, and the obtained polymers were extracted, purified and characterized. The used bacterial strain was isolated from commercial yoghurt and identified by molecular biology techniques as Weissella confusa. In order to select the potential medical applications of the biopolymer we conducted a series of biological assays: determination of cytotoxicity on fibroblasts, antibacterial testing and the antifungal susceptibility test against one of the most known pathogen nowadays, Candida albicans. 2. Materials and methods 2.1. Microorganisms The lactic acid bacteria strain coded PP29 was isolated from Romanian commercial yoghurt in the laboratories of Centre of Advanced Research in Bionanoconjugates and Biopolymers (IntelCentru) of the “Petru Poni” Institute of Macromolecular Chemistry, Iasi, and kept at −80 ◦ C in Man Rogosa Sharpe medium (MRS) supplemented with 20% glycerol. 2.2. Molecular identification of the bacterial strain The PP29 LAB strain was identifying by 16S rRNA gene sequence analysis. The bacterial DNA was extracted from 24 h cultures grown on MRS agar plates at 30 ◦ C. DNA purification was made in duplicate with the Genomic DNA Purification Kit (Thermo Scientific) and the elution was made in 100 L nuclease free water. The spectrophotometric quantification was made in NanoDrop. A 10 ng/L dilution was made for both samples and 5 L were used for the PCR reactions. The primer pair: 27F 5 AGAGTTTGATCMTGGCTCAG 3 and 1492R 5 TACGGYTACCTTGTTACGACTT 3 were used for 16 S rDNA gene amplification of [18]. The PCR reactions were performed in ® a total volume of 25 L (12.5 L GoTaq Master Mix, 2.5 L forward primer 10 mM, 2.5 L reverse primer 10 mM, 2.5 L nuclease free water and 5 L DNA). The reaction mixture was first incubated for 10 min at 95 ◦ C and then cycled for 35 times 30 s at 95 ◦ C followed by one cycle of 4 min at 60 ◦ C. The electrophoresis migration of the products was conducted in 2% agarose gel electrophoresis in order to verify the reaction and the possible contaminations. The PCR products were purified with Wizard SV Gel and PCR CleanUp Kit (Promega) and a new electrophoresis migration was made with the final products in order to verify the purification and to quantify the quantity to be sequenced. Sequencing reactions were prepared using primers 27F/1492R. DNA sequencing was carried out by using GenomeLabTM Dye Terminator Cycle Sequencing with Quick Start Kit (Beckman Coulter). The sequencing products were purified with glycogen, sodium acetate and Na2 -EDTA, as indi-
cated by the sequencing kit and were migrated in the sequencer GenomeLabTM GeXP Genetic Analysis System with the migration program LFR-a. The sequences were interpreted, exported in Chromas Lite program (version 2.01) and examined with deposited sequences by using nucleotide BLAST program (NCBI, http://www. ncbi. nlm.nih.gov) and BLAST search tools [19]. 2.3. Fermentations conditions The strain isolation and purification was made in Petri dishes using MRS agar supplemented with 1% CaCO3 , incubated at 30 ◦ C for 48 h [20]. For experimental fermentations were used three culture media denoted MDI, MDII and MDIII. The culture medium compositions were the following: MDI: MRS (55.3 g/L), fructose (40 g/L), glucose (40 g/L), dissolve in distillate water; MDII: MRS (55.3 g/L) and sucrose (80 g/L) dissolved in distillate water; MDIII: MRS (55.3 g/L) and sucrose (80 g/L) dissolved in UHT milk (with the following nutritional information/100 mL: energy value − 44 kcal, proteins − 3 g, lipids − 1.5 g (saturated fatty acids − 0.9 g), sugar − 4.5 g, calcium ions − 120 mg). All the fermentations were made in static (S) and dynamic conditions (D). The culture medium was sterilized at 110 ◦ C for 30 min and inoculated with 30% of fresh inoculum (24 h) with A600nm of 0.5 [21]. The samples were incubated at 33 ◦ C for 48 h without pH correction during fermentation, under static and dynamic conditions (at 100 rpm in an orbital incubator) [22]. Before performing the EPS extraction and purification, the culture was heated at 100 ◦ C for 15 min in order to inactivate the enzymatic equipment capable of degrading the biopolymer [23]. 2.4. EPS isolation and purification The cells and proteins were removed by precipitation with 20% trichloroacetic acid (TCA) followed by centrifugation at 10,000 rpm for 10 min at 4 ◦ C. The EPS were separated by precipitation with three volumes of cold ethanol for 24 h at 4 ◦ C [22]. The EPS were collected by centrifugation at 12,000 rpm for 15 min at 4 ◦ C, washed with ethanol three times, resuspended in double distilled water (DDW) and subjected to dialysis through a membrane with a porosity of 14,000 Da against DDW for three days at room temperature. For analysis, the EPS samples were coded as it follows: I-PP-29 − the PP29 strain fermented in MDI in dynamic conditions, I-PP-29-S − the PP29 strain fermented in MDI in static conditions, II-PP-29 − the PP29 strain fermented in MDII in dynamic conditions, II-PP-29S − the PP29 strain fermented in MDII in static conditions, III-PP-29 − the PP29 strain fermented in MDIII in dynamic conditions, III-PP29-S − the PP29 strain fermented in MDIII in static conditions and subjected to freeze drying process. The amount of the polymer was expressed in g of dry biopolymer per liter culture medium [24]. 2.5. Gel permeation chromatography analysis To estimate the distribution of the EPS molar masses, gel permeation chromatography (GPC) was used. Measurements (weight average of molecular weight number (Mw), the average molecular number (Mn) and the polydispersity index (PDI)) were recorded on a Polymer Laboratories System (PL-GPC 120, Varian) equipped with refractive index detector and three PL-aquagel packed columns filled with beads of porous gel composed of vinyl copolymers (cross-linked) with polymeric hydroxyl functionality (8 m particle size and 20, 40 and 60 Å pore type), connected in series and placed in the column oven at 30 ◦ C. The samples concentration was 0.8 mg/mL in H2 O (filtered through a cellulose filter with 0.45 m pore size) and 0.02 M NaNO3 solution was used as mobile phase with a flow rate of 1.0 mL/min. The calibration curve was made
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Fig. 1. The infrared spectra of EPS samples (from top to bottom).
on P-82 standard pullulan (Shodex Denko) with Mw 0.6 × 104 , 1 × 104 , 2.17 × 104 , 4.88 × 104 and 11.3 × 104 , 104 × 21, 36, 6 × 104 , 80.5 × 104 g/mole in H2 O, and 100 L injection volume was used. Data recording and processing were made with Cirrus GPC online and offline software. 2.6. Fourier-transform infrared spectroscopy (FTIR) The FTIR spectra were recorded in transmission on a Bruker Vertex 70 spectrometer (Bruker Optics, Germany) at a resolution of 2 cm−1 , on KBr pellets and for the data processing OPUS 6.5 software (Bruker Optics, Germany) was used. The interest regions were baseline-corrected by using the interactive concave rubber band method. In the curve fitting procedure, the position and the estimated number of the sub-bands were determined by using the second derivative spectrum. The mixed Gauss-Lorentz functions were used, where the peak position was maintained fixed and the intensity, shape and width were considered as variable parameters. 2.7. Nuclear magnetic resonance studies For the NMR analysis, the EPS sample was dissolved in deuterated water with TSP as internal standard. The spectra have been recorded at room temperature (approx. 24 ◦ C). Chemical shifts are reported in ppm and referred to TSP (ref. 1 H 0.00 ppm and 13 C 0.00 ppm). The NMR spectra had been recorded on a Bruker Avance III 400 MHz Spectrometer, equipped with a 5 mm inverse detection z-gradient probe, operating at 400.1 and 100.6 MHz for 1 H and 13 C nuclei. Total correlation spectroscopy (TOCSY) and heteronuclear single quantum coherence (HSQC) experiments have been recorded using standard pulse sequences in the version with z-gradients, as delivered by Bruker with TopSpin 2.1 PL6 spectrometer control and processing software. 2.8. Thermal analysis measurements − thermogravimetry (TGA) and differential scanning calorimetry (DSC) The thermogravimetric (TG) analysis of EPS samples were performed on STA 449F1 Jupiter NETZSCH equipment. DSC measurements were performed on a Maia F3 200 DSC device (Netzsch, Germany) using 10 mg of freeze-dried sample. Measurements were carried out in the 30–700 ◦ C temperature range, applying a heating rate of 10 ◦ C min−1 . Nitrogen purge gas was used as inert atmosphere at a flow rate of 50 mL/min. Samples were heated in open
Fig. 2. The infrared spectra of I-PP-29 (blue trace) and III-PP-29 (red trace). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Al2 O3 crucibles. The device was calibrated for temperature and sensitivity with indium, according to standard procedure.
2.9. Biological tests 2.9.1. Cytotoxicity assay To assess the cytotoxicity, the CellTiter 96sAQueous One Solution Cell Proliferation Assay, MTS (Promega) was performed on normal human dermal fibroblasts, NHDF (PromoCell), by following the protocol recommended by the manufacturer [25]. The cells were grown in DMEM: F12 medium (Lonza) supplemented with 10% fetal bovine serum (Gibco), 1 mM sodium pyruvate (Lonza) and 1% penicillin–streptomycin–amphotericin B mixture (10 K/10 K/25 mg in 100 mL, Lonza). The same medium was used to prepare serial dilutions of the EPS to be tested. To ensure cell adhesion, NHDF were seeded at 5 × 103 cells per well in 96-well tissue culture plates and incubated for 24 h at 37 ◦ C under a humidified atmosphere with 5% CO2 . The medium was then replaced with 100 mL per well of the previously prepared EPS dilutions and the plates were further incubated for 20 h. The MTS reagent was then added to each well and, after final 4 h incubation, the absorbance at 490 nm was recorded with an EnVisions plate reader (PerkinElmer). The experiment included 6 replicates and was repeated 3 times.
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Fig. 3. NMR spectra of III-PP-29 EPS sample produced by W. confusa, recorded in D2 O. (A) 1 H NMR spectra for the dextran produced by fermentation in comparison with 1 H NMR spectra of a commercial dextran, (B) 1 H, 1 H TOCSY spectrum, (C) 13 C NMR spectra of III-PP-29 sample, (D) 1 H, 13 C HSQC spectra of III-PP-29 sample.
2.9.2. The antifungal activity The in vitro susceptibility testing was performed following the EUCAST EDef 7.2 guidelines using Candida albicans SC5314 strain, which is known to form abundant biofilms with a complex structure. The stock solutions were prepared using water as a solvent. The active principle concentration range of the tested antifungals was 0.0156–3 mg L−1 . The minimum inhibitory concentrations (MICs) were determined using spectrophotometric technique on an iMark Microplate Reader (Bio-Rad Laboratories, USA) at 405 nm, after 24 h of incubation at 35 ◦ C. 2.9.3. Antimicrobial activity The antimicrobial activity was considered on three different reference strains, i.e. Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6583 and Pseudomonas aeruginosa ATCC27853. The antimicrobial activity was measured by the agar disk diffusion method which supposes the addition of the EPS on the culture medium pre-inoculated with the microbial suspension (0.5 McFarland standards optical turbidity in sterile saline solution, yielding a suspension containing approximately 1 × 108 CFU mL−1 for all three microorganisms), and measuring of the clear zone caused by growth inhibition around the film disks after 24 h of incubation. 3. Results and discussions 3.1. The influence of the fermentative conditions on the EPS biosynthesis yield The resulted sequences from PP29 LAB strain DNA sequencing showed 98.4% identities with those available in the nucleotide BLAST data base and led to the identification of the Weissella confusa bacterial species. The fermentations of the W. confusa strain in three different culture medium compositions, in static and dynamic conditions, lead
Table 1 The EPS amount extracted from the culture medium. Fermentations conditions
I-PP-29, g/L
II-PP-29, g/L
III-PP-29, g/L
Static fermentations Dynamic fermentations
– 2.8
– 5.18
25.2 17.4
to different amounts of EPS biosynthesized after 48 h of fermentative processes (Table 1). Our observations confirmed the literature data according to which the amount and the properties of the EPS depend entirely on the composition of the culture medium and on the fermentation conditions [26]. Dextran production by W. confusa is affected profoundly by nitrogen and carbon sources, and by inorganic salts present in the culture medium [27]. MDI and MDII in static conditions had inhibitory effect on EPS biosynthesis due to the low oxygenation of the culture media. In the case of dynamic fermentations, after 48 h we obtained small amounts of EPS. For MDI, the MRS carbon source was supplemented with fructose and glucose, which are the components of sucrose, but there were obtained only 2.8 g/L EPS. This denoted that the LAB enzymatic system does not recognized the components as sucrose but as single sugars. For the MDII, the MRS carbon source was supplemented with sucrose, and after 48 h of fermentation there were obtained almost double amounts of EPS (5.18 g/L EPS), which means that the proper sugar was sucrose. The MDIII culture medium was very productive, especially in static conditions there were obtained 25.2 g/L EPS. This amount was obtained due to the presence in the culture medium of milk, which supplements the MRS nitrogen source, and also due to the calcium ions which enhances the viscosity of dextran [28]. 3.2. Gel permeation chromatography analysis (GPC) In order to characterize the EPS, gel permeation chromatography was used to obtain information on the molecular weight
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I. Rosca et al. / International Journal of Biological Macromolecules xxx (2017) xxx–xxx Table 2 Average molecular number (Mn), average molecular weight (Mw), and polydispersity index (PDI) of the EPS biosynthesis. Sample
fraction
Mn, Da
Mw, Da
PDI
I-PP-29
1 2 1 2 1 2 3
2.4 * 105 4.5 * 104 7.4 * 105 8.7 * 104 9.9 * 105 1.4 * 105 1.3 * 104
4.5 * 105 6.6 * 104 8.7 * 105 1.2 * 105 1.2 * 106 2.5 * 105 1.6 * 104
1.86 1.46 1.17 1.40 1.24 1.78 1.25
II-PP-29 III-PP-29-S
expressed in fractions of different weight. Molecular weight is a very important variable that provides additional information on the EPS physical properties, their molecule being dependent on the ionic strength and therefore on their concentration in solution. The weight average of the numerical molecular weight (Mw) and the average molecular number (Mn) were estimated by comparison with standard pullulan calibration curve. Because the chromatographic method allows selecting molecules by size, an important parameter is the average molecular weight that can reasonably approximate the molecular weight of the polymer fragments separated at the spine level. The obtained values for the EPS compounds extracted are presented in Table 2. Analyzing the results, it can be concluded that the numeric molecular mass is influenced by the fermentation process, a phenomenon that determines the proportional increase of the molecular weight of the parent compound. For the EPS extracted from MDI culture media it was obtained a biopolymer with Mn lower than the biopolymer extracted from MDII culture media supplemented with sucrose. This means that the sugar type is very important for the obtained EPS with different Mn. In the MDIII and static conditions (III-PP-29-S) there were obtained three fractions with high molecular weight and an uniform PDI. Also, by supplementing the MRS nitrogen source, it was increased the amount and the molecular weight of the biosynthesized polymer comparatively with the fermentation made in MDII. 3.3. Fourier-transform infrared spectroscopy (FTIR) The FTIR spectra of samples I-PP-29 and II-PP-29, on one hand, and III-PP-29 and III-PP-29-S, on the other hand, were very similar with each other (Fig. 1). Within the spectra of samples I-PP-29 and II-PP-29, the overlapped absorption bands of hydroxyl stretching and NH amine stretching were noticed at 3394 cm−1 . The amide I and amide II bands (1668 cm−1 and 1545 cm−1 ) indicate the presence of proteic residues. The region between 1490 and 1308 cm−1 covers signals from the bending vibrations of CH, CH2 and OH groups. The broad band with the maximum around 1058 cm−1 is characteristics to glucopyranose fragments, being a superposition of vibrations given by the C O C stretching and C OH stretching and bending. Identification of glycosidic linkages, of the bonded and free hydroxyl groups in various positions was made with the help of the second derivative spectra. The sub-band at 1054 cm−1 , specific to ␣-d-glucoses, is mainly given by (C2-O2H) [29,30]. A sub-band observed at 1022 cm−1 can be assigned to the (C6-O6H) vibrations in H-bonded primary alcoholic groups. An intense peak at 1080 cm−1 arises from the overlapped (C6-O6H), ␦(C4-C5) and ␦ (C1-H) vibrations [30,31]. The medium-to-intense sub-band peaking at 1129 cm−1 is confidently assigned to (C4-O4H) stretching vibration [29]. The (C O C) in glycosidic linkages of ␣-d-glucopyranoses is observed at 1152 cm−1 and is further confirmed by the weaker subband at 832 cm−1 ((C O)), which indicates the ␣-(1→3) anomeric configuration [32,33]. The sub-band at 844 cm−1 is specific to ␣-
5
(1→6) linkages (Heyn, 1974). Therefore, the spectra of I-PP-29 and II-PP-29 are very close to that of dextran, but with traces of proteins and other polysaccharides. Instead, the FTIR spectra of III-PP-29 and III-PP-29-S do not show any signals from protein residues. The “sugar” band has a fine structure, where the vibrations corresponding to (C6 O6H· · ·) (1015 cm−1 ), (C3 O3H· · ·) (1040 cm−1 ), (C6-O6H) + ␦(C4-C5) (1080 cm−1 ), or as (C O C) and (C O) in glycosidic linkages (1155 and 840 cm−1 ) are clearly observed (Fig. 2). Comparison with a sample of pure dextran from commercial source (M = 40.000) led to the almost perfect matching of spectra. 3.4. NMR spectroscopy analysis In order to confirm the presence of dextran in the analysed sample and to obtain more structural details there were recorded several one- and two-dimensional NMR experiments. The 1 H NMR spectrum of III-PP-29-S (Fig. 3A) was recorded with suppression of the water signal, to reveal the less intense signals from the region 4.5-5.5 ppm. The most intense signals were assigned to the glucose units as it follows: ␦ = 3.53 (1H, t, J = 9.4 Hz, H-4,), 3.59 (1H, dd, J = 9.7, 3.1 Hz, H-2), 3.73 (1H, t, J = 9.3 Hz, H-3), 3.77 (1H, d, J = 9.8 Hz, H-6a or H-6b), 3.93 (1H, d, J = 9.1 Hz, H-5), 4.00 (1H, d, J = 7.1 Hz, H-6a or H-6b), 4.99 (1H, d, J = 2.9 Hz, H-1). The signal centered at 4.99 ppm has been previously assigned [34] to anomeric protons of ␣-(1 → 6)-linked glucose units, in the main chain. In the TOCSY spectrum presented in Fig. 3B it can be noticed the couplings of the anomeric proton from 4.99 ppm with H-2, H-3, H-4 and H-5 protons. These correlations confirmed that the investigated signals belong to the same spin system. In the anomeric spectral region, three less intense signals were observed: a doublet centered at 4.67 ppm (J = 7.8 Hz), another doublet centered at 5.23 ppm (J = 3.2 Hz) and the third doublet centered at 5.34 ppm (J = 3.7 Hz). The first two doublets belong to free glucose remained from the culture medium, 4.67 ppm − anomeric proton from beta glucose and 5.23 ppm − anomeric proton from alpha glucose. The third doublet, from 5.34 ppm, has been previously assigned [34] to anomeric protons of ␣-(1 → 3)-linked glucose units, as branches of the main chain. The rest of the signals for the branch glucose units could not be assigned because of their low intensity and overlap with the signals from the main chain units. Based on the two anomeric signals and the ratio of their integrals, the percentage of glycosidic linkages was establish as being 96.2% for ␣-(1 → 6) and 3.8% for ␣-(1 → 3). Information about carbon chemical shifts was obtained from the 1D 13 C NMR and 2D 1 H, 13 C HSQC spectra (Fig. 3C and D). Based on the 1 H–13 C correlations from 1 H, 13 C HSQC spectrum, there were assigned the six intense signals as it follows: 68.5 (C-6), 72.5 (C-4), 73.1 (C-5), 74.4 (C-2), 76.4 (C-3), 100.7 (C-1). All these signals belong to ␣-(1 → 6)-linked glucose units, in the main chain. From the same HSQC spectrum it was assigned the C-1 from the branch ␣-(1 → 3)-linked glucose units at 102.6 ppm. The obtained NMR data indicated the obtaining of an exopolysaccharide. Based on the reported data from literature [34] it was deduced that the obtained exopolysaccharide is dextran, results supported by the FTIR analysis as well. 3.5. Thermal analysis measurements − thermogravimetry (TGA) and differential scanning calorimetry (DSC) By analyzing the thermal degradation curves, it can notice that all four samples displayed comparable thermal degradation tendency in two stages (Fig. 4, Table 3). Since there is a direct relationship between carboxyl group content and the material hydrophobicity, the initial material moisture content is given by the increased carboxyl group quantity. Regard-
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Fig. 4. TG and DTG curves of the studied samples.
Table 3 Thermal characteristics of the studied samples. Sample
I-PP-29 ◦
Stage I Stage II Wrez , %
II-PP-29 ◦
◦
◦
III-PP-29 ◦
◦
◦
III-PP-29-S ◦
◦
Ti , C
Tmax , C
Tf , C
w , %
Ti , C
Tmax , C
Tf , C
w , %
Ti , C
Tmax , C
Tf , C
w , %
Ti , ◦ C
Tmax , ◦ C
Tf , ◦ C
w , %
57 260 40.56
71 304
95 332
6.3 53.14
57 265 37.33
76 302
88 331
7.08 55.59
48 269 27.19
78 307
108 322
4.77 68.04
55 267 21.37
80 305
114 323
8.1 70.53
Ti − initial thermal degradation temperature; Tmax − temperature corresponding to the maximum rate of decomposition for each stage, evaluated from the peaks of the DTG curves; Tf − final thermal degradation temperature; Wm − weight loss rate corresponding to the Tmax values; Wrez − percentage of residue remained at the end of thermal degradation (700 ◦ C).
3.6. Biological tests
Fig. 5. Second heating curves of the studied samples.
ing to this, the samples structures suffer physical and chemical moisture loss during the first thermal degradation stage, between 71 and 80 ◦ C with a mass loss from 4.7-8.1% (Table 3) [35]. The second and principal degradation step is from 302 to 307 ◦ C recording an important mass loss of 53.17–70.53% [36], corresponding to random polymer backbone cleavages and depolymerisation, with remaining of inorganic material [37]. Fig. 5 depicts the second heating scans of the studied samples. All samples showed a glass transition temperature domain (Tg) between 217 and 223 ◦ C. This aspect is in accordance with the data reported by Scandola [38] reporting a Tg value of 223 ◦ C for natural dextran. The producing of a more amorphous (i.e. enhanced segmental chain mobility) dextran could explain the lower Tg domain values, down to 217 ◦ C.
3.6.1. Cytotoxicity assay By testing the biocompatibility it was proved that III-PP-29-S sample, dextran-based, does not have cytotoxic effect on fibroblasts up to a maximum concentration of 3 mg/mL, when cell viability drops below 70% (Fig. 6). Other results have been reported for EPS based on glucose and mannose, biosynthesized by Lactobacillus plantarum, where the fibroblast cell viability decreased to 70%, at a concentration of 100 mg/mL EPS [39]. These differences between results may be related to the different type of lactic acid bacteria strain and on the different type of EPS resulted from fermentation. For example, EPS based on galactose and glucose at a concentration of 5–50 g/L did not have cytotoxic effect on the normal intestinal cells [40]. EPS based on galactose and glucose, at a dosage of 5–50 g/mL showed no cytotoxic effect on normal intestine 407 cells [40]. 3.6.2. The antifungal activity Antifungal susceptibility testing using the species Candida albicans showed that III-PP-29-S can break by up to 70% of the biofilms formed by the pathogenic fungus to a maximum concentration of 3 mg/mL (Fig. 7), results that have not been reported yet in the literature. 3.6.3. EPS antimicrobial activity EPS based on dextran showed no antimicrobial activity against the reference strains. However, EPS based on small amounts of sulphate (1.43%), uronic acids (2.98%), proteins (4.08%), galactose, glucose and mannose, in a concentration of 4 mg/L, inhibited 65.82% of S. aureus biofilm formation, 43.58% P. aeruginosa and 33.41% of E. coli [41]. In the same time, EPS based on glucose had antibacterial activity against E. coli ATCC8739 and S. aureus ATCC6538 [42]. How-
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Fig. 6. Relative cell viability at 48 h. The relative cell viability of NHDF cells was determined by MTS assay after 48 h of treatment with EPS. Data is represented as means ± SD (n = 3).
Fig. 7. EPS antifungal assay.
ever, EPS produced by this particular strain have no antimicrobial effect against the reference pathogens. 4. Conclusions FTIR and NMR analyses confirm the dextran structure of the EPS extracted from experiment III-PP-29-S. According to TGA, the polymer has a very stable structure with high purity and its amorphous properties were demonstrated by DSC analysis. Because the EPS proved to have antifungal properties, the next step will be to load together with this compound an antifungal drug in order to test its capacity to destroy the biofilm formation and the yeast cells of Candida albicans, which is nowadays an important health issue. Statement of conflict of interest The authors declare no competing financial interest. Acknowledgements This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCS − UEFISCDI, project number PN-II-RU-TE-2014-4-0558. PhD I. Ros¸ca and MD PhD D. Peptanariu are grateful also for the financial support from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 667387 WIDESPREAD 2-2014 SupraChem Lab. References [1] P.B. Devi, D. Kavitake, P.H. Shetty, Physico-chemical characterization of galactan exopolysaccharide produced by Weissella confusa KR780676, Int. J. Biol. Macromol. 93 (2016) 822–828. [2] D. Kavitake, P.B. Devi, S.P. Singh, P.H. Shetty, Characterization of a novel galactan produced by Weissella confusa KR780676 from an acidic fermented food, Int. J. Biol. Macromol. 86 (2016) 681–689.
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