Fermentation Parameters and Conditions Affecting Levan

Bioorganic Chemistry 93 (2019) 102787

Contents lists available at ScienceDirect

Bioorganic Chemistry

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

Fermentation parameters and conditions affecting levan production and its T potential applications in cosmetics ⁎ Marta Domżał-Kędziaa, Agnieszka Lewińskab, , Anna Jarominc, Marek Weselskib, ⁎ Robert Pluskotad, Marcin Łukaszewicza, a Department of Biotransformation, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland b Faculty of Chemistry, University of Wroclaw, Joliot-Curie 14, 50-383 Wroclaw, Poland c Department of Lipids and Liposomes, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14A, 50-383 Wroclaw, Poland d InventionBio Sp. z o.o., Wojska Polskiego 65 st., 85-825 Bydgoszcz, Poland

ARTICLE INFO ABSTRACT

Keywords: Levan is a polysaccharide composed of fructose units with β-2,6-glycoside bonds. Microorganisms synthesize Biocatalysis levan by levansucrase as a mixture of low- and high-molecular-weight fractions. Due to its properties, it has a Bacillus subtilis wide range of applications in cosmetics, pharmaceuticals, food and medicine; it appears that the molecular Levan weight of levan might impact its industrial use. To obtain one fraction of levan after biotransformation, ethanol Isolation precipitation with an increasing volume of alcohol was conducted. This precipitation process was also optimized. Purification Several types of analyses were used. Low-molecular-weight levan was evaluated for toxicity in a normal human Structural characterization Cytotoxicity dermal fibroblast cell line and hemolytic potential on human erythrocytes. Levan was found to be non-cytotoxic Hemolysis and non-hemolytic in concentrations ranging from 0.01 to 1.00 mg/ml. Moreover, levan demonstrated anti- Antioxidant activity oxidant potential expressed as an ability to inhibit of oil/water emulsion oxidation and DPPH radical scavenging.

1. Introduction keratinocyte proliferation [3], it shown no-toxic sensitization for skin or eyes in The Human Repeated Insulin Patch (HRIPT) and Chorioallantoic The current era of human society raised the application of natural Membrane Vascular Assay (CAMVA) tests [4], and it soothes skin irri- sources in personal care especially in cosmetics, which contact with the tations caused by skin irritants such as sodium laureth sulfate (SLS) epidermis, hair system, oral cavity, nails and plays a vital role in en- [3,5,6]. Levan has potential in discolouration products because it can hancing human body appearance, therefore their active ingredients prevent melanin production by reducing tyrosinase activity [7]. The should be biocompatible. Several bacterial polysaccharides possess addition of ascorbic acid to such a levan product results in increased properties that render them suitable for use in cosmetic applications. stability to oxidation [8]. It can also co-create a polymer matrix in The most relevant are xanthan gum and gellan gum that are mostly cosmetics in the form of a solid foil that disappears when applied to the used as base substances in cosmetic vehicles and bacterial cellulose, skin [9]. Levan has high solubility in oil, low viscosity and it is stable in hyaluronic acid, and levan. Biodegradable polymers, especially poly- high temperature [10,11]. Due to the ability of levan to create films and saccharides, attract the attention of scientists looking for new com- its low viscosity, it can be used in hair-fixing products [4,12,13]. The pounds with cosmetic properties, because they can be eliminated by Cosmetic Ingredient Review (CIR) Expert Panels have concluded levan normal metabolic pathways [1,2]. According to the research of many to be safe for use in cosmetic formulations [14]. scientists, levan can be an attractive raw material for cosmetic for- Levan is a polysaccharide consisting of fructose units linked with β- mulations for various purposes. These tests were performed on levan 2,6-glycoside bonds in its main chain and β-2,1 in its branches. It is from other microorganism cultures than B. subtilis, but there is a lack of synthesized from sucrose by a wide range of organisms such as bacteria information about the usefulness of levan in cosmetics of a specific (Bacillus subtilis, Zymomonas mobilis, Erwinia herbicola) and fungi molecular weight [3]. The use of levan in cosmetics, due to its prop- (Aspergillus sydowii, A. versicolor) [15–17]. Levan was described for the erties can be extensive. According to the Transepidermal Water Loss first time in 1881 by Lipmann, who used the term lävulan. The nameof (TEWL) test, the moisturizing effect of levan is comparable to that after the polymer we know today—levan—was proposed in 1901 by bac- using hyaluronic acid and stimulates human fibroblasts and teriologist Greig-Smith [18]. He found a Bacillus strain that grown on

⁎ Corresponding authors. E-mail addresses: [email protected] (A. Lewińska), [email protected] (M. Łukaszewicz). https://doi.org/10.1016/j.bioorg.2019.02.012 Received 14 December 2018; Received in revised form 2 February 2019; Accepted 4 February 2019 Available online 11 February 2019 0045-2068/ © 2019 Published by Elsevier Inc. M. Domżał-Kędzia, et al. Bioorganic Chemistry 93 (2019) 102787 sucrose and produced a polymer. Greig-Smith named the strain levan from BioShop®. Levan standard (E. herbicola), sorbitan sesquioleate, for its levorotation of polarized light and its properties, which are si- fetal bovine serum, 1,1-diphenyl-2-pycryl-hydrazyl (DPPH) and MTT milar to those of dextran [18,19]. Levans of microbial origin are (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) were characterized by a high molecular weight exceeding 500 kDa; they can purchased from Sigma-Aldrich (Poznan, Poland). Flaxseed oil was from be linear or branched and have a wide spectrum of applications [16]. Oleofarm (Wroclaw, Poland). L-glutamine, penicillin, streptomycin and Microbial levan is synthesized by levansucrase, an extracellular en- amphotericin B were from Life Technologies (Warsaw, Poland). Normal zyme, which carries out sucrose hydrolysis as well as transfructosyla- human dermal fibroblast cell line and minimum essential medium eagle tion reactions with sucrose molecule or fructooligosaccharide chains as alpha modifications medium were purchased from Lonza (Warsaw, acceptors [20,21]. It was previously reported that the biosynthesis of Poland). All chemicals and reagents not specified in the text were of low- and high-molecular-weight (LMW and HMW, respectively) levan analytical grade. Deionized double distilled water was used for all of co-occurs [22]. The mechanisms by which LMW and HMW levan is the experiments. produced have not yet been determined [23]. High-molecular-weight levan possesses antitumor activity [28,29]. High-molecular-weight 2.1. Bacterial strain and culture conditions particles contribute to lowering blood cholesterol [30]. The use of LMW polymer results in more efficient encapsulation capacity due to the slow B. subtilis natto KB1 strain was isolated from “Soybean Powder” and constant drug release and HMW polymer has a poorer encapsula- Natto Culture Starter produced by Proster®(Korea) [34]. The pre-cul- tion capacity and impairs the release efficiency of the active ingredient ture was incubated for 24 h at 37 °C in 10 ml of LB medium (10 g/L contained in the capsules [14,31]. Levan, a product of levansucrase bacto-tryptone, 5 g/L bacto-yeast extract, 10 g/L NaCl; BioShop® (Ca- biocatalysis, has many applications in biomedicine and health as, for nada); Lab Empire® (Poland) with agitation (180 rpm). For levan pro- example, a drug carrier and a prebiotic [24]. Levan can self-assemble in duction, the KB1 strain was grown in a sucrose-rich medium proposed water into nanoparticles, which makes it an interesting encapsulation by Shih and Yu [35] that consisted of MgSO4·7H2O (0.5 g/L) agent [25,26]. Levan is also used as a color and color enhancer in the NaH2PO4·2H2O (3 g/L), Na2HPO4·12H2O (3 g/L), sucrose (50 g/L) and production of tablets and capsules [27]. The addition of levan to a sodium salt of L-glutamic acid (15 g/L). B. subtilis natto KB1 was culti- matrix supporting capsules enables faster dissolution and a faster vated for 9 days at 37 °C in 50 ml of medium with agitation (180 rpm). therapeutic effect of the active ingredient. These compositions are mainly used in drug formulations that are intended to release the active 2.2. Optimization of the precipitation process ingredient in the oral cavity [28]. Levan production on an industrial scale is associated with many The precipitation process was optimized by setting optimal para- problems such as low process efficiency and pollution of the products meters such as the temperature of the precipitating agent, the rate of its obtained [29]. On the laboratory scale, levan is purified in several ways addition and the speed of the mixing during the precipitation. Three by, for example, re-dissolving the precipitate, removing insoluble par- temperatures of the precipitating agent were tested: 22 °C; +8 ÷ +4°C; ticles from it and re-precipitating, dialyzing the dissolved precipitate −23 °C. The mixing speed of the entire system during the ethanol ad- and ultrafiltration [30–32]. The lack of a suitable way to purify the dition was tested over a range of 200–600 rpm. The speed of ethanol product on a large scale presents a challenge. One must also note a large incorporation was also determined. amount of ethanol left after precipitation from the culture medium. Lyophilization and vacuum drying of obtained levan are commonly 2.3. Fourier-transform infrared analysis employed on a laboratory scale [27,33], but it is not possible to scale up these methods to industrial production. Functional groups and chemical bonds of the obtained levan were This study aimed to obtain levan with a low molecular weight and to determined using Fourier-transform infrared (FT-IR) spectroscopy on determine an easy way to separate LMW and HMW molecules. To separate Spektrometr Bruker Vertex 70 FT-IR. The sample was prepared as a KBr LMW and HMW polymers, fractionation with an increasing amount of pellet. All of the samples were scanned over a wavelength range of ethanol was used. This technique was already described as an efficient 4000–400 cm−1. The spectrum obtained was compared with the spec- method for LMW and HMW molecule separation [9]. The precipitation trum of a levan standard from Sigma-Aldrich (Poznan, Poland) and with process was optimized by setting optimal parameters such as the tem- literature data. perature of the precipitating agent, the rate of its addition and the speed of the mixing during the precipitation. Many techniques have been used to 2.4. Nuclear magnetic resonance analysis characterize the levan polymer, such as Fourier Transform Infrared (FT- IR), 1Hydrogen-Nuclear Magnetic Resonance and 13Carbon-Nuclear Mag- 13C and 1H nuclear magnetic resonance (NMR) spectra were re- netic Resonance (1H and 13C NMR spectroscopy), Scanning electron mi- corded using an AVANCE III NMR 500 MHz spectrometer (Brucker Co., croscope analysis (SEM), Thermal analysis (TGA) and Gel Permeation Billerica, MA, USA) at 25 °C. Ten milligrams of a lyophilized sample was

Chromatography (GPC). We also conducted preliminary tests to determine dissolved in 1 ml of deuterium oxide (D2O). The chemical shifts (δ) the usefulness of levan in the production of cosmetics, and we specify were obtained as ppm. The spectrum obtained was compared with a parameters such as Water-holding capacity (WHC) and Water-solubility spectrum of a levan standard from Sigma-Aldrich and with literature index (WSI). Any material used in the application must be biocompatible data. 1H NMR was conducted for all of the collected samples. For the and exhibit excellent in vitro cytotoxicity and blood-compatibility behavior 13C NMR analysis, we used a sample precipitated from 9 days of liquid [24]. Therefore, in this study, we examined the changes in cell viability of cultivation after adding a third portion of ethanol. normal human dermal fibroblast (NHDF) cell lines and possible human erythrocyte membrane damage after treatment with isolated levan. We 2.5. Scanning electron microscope analysis also focused on the antioxidant properties of produced biopolymers, especially in light of the many encouraging reports describing these ac- The surface morphology microstructure of the levan was analyzed tivities for levans obtained by microbial production [25–27]. using scanning electron microscopy (SEM) with a Hitachi S-3400N with energy dispersive X-ray spectrometry (EDS) with a Thermo Scientific 2. Materials and methods Ultra Dry at an acceleration voltage of 10 kV and a magnification of 5–500 µm. The lyophilized samples were fixed and coated with a layer Ethanol and the minerals were purchased from Chempur (Poland), of Au roughly 10 nm thick using a sputtering apparatus (Cressington and the sucrose and sodium salt of L – glutamic acid were purchased 108A; (Cressington Scientific Instruments, United Kingdom).

2 M. Domżał-Kędzia, et al. Bioorganic Chemistry 93 (2019) 102787

2.6. Thermal analysis fibroblasts (NHDF) were used. The cell line was inoculated on a96-well plate supplemented with 100 µl of α-MEM (Minimum Essential Medium The thermal stability of the purified levan was determined using a Eagle) for NHDF with a density 6 × 103 cells per well. The plates were thermogravimeter analyzer (TG-DTA Setaram SETSYS 16/18; (Setaram incubated for 24 h at 37 °C. After the addition of levan solution, the cells Instrumentation, France). The thermograms were obtained over a were incubated for 3 days in 37 °C. The viability of the cells was mea- temperature range of 40–800 °C with a ramp rate of 10 °C/min in a sured using an MTT assay. MTT solution (50 µl) was added to each well, nitrogen atmosphere. which was then incubated for 4 h. The absorbance was measured at 570 nm. The cell viability was calculated as: 2.7. Molecular weight measurement on gel permeation chromatography Absorbance570 nm (sample) Cell viability (%) = × 100% Absorbance The molecular weight of levan was measured using a Malvern Omnisec 570 nm (control) (Malvern Panalytical, United Kingdom) gel permeation chromatography Assays were performed in triplicate, and the data were expressed as system, integrated with multi-detector. Diode-array detector (DAD), right- mean values ± standard deviations. angle laser light scattering (RALS), low-angle laser light scattering (LALS), refractive index detector (RI), inlet pressure viscometer (IP) and differ- 3.2. Hemolytic assay ential pressure viscometer (DP) detectors were used. The gel permeation chromatography (GPC) analyses were performed using two aqueous col- The study protocol was approved by the Bioethics Commission at umns (A7000 – 35 µm, 300.0 × 8.0 mm; A5000 – 13 µm, the Lower Silesian Chamber of Physicians and Dentists (1/PNHAB/ 300.0 × 8.0 mm; Malvern), one pre-column and one guard-column with a 2018). The hemolytic activity was tested by determining the extent of filter (0.22 µm). The mobile phase consisted of 0.5 M NaCl (Chempur) hemoglobin release from human erythrocyte suspensions followed the with 0.2% NaN (POCh) (eluent A) and 0.1 M phosphate buffer pH 8.1 3 same method as described in a previous study [39]. The freshly col- (K HPO and KH PO Sigma-Aldrich) (eluent B) in a 1:1 ratio. The eluent 2 4 2 4 lected blood was mixed with the anticoagulant solution and centrifuged flow rate was 1 ml/min. The detectors and columns were thermo- at 650g for 10 min. The supernatant was discarded, and the ery- regulated, and the temperature was 23 and 40 °C, respectively. The in- throcytes were resuspended in phosphate buffered saline (PBS; 5mM jection volume was set at 150 µl. Dextran (Malvern) was used as a material phosphate, 150 mM NaCl, pH = 7.4). Next, the erythrocytes were wa- for system calibration. The protein content in the samples was determined shed three times with PBS, and the upper phase with a buffy coat using the Bradford method [36]. The tested samples were dissolved in the containing precipitated debris and serum proteins was carefully re- mobile phase and filtered through a 0.22-µm filter. The data wereana- moved at each wash step. After the last washing, the packed cells were lyzed using dedicated software for this device (Omnisec V10). suspended in a buffer to a hematocrit of 50%. This suspension wasused within 24 h after collection. To determine the hemolytic effects, mi- 2.8. Water solubility index croliter amounts of an aqueous solution of levan were added to PBS containing 20 μl of erythrocyte suspension (hematocrit of 50%) and The water solubility index (WSI) determines the degree of solubility filled with buffer to a final volume of 4 ml (final concentration: 0.01, of a given material in water. The WSI of levan was determined using the 0.1 and 1 mg/ml, respectively). The samples were then stirred and in- method described by Anderson et al. [37]. Two hundred mg of the cubated for 30 min at 37 °C. After centrifugation at 650g for 10 min, the sample was dissolved in 5 ml of Milli-Q water and stirred for 40 min in a amount of the released hemoglobin was measured at 540 nm. The he- water bath at 40 °C to obtain a uniform suspension. Then, the sample molytic effect, measured as the percent of hemolysis (H), wasde- was centrifuged at 4000g for 10 min and the supernatant was placed in termined spectrophotometrically against a corresponding blank sample a Petri dish and dried at 105 °C for 4 h to obtain a dry solid weight. by the following equation: Then, the WSI was calculated based on the following equation: Absorbancesample Absorbancemechanical hemolysis dry weight of solids in supernatant H (%) = × 100% WSI (%) = × 100% Absorbance Absorbance weight of dry sample 100% hemolysis mechanical hemolysis where mechanical hemolysis (erythrocytes in PBS buffer), and the 2.9. Water-holding capacity 100% hemolysis (erythrocytes in double-distilled water).

The water-holding capacity (WHC) represents the quantity of water 3.3. Antioxidant activity of levan material is apt to retain. The result is given as the amount of water the polymer is capable of retaining in its molecule. The WHC of collected 3.3.1. Determination of inhibition of oxidation of o/w emulsion levan samples was determined as per the method of Feng et al. [38]. The emulsion containing 15 mg of sorbitan sesquioleate, 50 mg of Two hundred mg of sample was dissolved in 10 ml of Milli-Q water and flaxseed oil, and 2 ml of 10 mM Tris-HCl buffer, pH 7.4 was prepared kept at 40 °C for 10 min. Then, the sample was centrifuged at 14,000g using a previously reported method [40,41]. Determination of the for 30 min, and the supernatant was then dumped. The pellet was then oxidation inhibition of the o/w emulsion was carried out according to a put on pre-weighed filter paper to remove the water. The weight ofthe procedure described in detail in [42], using 10 μl of the 1:1 (v/v) precipitated sample was noted. The percentage of WHC was calculated 10 mM Tris-HCl buffer, pH 7.4-diluted emulsion, an aqueous solution of according to the following equation: levan (final concentration 0.1 mg/ml), and ammonium iron(II) sulfate Total sample weight after water absorption hexahydrate (Mohr’s salt; 2 mM). Thiobarbituric acid reacting sub- WHC (%) = × 100% stances (TBARS) were determined according to Buege and Aust [43]. Total dry sample weight

3.3.2. DPPH assay 3. Biological activity The antioxidant capacity of levan was evaluated by its scavenging activity against DPPH radicals, using the method of Srikanth et al. [44]. 3.1. Viability assay Briefly, a DPPH reagent solution was prepared by dissolving DPPHin ethanol to obtain a 0.1 mM concentration. Levan test solutions were To determine the viability of cells in the presence of levan, a 1 mg/ prepared by dissolving levan in water (final concentration: 0.1 mg/ml). ml solution of levan from B. subtilis KB1 was prepared. Human Then, 300 μl of freshly prepared DPPH reagent was added to 1 ml of

3 M. Domżał-Kędzia, et al. Bioorganic Chemistry 93 (2019) 102787 each levan solution. The reaction was carried out in the dark for 30 min 1), which disappeared in a lyophilized sample after two days dried with at room temperature (RT). Finally, the absorbance was measured at the steam of dry nitrogen at 100 °C (SM 2). All of the peaks were be- 517 nm. A mixture of DPPH and ethanol was served as a control, and a tween 4.20 and 3.40 ppm. Levan was identified, based on 1H NMR, in mixture of each levan solution and ethanol served as a blank for the all samples collected from liquid cultures. Using this method we were sample. The % inhibition was calculated using the following formula: not able to identify the occurrence of branches, despite literature data in which spectra of slightly branched bacterial levans showed a weak C- Inhibition(%) 3 signal about 0.2 ppm downfield from the major levan C-3 signal [46]. Absorbance of the control Absorbance of the sample = × 100% This could be due to the small number of branches in the molecule and Absorbance of the control a large number of D-fructofuranose units between the β(2 → 1) bonds. 13C and 1H NMR spectrums of levan from B. subtilis KB1 are found in Supplementary Materials. 4. Results and discussion The 13C and 1H spectra are unique to each compound and often quite predictable, in particular for small molecules [47]. With this To develop and optimize methods of levan production, purification method, it is possible to determine the primary structure together with and fractionation according to chemical structure (molecular weight the stereochemical relationships, the saccharide conformation, and the and branching), appropriate analytical methods are necessary. These presence of substituents. 13C NMR spectroscopy distinguishes branched methods indirectly contribute to the development of production, pur- and linear levan of plant origin [48,49]. Unfortunately, with this ification and fractionation methods to obtain a polymer with specific method ethanol remains, even after a few days of lyophilization. This properties. The simultaneous use of several methods allows one to ob- situation is a problem because it disturbs the spectra, particularly 13C tain information about the structure of the molecule, its molecular spectra. On laboratory scales, ethanol is removed from precipitated weight and distribution of masses and the occurrence of branching. levan by dialysis or drying, which is not used on an industrial scale. FT-IR was used to investigate the nature of the functional groups of 4.1. Isolation and characterization of levan levan regarding monomeric units and their linkages. In the current study, the spectral data obtained for levan produced from Bacillus After cultivation of the liquid culture, it was centrifuged to remove subtilis KB1 exhibited major characteristic peaks. The FT-IR spectrum the bacterial cells. Next, the levan was harvested by precipitation with shown in Fig. 2 exhibits a strong band at 3430 cm−1, which was as- the addition of 96% cold ethanol. Ethanol was gradually added in four signed to the hydroxyl (OH) stretching vibrations of the polysaccharide. portions. After each addition, it was stored at 4 °C overnight, then the Bands from the carbon-hydrogen (CeH) stretching vibration were precipitate was centrifuged and lyophilized. around 2900 cm−1, which confirmed the existence of fructose residue The maximum yield of levan was obtained with a 1:4 supernatant to [50]. The band at 1410 cm−1 was assigned to CeH. The bands around alcohol ratio. The optimal temperature for added ethanol is 4 °C. To 1076 cm−1 were assigned to stretching vibrations of the glycosidic achieve maximum efficiency, the system should be mixed during pre- linkage CeOeC and CeOH groups [51]. The absorption around cipitation at 500 rpm, and the ethanol should be introduced con- 862 cm−1 and 920 cm−1 was assigned to the stretching vibrations of tinuously at a rate of roughly 15 ml/min. These parameters were used the pyran ring [52]. The band around 1634 cm−1 was evidence of to precipitate levan from the supernatant from the liquid fermentation. bound water [53]. The FT-IR results indicated that levan was present in The maximum yield of levan was precipitated when the ratio of su- all obtained samples from the liquid cultures. Levan was identified in all pernatant to ethanol was 1:4 [23]. Thus far, there have been no pub- samples collected after 9 days cultivation. The FT-IR analysis of levan in lished data as to whether the yield of precipitated levan increases when this study was comparable to that of previously reported data (Table SM a system is mixed during the introduction of ethanol. 2), which confirmed that levan was produced. The FT-IR spectrum of Using the technique of gradual precipitation, levan fractionation levan from B. subtilis KB1 is found in Supplementary Materials (SM 3). was achieved due to differences in molecular weight. A possible ex- We used infrared spectroscopy to characterize levan and identify other planation for this fact is the different solubility of individual levan sugars in the sample. Levan exhibits hydroxyl stretching vibrations of fractions in water. Furthermore, the addition of additional portions of CeH, stretching vibrations from glycosidic linkage CeOeC and the alcohol precipitate fractions with even better solubility in water; this presence of fructose units. The biggest problem in the FT-IR analysis is property is determined by, for example, molecular weight. Stepwise the presence of water in the sample. Fructans can retain water in their precipitation additionally enables the purification of selected levan structure and particles formed; plants use this property to increase their fractions indirectly because proteins largely precipitate during the ad- resistance to cold and drought [54–56] and bacteria and to protect dition of the first ethanol fraction. Therefore, the remaining fractions them from environmental factors such as high salt concentration or are not co-precipitated with proteins, or there are fewer of them. As a temperature [57,58]. result, precipitation with portions of alcohol is one method of levan Scanning electron microscopy analyses enable particles and nano- purification from protein impurities. particles, fracture surfaces, surface morphologies, composites and their Spectroscopic methods such as NMR spectroscopy and infrared constituents, and microstructures of materials to be examined and spectroscopy make it possible to determine the molecular structure of a characterized [59]. With the addition of EDS, we can simultaneously given compound based on specific chemical bonds, functional groups determine the elemental composition and morphology of different and stoichiometry of a molecule [45]. Nuclear magnetic resonance materials [60,61]. We used SEM to observe the microstructure of levan spectroscopy is the most important technique for obtaining detailed (Fig. 1). The sample exhibited an irregular polysaccharide structure. We structural information about levan. Our 13C NMR spectrum exhibited noted a highly porous structure, which suggests that levan will be a six signals corresponding to levan: 104.84 (C-2), 80.91 (C-5), 76.93 (C- suitable material for emulgation, thickening and gelling processes. In 3), 75.82 (C-4), 64.03 (C-6), and 60.54 (C-1) ppm. Two other signals, at comparison with the polymer reported by Feng, the structure of levan 58.06 and 17.44 ppm, corresponded to ethanol in the sample and were from B. subtilis natto KB1 was more porous, and the B-2 EPS structure used as a reference. The chemical shifts from levan were comparable presented irregular sheets and a smooth surface [38,62]. A porous with previously reported data (Table SM 1), which confirmed that levan structure is necessary for WHC, which makes it an interesting compo- was produced. According to the 1H NMR spectrum, seven signals of nent for cosmetics production. Similar properties were observed in EPS chemical shifts were observed at 4.17 (H-3), 4.07 (H-4), 3.93 (H-5), from Leuconostoc lactis KC117496 [63]. 3.88 (H-6a), 3.75 (H-1a), 3.65 (H-1b), and 3.54 (H-6b) ppm. The ad- Thermal Gravimetric Assay (TGA) makes it possible to determine ditional peak at 3.64 ppm belongs to the CH2-group from ethanol (SM the individual components of a test sample. In studies of thermal

4 M. Domżał-Kędzia, et al. Bioorganic Chemistry 93 (2019) 102787

Fig. 1. Scanning electron micrograph of the levan produced from Bacillus subtilis KB1. stability of polymers, it is deduced from the loss of its mass [64]. Results samples, which was expected and was consistent with the literature from the thermogravimetric analysis of levan samples obtained by cold [23]. In the first precipitated sample, two fractions with different mo- ethanol precipitation are shown in Fig. 2. The thermal degradation of lecular weights were observed. The average weights were 582.3 kDa levan samples occurred in two distinct stages preceded by a drying (13.51%) and 50330 kDa (86.49%), respectively. There were proteins stage. In the first stage between RT and ∼135 °C, samples 1 and2lost detected in the sample that interfered with the determination of the 5.6 and 4.0% of their weight, respectively. These results are connected sample’s molecular weight. The second precipitated sample also con- with the loss of water molecules absorbed and bonded by H-bonds as tained two fractions, but the LMW polymer constituted 99.69% with a well as the loss of ethanol molecules (corresponding with the 1H and molecular weight of 37.62 kDa. High-molecular-weight fraction con- 13C NMR analysis). The primary difference in the mass changes in this stituted only 0.31% with a molecular weight of 50,670 kDa. No signals stage is a result of sample drying. In sample 3, this stage as not well were corresponding to the branching of levan molecules which can be separated from the second one. As the sample was heated more, to over related to the small amount of branching in the molecule and a large 135 °C, decomposition occurred. In our case, the first step was observed amount of D-fructofuranose particles between β(2 → 1) branching. No near 140–150 and 210 °C; roughly 4 (sample 1), 7 (sample 2) and 6% proteins were detected in this sample. Due to its good solubility, it was (sample 3) of the sample mass was lost. Over a temperature range of selected for use in subsequent tests. 220–300 °C, 12 (sample 1), 23 (sample 2) and 22% (sample 3) of the mass was lost. Over 300 °C—the last stage—we noted a mass loss of 8 4.2. Water solubility index and water-holding capacity as moisture (sample 1), 24 (sample 2) and 12% (sample 3), which indicated com- indicators plete decomposition. Some authors [65] have connected decomposition between 200 and Water solubility index (WSI) determines the degree of solubility of a 300 °C with gradual degradation of levan polymer. In first step β(2 →1) given material in water. Water holding capacity (WHC) represents the bonds, which is responsible for branch linkages and bonds in the ring. quantity of water the material is apt to retain. The results are expressed Then, above 300 °C, the main chain β(2 → 6) bonds break. According to as the number of times the proteins powder can retain its weight of our team and other authors results, the primary differences between water. The WSI (Fig. 4) and WHC (Table 1) were determined for all levan from B. subtilis KB1 might arise from the different molecular samples containing crude levan. After 9 days of liquid culture, the first structures of fructans [65,66]. precipitate sample contained two fractions of levan and the second The most complex method used in the study of polymers is GPC, precipitated sample contained only almost one fraction. The WSI and which enables the determination of relevant parameters such as the WHC for the first precipitated sample were 88.34 ± 3.01% and average molecular weight and its distribution, the dispersal coefficient 99.16 ± 6.71%, respectively. The second precipitated sample mainly or intrinsic viscosity and the occurrence of branching. This method consisted of the LMW fraction of levan. The WSI for this sample was makes it possible to determine the quantity and quality of levan high: 99.30 ± 4.30. The WHC for this sample was 100.61 ± 0.43. The [23,67,68]. Gel permeation chromatography was performed to de- WSI was 90.94 ± 2.73%. The WSI and WHC determined for the levan termine the molecular weight of levan in each sample from 9 days of standard from Erwinia herbicola (Sigma-Aldrich) were 86.30 ± 3.30% cultivation (Fig. 3). Low-molecular-weight polymer dominated in all and 1527.20 ± 42.60%. These results indicate that levan from B. subtilis natto KB1 has the potential to hold a large amount of water in its hydrogen bonds. The surface morphology of levan from B. subtilis KB1 confirmed this con- clusion. Saravan and Shetty reported WSI and WHC values of 14.20 ± 0.21% and 117.00 ± 7.50%.

4.3. Biological activity

4.3.1. Effect on NHDF cell viability Assessments of fibroblast toxicity in vitro are commonly used to estimate the cytotoxicity of many compounds [69–71]. For these rea- sons, we selected NHDF cells as a model in our experiment. We in- vestigated cytotoxicity by measuring the cellular metabolic activity. The results of the MTT viability assay (in Supplementary Materials as Fig. 2. Thermogravimetric analysis of levan obtained by cold ethanol pre- SM 4) showed that LMW levan was not cytotoxic to NHDF cells when cipitation. One (black line), two (red line) and three (blue line) volumes of applied at concentrations ranging from 0.98 to 1000 µg/ml for 72 h. ethanol, respectively. The cells viabilities were close to 100%, indicating that the addition of

5 M. Domżał-Kędzia, et al. Bioorganic Chemistry 93 (2019) 102787

Fig. 3. Chromatogram of levan.

properties of polymers. It is interesting to note that all samples of levan, tested over the same range of concentration as in NHDF cell culture experiments (0.98–1000 µg/ml), had a very small hemolytic effect on the level of mechanical hemolysis (erythrocytes in PBS). Because no detectable disturbances of the red blood cell membranes were identified, we suggest that levan from B. subtilis natto KB1 is safe for use in mammalian cells.

4.3.3. Antioxidant activity of levan In order to evaluate the antioxidant activity of levan, we performed two assays. Many authors have suggested confirming levan’s properties Fig. 4. Water solubility index of the samples after 9 days of cultivation. using at least two different methods because the results depend onthe applied approach [73]. We decided to carry out the determinations Table 1 using well-recognized methods: lipid emulsion oxidation and the DPPH Water-holding capacity for samples after 9 days of assays for a levan concentration of 0.1 mg/ml in the sample. As shown cultivation. in Table 2, levan collected from 9 days of liquid culture exhibited effective antioxidant activity. The LMW fraction suppressed the lipid Sample WHC (%) peroxidation of flax seed oil emulsion after treatment with the inductor 2+ 1V EtOH 99.10 ± 6.7 (Fe ) at the level of 57.35%. Surprisingly, levan produced by E. her- 2V EtOH 100.61 ± 0.43 bicola had no activity in the test conditions. This finding can be ex- a 3V EtOH – plained by the fact that the structural features of levan—like its chain E. herbicola 1527.20 ± 42.6 length or degree of branching—depend on its biological source and a No activity at the tested concentration. therefore affect its activity. However, this tendency is not visible in DPPH free radical scavenging assays. In all cases, we observed a de- such material permitted normal cell growth, even after a long period. crease in the DPPH solution absorbance, which demonstrated which These results correspond well with findings reported by Kim et al. [3] demonstrated DPPH radical scavenging ability (22.27–31.70%). It is indicating that levan from Z. mobilis exhibits no cytotoxicity in human worth noting that our results are in agreement with data reported for fibroblast cell lines. These findings are also comparable with experi- levan from Acetobacter xylinum for the same concentration [44]. ments performed by Dos Santos et al. [72] on Chinese hamster ovary In summary, we can conclude that LMW levan produced by B. (CHO-K1) and Kang et al. [29] on cell lines. Levan did not affect the subtilis natto KB1 is non-toxic to mammalian cells and exerts strong viability of cells incubated with polysaccharide with concentrations antioxidant activity. It, therefore, has the potential to be used in dif- ranging from 80 to 1000 μg/mL of the exopolysaccharide. In these ferent fields of biomedicine. studies, the viability assays were also evaluated by MTT. The re- searchers observed no mortality after incubation of the cells with levan; Table 2 it was not possible to determine LD50. Antioxidant activity of levan from B. subtilis natto KB1 and E. herbicola determined at 0.1 mg/ml.

4.3.2. Hemolytic activity Inhibition of o/w emulsion DPPH scavenging (%) The cytotoxicity results are even more encouraging when they are oxidation (%) compared with the effects of levan on human erythrocytes. The inter- B. subtilis natto KB1 57.35 ± 4.60 31.70 ± 2.13 actions of levan collected from 9 days of liquid culture as well as levan E. herbicola n.a.a 22.27 ± 2.68 from E. herbicola were determined based on measurements of the release of hemoglobin; this process reflects the membrane-damaging a No protection detected.

6 M. Domżał-Kędzia, et al. Bioorganic Chemistry 93 (2019) 102787

5. Conclusions ch013. [17] F. Freitas, M.A.M. Reis, U.N. De Lisboa, U. De Lisboa, Polysaccharides (2014) 1–24, https://doi.org/10.1007/978-3-319-03751-6. In this study, we determined the biological properties of levan from [18] R. Greig-Smith, The gum fermentation of sugar cane juice, Linnean Society of New Bacillus subtilis KB1, including antioxidant and hemolytic activity and South Wales, 1902. < https://www.biodiversitylibrary.org/part/32523 > (ac- cell cytotoxicity. We also evaluated WHC and WSI. Our results indicate cessed July 5, 2018). [19] R. Greig-Smith, T. Steel, Levan: a new bacterial gum from sugar, Scottish Sect. J. that levan might be useful and safe in cosmetics and biomedical fields. Soc. Chem. Ind. (1902) 1381. Levan fractionation with an increasing volume of ethanol was proven to [20] K. Mardo, T. Visnapuu, M. Gromkova, A. Aasamets, K. Viigand, H. Vija, T. Alamäe, be useful for separating LMW and HMW polymers. By optimizing the High-throughput assay of levansucrase variants in search of feasible catalysts for the parameters of levan precipitation, we obtained the most effective yield synthesis of fructooligosaccharides and levan, Molecules 19 (2014) 8434–8455, https://doi.org/10.3390/molecules19068434. during the precipitation process. We used several analytical methods, [21] B.C.M. Goncalves, C. Baldo, M.A.P.C. Celligoi, Levan and levansucrase-a mini re- and they all proved to be essential for determining the structural view, Int. J. Sci. Technol. Res. 4 (2015) 100–104 www.ijstr.org. characterization of the polymers. [22] T. Tanaka, S. Oi, T. Yamamoto, The molecular structure of low and high molecular weight levans synthesized by levansucrase, J. Biochem. 87 (1980) 297–303. [23] I.-L. Shih, Y.-T. Yu, C.-J. Shieh, C.-Y. Hsieh, Selective production and character- Acknowledgements ization of levan by bacillus subtilis (natto) takahashi, J. Agric. Food Chem. 53 (2005) 8211–8215, https://doi.org/10.1021/jf058084o. [24] Á. González-Garcinuño, A. Tabernero, Á. Domínguez, M.A. Galán, E.M. Martin del The project was supported in part by a statutory activity of subsidy Valle, Levan and levansucrases: polymer, enzyme, micro-organisms and biomedical from the Polish Ministry of Science and Higher Education for the applications, Biocatal. Biotransformation. 2422 (2017) 1–12, https://doi.org/10. Faculty of Biotechnology and Faculty of Chemistry of University of 1080/10242422.2017.1314467. [25] A.D. Sezer, H. Kazak Sarılmışer, E. Rayaman, A. Çevikbaş, E.T. Öner, J. Akbuğa, Wroclaw, by The National Center for Research and Development, Development and characterization of vancomycin-loaded levan-based micro- Poland (NCBiR) POIR.01.01.02-00-0128/16-00, POIR.01.02.00-00- particular system for drug delivery, Pharm. Dev. Technol. 22 (2017), https://doi. 0064/17 and Wroclaw Center of Biotechnology, Poland (WCB) program org/10.3109/10837450.2015.1116564. [26] S.J. Kim, P.K. Bae, B.H. Chung, Self-assembled levan nanoparticles for targeted The Leading National Research Center, Poland (KNOW) for years breast cancer imaging, Chem. Commun. (2015), https://doi.org/10.1039/ 2014–2018. c4cc07679f. [27] Y.W. Han, Microbial levan, Adv. Appl. Microbiol. 35 (1990) 171–194. Appendix A. Supplementary material [28] I. Makino Tadashi, K. Masayuki Yamada, K. Jun-ichi, Fast dissolving tablet and production, US5501861A US Grant, 1996. [29] S.A. Kang, K.-H. Jang, J.-W. Seo, K.H. Kim, Y.H. Kim, D. Rairakhwada, M.Y. Seo, Supplementary data to this article can be found online at https:// J.O. Lee, S. Do Ha, C.-H. Kim, S.-K. Rhee, Levan: applications and perspectives, doi.org/10.1016/j.bioorg.2019.02.012. Microb. Prod. Biopolym. Polym. Precursors. (2009) 145–161, https://doi.org/10. 1017/s1049023x11006789. [30] I.L. Shih, L.D. Chen, T.C. Wang, J.Y. Wu, K.S. Liaw, Tandem production of levan and References ethanol by microbial fermentation, Green Chem. 12 (2010) 1242–1247, https://doi. org/10.1039/b924765c. [31] I.L. Shih, T.C. Wang, S.Z. Chou, G. Der Lee, Sequential production of two biopo- [1] C. Caballero-George, M.I. Marin, C. Briceño, Critical evaluation of biodegradable lymers-levan and poly-lysine by microbial fermentation, Bioresour. Technol. polymers used in nanodrugs, Int. J. Nanomed. 8 (2013) 3071, https://doi.org/10. (2011), https://doi.org/10.1016/j.biortech.2010.11.066. 2147/IJN.S47186. [32] Y.W. Han, M.A. Clarke, Production and characterization of microbial levan, J. [2] J. Joshi, R. Patel, Role of biodegradable polymers in drug delivery, Int. J. Curr. Agric. Food Chem. (1990), https://doi.org/10.1021/jf00092a011. Pharm. Res. 4 (2012) 74–81. [33] H. Kazak Sarilmiser, O. Ates, G. Ozdemir, K.Y. Arga, E. Toksoy Oner, Effective [3] K.H. Kim, C.B. Chung, Y.H. Kim, K.S. Kim, C.S. Han, C.H. Kim, Cosmeceutical stimulating factors for microbial levan production by Halomonas smyrnensis properties of levan produced by Zymomonas mobilis, Int. J. Cosmet. Sci. 28 (2006) AAD6T, J. Biosci. Bioeng. (2015), https://doi.org/10.1016/j.jbiosc.2014.09.019. 231 doi:ICS314_2 [pii]\r10.1111/j.1467-2494.2006.00314_2.x. [34] P. Jajor, D. Piłakowska-Pietras, A. Krasowska, M. Łukaszewicz, Surfactin analogues [4] E.T. Oner, L. Hernandez, J. Combie, Review of Levan polysaccharide: from a cen- produced by Bacillus subtilis strains grown on rapeseed cake, J. Mol. Struct. 1126 tury of past experiences to future prospects, Biotechnol. Adv. 34 (2016) 827–844, (2016) 141–146, https://doi.org/10.1016/j.molstruc.2016.02.014. https://doi.org/10.1016/j.biotechadv.2016.05.002. [35] I.L. Shih, Y.T. Yu, Simultaneous and selective production of levan and poly(γ-glu- [5] T. Kim, K. Kim, K. Ryo, O. Lee, T. Kim, Cosmetic composition containing levan tamic acid) by Bacillus subtilis, Biotechnol. Lett. (2005), https://doi.org/10.1007/ having cell proliferation, skin-moisturizing and irritation-alleviating effects, s10529-004-6936-z. Japanese Patent 2003277225, 2003. [36] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram [6] O.M. Bondarenko, A. Ivask, A. Kahru, H. Vija, T. Titma, M. Visnapuu, U. Joost, quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. K. Pudova, S. Adamberg, T. Visnapuu, T. Alamäe, Bacterial polysaccharide levan as 72 (1976) 248–254, https://doi.org/10.1016/0003-2697(76)90527-3. stabilizing, non-toxic and functional coating material for microelement-nano- [37] R.A. Anderson, H.F. Conway, A.J. Peplinski, gelatinization of corn grits by roll particles, Carbohydr. Polym. 136 (2015) 710–720, https://doi.org/10.1016/j. cooking extrusion, cooking and steaming, Starch - Stärke 22 (1970) 130–135, carbpol.2015.09.093. https://doi.org/10.1002/star.19700220408. [7] F. Masayo, T. Takayuki, Beautifully whitening agent, JP2004000232887, 2006. [38] F. Feng, Q. Zhou, Y. Yang, F. Zhao, R. Du, Y. Han, H. Xiao, Z. Zhou, Characterization [8] K.H. Kim, S.K. Rhee, C.H. Kim, S.N. Park, S.M. Lee, Cosmetic composition com- of highly branched dextran produced by Leuconostoc citreum B-2 from pineapple prising beta-fructosyl-L-ascorbic acid for skin whitening, US 7,932,232 B2, 2006. fermented product, Int. J. Biol. Macromol. 113 (2018) 45–50, https://doi.org/10. < http://doi.org/10.1038/incomms1464 > . 1016/j.ijbiomac.2018.02.119. [9] E.L. Evans, T.C. Gavrilovich, E. Mihai, R.C. Isbasescu, I D. Thelen, J.A. Martin, S.M. [39] K. Sidoryk, M. Świtalska, A. Jaromin, P. Cmoch, I. Bujak, M. Kaczmarska, Allen, S. SA, Delivery system for cosmetic and skincare products, US 2008/0069864 J. Wietrzyk, E.G. Dominguez, R. Żarnowski, D.R. Andes, K. Bańkowski, M. Cybulski, A1, 2014. < http://doi.org/10.1037/t24245-000 > . Ł. Kaczmarek, The synthesis of indolo[2,3-b]quinoline derivatives with a guanidine [10] A.M. Abdel-Fattah, A.M. Gamal-Eldeen, W.A. Helmy, M.A. Esawy, Antitumor and group: highly selective cytotoxic agents, Eur. J. Med. Chem. 105 (2015) 208–219, antioxidant activities of levan and its derivative from the isolate Bacillus subtilis https://doi.org/10.1016/J.EJMECH.2015.10.022. NRC1aza, Carbohydr. Polym. 89 (2012) 314–322, https://doi.org/10.1016/J. [40] R. Zarnowski, A. Jaromin, M. Certik, T. Czabany, J. Fontaine, T. Jakubik, CARBPOL.2012.02.041. M.C.M. Iqbal, A. Grandmougin-Ferjani, A. Kozubek, S.J. Pietr, The oil of [11] F. Sima, E.C. Mutlu, M.S. Eroglu, L.E. Sima, N. Serban, C. Ristoscu, S.M. Petrescu, Adenanthera pavonina L. seeds and its emulsions, Zeitschrift Fur Naturforsch. - Sect. E.T. Oner, I.N. Mihailescu, Levan nanostructured thin films by MAPLE assembling, C J. Biosci. 59 (2004) 321–326, https://doi.org/10.1515/znc-2004-5-605. Biomacromolecules 12 (2011) 2251–2256, https://doi.org/10.1021/bm200340b. [41] A. Jaromin, R. Zarnowski, A. Kozubek, Emulsions of oil from Adenanthera pavonina [12] J. Harris, Hair product with stable viscosity, US 2015/0342861 A1, 2015. L. seeds and their protective effect, Cell. Mol. Biol. Lett. 11 (2006) 438–448, [13] E. Naiberk, V. Decarlo, A. Botto, A. Suleiman, A. Mahadeshwar, Leave-on hair https://doi.org/10.2478/s11658-006-0037-z. styling compositions and methods of use, US 9,918,923 B1, 2018. [42] A. Jaromin, R. Zarnowski, M. Piȩtka-Ottlik, D.R. Andes, J. Gubernator, Topical [14] M.M. Fiume, B. Heldreth, W.F. Bergfeld, D.V. Belsito, R.A. Hill, C.D. Klaassen, delivery of ebselen encapsulated in biopolymeric nanocapsules: drug repurposing D.C. Liebler, J.G. Marks, R.C. Shank, T.J. Slaga, P.W. Snyder, F.A. Andersen, enhanced antifungal activity, Nanomedicine 13 (2018) 1139–1155, https://doi. L.J. Gill, Safety assessment of microbial polysaccharide gums as used in cosmetics, org/10.2217/nnm-2017-0337. Int. J. Toxicol. 35 (2016) 5S–49S, https://doi.org/10.1177/1091581816651606. [43] J.A. Buege, S.D. Aust, Microsomal lipid peroxidation, Methods Enzymol. 52 (1978) [15] S.K. Gupta, P. Das, S.K. Singh, M.S. Akhtar, D.K. Meena, S.C. Mandal, Microbial 302–310, https://doi.org/10.1016/S0076-6879(78)52032-6. levan, an ideal prebiotic and immunonutrient in aquaculture, World Aquac. (1990) [44] R. Srikanth, G. Siddartha, C.H.S.S. Sundhar Reddy, B.S. Harish, M. Janaki Ramaiah, 61–64, https://doi.org/10.1142/9789814360807. K.B. Uppuluri, Antioxidant and anti-inflammatory levan produced from Acetobacter [16] J. Combie, Properties of levan and potential medical uses, Polysaccharides Drug xylinum NCIM2526 and its statistical optimization, Carbohydr. Polym. 123 (2015) Deliv. Pharm. Appl. 934 (2006) 13–263, https://doi.org/10.1021/bk-2006-0934. 8–16, https://doi.org/10.1016/j.carbpol.2014.12.079.

7 M. Domżał-Kędzia, et al. Bioorganic Chemistry 93 (2019) 102787

[45] N.R. Jathore, M.V. Bule, A.V. Tilay, U.S. Annapure, Microbial levan from [60] R. Shukla, A. Goyal, Elucidation of structure and biocompatibility of levan from Pseudomonas fluorescens: characterization and medium optimization for enhanced Leuconostoc mesenteroides NRRL B-1149, Curr. Trends Biotechnol. Pharm. 7 (2013) production, Food Sci. Biotechnol. 21 (2012) 1045–1053, https://doi.org/10.1007/ 635–643. s10068-012-0136-8. [61] U.J. Yun, H.D. Park, Physical properties of an extracellular polysaccharide pro- [46] F.R. Seymour, R.D. Knapp, J.E. Zweig, S.H. Bishop, 13C-nuclear magnetic resonance duced by Bacillus sp. CP912. Lett. Appl. Microbiol. 36 (2003) 282–287, https://doi. spectra of compounds containing β-D-fructofuranosyl groups or residues, org/10.1046/j.1472-765X.2003.01309.x. Carbohydr. Res. 72 (1979) 57–69, https://doi.org/10.1016/S0008-6215(00) [62] Y. Yang, Q. Peng, Y. Guo, Y. Han, H. Xiao, Z. Zhou, Isolation and characterization of 83923-3. dextran produced by Leuconostoc citreum NM105 from manchurian sauerkraut, [47] R. Srikanth, C.H.S.S. Sundhar Reddy, G. Siddartha, M. Janaki Ramaiah, K. Babu Carbohydr. Polym. 133 (2015) 365–372, https://doi.org/10.1016/J.CARBPOL. Uppuluri, Review on production, characterization and applications of microbial 2015.07.061. levan, Carbohydr. Polym. 120 (2015) 102–114, https://doi.org/10.1016/j.carbpol. [63] C. Saravanan, P.K.H. Shetty, Isolation and characterization of exopolysaccharide 2014.12.003. from Leuconostoc lactis KC117496 isolated from idli batter, Int. J. Biol. Macromol. [48] H. Hammer, S. Morgenlie, Classification of grass fructans by C NMR Spectroscopy. 90 (2016) 100–106, https://doi.org/10.1016/j.ijbiomac.2015.02.007. pdf, (n.d.). [64] Mamay, D. Wahyuningrum, R. Hertadi, Isolation and characterization of levan from [49] J. Tomašić, H.J. Jennings, C.P.J. Glaudemans, Evidence for a single type of linkage moderate halophilic bacteria bacillus licheniformis BK AG21, Procedia Chem. 16 in a fructofuranan from Lolium perenne, Carbohydr. Res. 62 (1978) 127–133, (2015) 292–298, https://doi.org/10.1016/j.proche.2015.12.055. https://doi.org/10.1016/S0008-6215(00)83384-4. [65] W. Xu, Q. Liu, Y. Bai, S. Yu, T. Zhang, B. Jiang, W. Mu, Physicochemical properties [50] C. Liu, J. Lu, L. Lu, Y. Liu, F. Wang, M. Xiao, Isolation, structural characterization of a high molecular weight levan from Brenneria sp. Eni D312, Int. J. Biol. and immunological activity of an exopolysaccharide produced by Bacillus licheni- Macromol. 109 (2018) 810–818, https://doi.org/10.1016/j.ijbiomac.2017.11.056. formis 8–37-0-1, Bioresour. Technol. 101 (2010) 5528–5533, https://doi.org/10. [66] H. Espinosa-Andrews, R. Rodríguez-Rodríguez, Water state diagram and thermal 1016/j.biortech.2010.01.151. properties of fructans powders, J. Therm. Anal. Calorim. 132 (2018) 197–204, [51] X. Xu, C. Gao, Z. Liu, J. Wu, J. Han, M. Yan, Z. Wu, Characterization of the levan https://doi.org/10.1007/s10973-017-6868-1. produced by Paenibacillus bovis sp. nov BD3526 and its immunological activity, [67] J.R. Porras-Domínguez, Á. Ávila-Fernández, A. Miranda-Molina, M.E. Rodríguez- Carbohydr. Polym. 144 (2016) 178–186, https://doi.org/10.1016/j.carbpol.2016. Alegría, A.L. Munguía, Bacillus subtilis 168 levansucrase (SacB) activity affects 02.049. average levan molecular weight, Carbohydr. Polym. 132 (2015) 338–344, https:// [52] M. Schwanninger, J.C. Rodrigues, H. Pereira, B. Hinterstoisser, Effects of short-time doi.org/10.1016/j.carbpol.2015.06.056. vibratory ball milling on the shape of FT-IR spectra of wood and cellulose, Vib. [68] F.-C. Wu, S.-Z. Chou, I.-L. Shih, Factors affecting the production and molecular Spectrosc. 36 (2004) 23–40, https://doi.org/10.1016/J.VIBSPEC.2004.02.003. weight of levan of Bacillus subtilis natto in batch and fed-batch culture in fermenter, [53] X. Yu, L. Li, J. Zhang, Z. Shen, C. Zhu, P. Wang, X. Jiang, Structural analysis of J. Taiwan Inst. Chem. Eng. 44 (2013) 846–853, https://doi.org/10.1016/j.jtice. macromolecular levan produced by Bacillus megaterium GJT321 based on enzymatic 2013.03.009. method, Int. J. Biol. Macromol. 93 (2016) 1080–1089, https://doi.org/10.1016/j. [69] J.K. Lee, D.B. Kim, J. Il Kim, P.Y. Kim, In vitro cytotoxicity tests on cultured human ijbiomac.2016.09.086. skin fibroblasts to predict skin irritation potential of surfactants, Toxicol. Vitr.14 [54] H.G. Pontis, Fructans and cold stress, J. Plant Physiol. 134 (1989) 148–150, https:// (2000) 345–349, https://doi.org/10.1016/S0887-2333(00)00028-X. doi.org/10.1016/S0176-1617(89)80047-1. [70] O. Damour, S. Zhi Hua, F. Lasne, M. Villain, P. Rousselle, C. Collombel, Cytotoxicity [55] R. Valluru, W. Van den Ende, Plant fructans in stress environments: emerging evaluation of antiseptics and antibiotics on cultured human fibroblasts and kera- concepts and future prospects, J. Exp. Bot. 59 (2008) 2905–2916, https://doi.org/ tinocytes, Burns 18 (1992) 479–485, https://doi.org/10.1016/0305-4179(92) 10.1093/jxb/ern164. 90180-3. [56] D.P. Livingston, D.K. Hincha, A.G. Heyer, Fructan and its relationship to abiotic [71] M.M. Soltan-Dallal, M. Validi, M. Douraghi, J. Fallah-Mehrabadi, stress tolerance in plants, Cell. Mol. Life Sci. 66 (2009) 2007–2023, https://doi.org/ L. Lormohammadi, Evaluation the cytotoxic effect of cytotoxin-producing Klebsiella 10.1007/s00018-009-0002-x. oxytoca isolates on the HEp-2 cell line by MTT assay, Microb. Pathog. 113 (2017) [57] P. Gilbert, J. Das, I. Foley, Biofilm susceptibility to antimicrobials, Adv. Dent. Res. 416–420, https://doi.org/10.1016/J.MICPATH.2017.11.003. 11 (1997) 160–167, https://doi.org/10.1177/08959374970110010701. [72] L.F. Dos Santos, F.C. Bazani Cabral De Melo, W.J. Martins Paiva, D. Borsato, [58] H.C. Flemming, Biofilms and environmental protection, Water Sci. Technol. 27(27) M.D.L. Corradi Custódio Da Silva, M.A.P. Colabone Celligoi, Characterization and (1993) 1–10. optimization of levan production by Bacillus subtilis NATTO, Rom. Biotechnol. Lett. [59] B. Kekez, G. Gojgić-Cvijović, D. Jakovljević, V. Pavlović, V. Beškoski, A. Popović, 18 (2013) 8413–8422. M.M. Vrvić, V. Nikolić, Synthesis and characterization of a new type of levan-graft- [73] A. Janaszewska, G. Bartosz, Assay of total antioxidant capacity: comparison of four polystyrene copolymer, Carbohydr. Polym. 154 (2016) 20–29, https://doi.org/10. methods as applied to human blood plasma, Scand. J. Clin. Lab. Invest. 62 (2002) 1016/J.CARBPOL.2016.08.001. 231–236, https://doi.org/10.1080/003655102317475498.