Bacillus amyloliquefaciens levan and its silver nanoparticles with antimicrobial properties

Abstract In this study, the exopolysaccharide from Bacillus amyloliquefaciens PB6 was purified and identified as levan. To explore the application of this levan in nanotechnology, its silver nanoparticles (AgNPs) were prepared in a simple and green method. Transmission electron microscopy analysis revealed that the size of the synthesised AgNPs was in the range of 19.47 ± 2.45 nm. X-ray powder diffraction analysis confirmed the face-centred cubic crystalline structure of metallic silver. Moreover, the AgNPs at the concentration of 0–12 μg/mL exhibited a significant inhibitive effect on Staphylococcus aureus and Escherichia coli. Tested by tetrazolium-based colourimetric assay, the viability of RAW 264.7 cells was higher than 75% in the presence of 20 μg/mL AgNPs. The results not only proved the feasibility of producing levan from B. amyloliquefaciens with high safety and a high yield, but also demonstrated the simple and cost-efficient approach of AgNPs synthesis as potential antibacterial agents from levan.


Introduction
Several levansucrases from the generally regarded as safe (GRAS) strain of Bacillus amyloliquefaciens have been purified, characterised and heterologously expressed, for use in the biosynthesis of levan [1,2]. Some exopolysaccharides also have been produced from B. amyloliquefaciens [3,4]. However, to the best of our knowledge, there have been few reports on the direct production of levan in a high yield from B. amyloliquefaciens [5,6]. In the past, we optimised the fermentation of an exopolysaccharide from B. amyloliquefaciens PB6 at a high yield of 104 g/L, but the structure of the produced exopolysaccharide was not identified [6].
Levan is a natural homopolysaccharide composed of D-fructo-furanosyl residues linked by b-(2,6) glycosidic linkage as the main chain. In the last few years, levan and its derivatives have attracted significant attention in the biotech and the chemical industry [7,8]. At the same time, silver nanoparticles (AgNPs) have shown great application potential in the research of catalysis, biosensing, drug delivery and medical textiles, due to their unique optical, electrical and biological properties [9][10][11]. Polysaccharides have been widely used in the synthesis of AgNPs as ecofriendly reducing and capping agents, and therein exopolysaccharides are fascinating in nanocomposites, attributed to their feasibility of large-scale or mass industrial production [12][13][14][15]. As for levan, besides antibacterial iron oxide nanoparticles prepared from levan of Bacillus polymyxa PTCC1020 [16], only AgNPs from levan of Acetobacter xylinum with catalytic ability have been reported by Ahmed et al. [17]. Moreover, with the optimised variables from the statistical methods, the highest yield of levan produced from B. polymyxa PTCC1020 and A. xylinum NCIM2526 was only 27 g/L and 13.25 g/L, respectively [18], indicating that levan from the two strains was not suitable for the large-scale industrial production of AgNPs.
In this article, the exopolysaccharide produced from B. amyloliquefaciens PB6 was identified as levan and its AgNPs were further prepared. Moreover, the antimicrobial and cytotoxic effects of the prepared AgNPs were studied.
The exopolysaccharide-producing B. amyloliquefaciens PB6 is a strain maintained in the National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences.
RAW 246.7, a murine macrophage cell line was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China).

Production of exopolysaccharide from B. amyloliquefaciens PB6 by fermentation
According to our reported procedure [6], the fermentation production of the exopolysaccharide from B. amyloliquefaciens PB6 was carried out under the optimal culture medium (400 g/L sucrose, 2.5 g/L yeast extract, 10 g/L sodium chloride) and the optimal fermentation conditions (initial pH 7.0, 4% inoculation amount, 30 C, 150 r/min) for 26 h. Then the culture medium was centrifuged to remove the bacterial cells, and the exopolysaccharide was harvested from the culture broth by ethanol precipitation, followed by deproteinization with Sevag reagent and dialysis through a membrane with a 10-kDa cutoff. Finally, after freeze drying, the exopolysaccharide was obtained as white powder.
Molecular weight distribution. The molecular weight distribution was determined by gel permeation chromatography on an Agilent 1200 Series HPLC (high performance liquid chromatography) system (Agilent Technologies, Palo Alto, CA, USA) equipped with a refractive index detector and an Agilent PL aquagel-OH MIXED-H column (300 Â 7.5 mm, 8 mm), using standard dextrans as references.
Sugar composition and methyl analysis. The sugar composition and methyl analysis were carried out strictly as described by Dong et al. [19].
Nuclear magnetic resonance (NMR) analysis. 1 H NMR and 13 C NMR spectra were recorded on a Brucker AVANCE III spectrometer (Bruker-Biospin, Rheinstetten, Germany) using D 2 O as solvent.
Preparation of AgNPs. According to the method of Ahmed et al. [17], 20 mg levan was dissolved in 49 mL of 0.2% sodium hydroxide solution, by subsequent addition of 1 mL silver nitrate aqueous solution (1 mmol/L final concentration). The mixture was allowed to stir at room temperature for 5 min and then heated to 100 C for 30 min. Then the synthesised AgNPs were dialysed with a 3.5-kDa cutoff membrane for 24 h in darkness and were freeze-dried.
Characterization of AgNPs. The ultraviolet (UV)-vis spectrum was measured using a UV 1800 spectrophotometer (Shimadzu, Tokyo, Japan). X-ray powder diffraction (XRD) measurement was performed at room temperature by a Bruker D8 Advance diffractometer (Bruker Optik GMBH, Karlsruhe, Germany) at an accelerating voltage of 40 kV using CuKa (a ¼ 1.5406A˚) as an X-ray source. The shape, size and dispersity of AgNPs were monitored by transmission electron microscopy (TEM; H-7500, Hitachi, Tokyo, Japan) at an accelerating voltage of 80 kV. Silver content was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES; Agilent Technologies 700 Series).
Antibacterial assay. The antibacterial activity of AgNPs was studied by the growth curve method with different concentrations of AgNPs [20]. Log phase bacterial cultures in Luria-Bertani (LB) medium with an initial 0.1 absorbance at OD 600 were incubated at 37 C at 200 rpm, after the addition of AgNPs. The bacterial growth was monitored at an interval of 2 h by the absorbance of the culture media at 600 nm (OD 600 ) .
Cell viability analysis. Cell viability was measured with tetrazolium-based colourimetric assay in RAW 264.7 cells. Briefly, RAW 264.7 cells (100 lL, 5 Â 10 4 cell/mL) were incubated in a 96-well plate for 24 h at 37 C and 5% CO 2 conditions. Then the cells were washed with fresh Dulbecco's Modified Eagle Medium (DMEM medium) and treated with AgNPs at final concentrations of 0-20 mg/mL for 24 h. After that, 10 lL of 5 mg/mL MTT was added into each well and the plate was incubated at 37 C for 4 h. Finally, the medium was replaced with 150 mL of dimethyl sulphoxide to dissolve the formazan crystals, and the absorbance of the solution was measured at 570 nm. DMEM medium with only RAW 264.7 cells served as the control.

Data analysis
Data are expressed as mean values with standard deviation (±SD) from three independent experiments. Statistical comparisons were performed using analysis of variance (ANOVA), and differences at P < 0.05 were considered statistically significant.

Identification of B. amyloliquefaciens levan
The purified exopolysaccharide from B. amyloliquefaciens PB6 had only one single symmetrical peak on gel permeation chromatography (Figure 1(a)), indicating its molecular weight homogeneity. The average molecular weight of the exopolysaccharide was estimated as 6.02 kDa. In the sugar composition analysis, the exopolysaccharide was determined to predominantly have fructose (97.7%) with some glucose (2.3%), as seen in Figure 1(b). Methyl analysis confirmed the existence of major (2!6)-linked Fruf (90.02%) units and a small amount of (1,2!6)linked Fruf (2.14%), t-Glcp (2.29%) as well as t-Fruf (5.55%) units in the exopolysaccharide. In Fourier transform infrared spectroscopy (FTIR), the exopolysaccharide showed a broad peak centred at 3400 cm À1 from the O-H stretching, a peak at 2937 cm À1 from the C-H stretching, three peaks at 1128, 1061 and 1019 cm À1 from the glycosidic linkage (C-O-C) stretching and two peaks at 928 and 814 cm À1 representative of the furanoid fructose ( Figure 2). The results from 1 H NMR and 13 C NMR are shown in Figure 1(c,d), respectively. In the 13 C NMR spectrum, the chemical shift at 104.2, 103.8 and 103.6 ppm indicating b-type glycosidic linkages corresponded to the fructosyl anomeric carbon in (2!6), (1,2!6) linkage and at the terminus, respectively, whereas the chemical shifts at 80.3, 76.3, 75.2, 63.3 and 60.1 ppm were representative of C-5, C-3, C-4, C-6 and C-1 of the fructosyl unit, respectively.  Consistent with other reports on microbial levans [21,22], the above analysis suggested that the exopolysaccharide from B. amyloliquefaciens PB6 existed as a levan structure having b-(2,6) linked fructofuranosyl backbone and some b-(1,2!6) linked fructofuranosyl branches with a D-fructosyl or D-glucosyl residue at the end of the main chain. Gu et al. [23] reported that, through metabolic optimisation of regulatory elements, the levan production in the recombinant B. amyloliquefaciens strain could be improved to 102 g/L, which was close to the reported highest levan production level in the literature. Considering that B. amyloliquefaciens PB6 is a non-engineered strain and its exopolysaccharide yield was 104 g/L, the confirmation of levan structure in this study implied that levan could be produced from B. amyloliquefaciens PB6 with high safety and a high yield.

Characterisation of AgNPs
Attributed to the excitation of surface plasmon resonance (SPR) in the metal nanoparticles, the formation of AgNPs from levan was preliminarily detected by the colour change of the reaction solution from colourless to yellow-brown [24]. A typical SPR band of AgNPs around 409 nm was also observed in the UV-vis spectrum (Figure 3(a)). According to Yang et al. [25], the SPR band at 409 nm indicated that the size of the AgNPs was approximately between 4 and 20 nm. The TEM image showed that the AgNPs from levan were almost spherical with an average diameter of 19.47 ± 2.45 nm (Figure 3(b,c)). Though the size was larger than that reported by Ahmed et al. [17], our AgNPs from levan had a smaller diameter than the AgNPs synthesised from exopolysaccharides of Leuconostoc lactis KC117496, Streptomyces violaceus MM72 and Cs-Hk fungal cultures [26][27][28]. It was speculated the molecular dimension of these exopolysaccharides might be one cause of the different size of their AgNPs. In the XRD pattern analysis, five main characteristic peaks at 2h values of 38 , 44 , 64 , 77 and 81 corresponding to 111, 200, 220, 311 and 222 planes for silver (Figure 3(d)), confirmed the face-centred cubic silver in the AgNPs from levan [29]. In the FTIR spectrum of AgNPs (Figure 2), all peaks of levan were still observed and the stretching vibration absorption peak of O-H at 3400 cm À1 in levan was shifted to a lower wavenumber round 3320 cm À1 , suggesting interactions between the hydroxyl groups of the levan and the AgNPs [30].

Antibacterial and cytotoxic activity of AgNPs
The results from the antibacterial test are shown in Figure 4. AgNPs exhibited a dose-dependent growth inhibition activity against both Gram-positive and Gram-negative bacteria, including K. pneumoniae and S. typhimurium. The minimum inhibitory concentration (MIC) value of the AgNPs for E. coli was determined to be 12.8 mg/mL, and its best antimicrobial activity was detected in S. aureus: the growth of this strain was almost inhibited in 10 h with the AgNPs concentration at or higher than 9.6 mg/mL. Our results had some differences from the report of Sivasankar et al. [25], in which exopolysaccharide mediated AgNPs synthesised by S. violaceus MM72 showed MIC values of 6.15 and 14.6 lg/mL against E.coli and S. aureus, respectively. However, the high sensitivity of S. aureus to AgNPs was not only observed by us, but also reported in the study of AgNPs prepared from exopolysaccharide of Cordyceps militaris Hk fungal cultures [26]. The reason for this observation was unclear and needs more investigation. Despite of its potent antibacterial activity and wide biological applications, the use of AgNPs as a therapeutic agent is limited due to their potential cytotoxic activity against mammalian cells [31]. Thus, the effect of AgNPs on the viability of macrophage RAW 264.7 cells was also tested by tetrazolium-based colourimetric assay. The result showed that the cell inhibition was below 25% at the presence of 20 lg/mL AgNPs ( Figure 5). Freire et al. [32] thought that colloidal chitosan-silver nanoparticle-fluoride nanocomposites were non-toxic at a concentration of 50 mg/mL, since in their presence, the RAW 264.7 cell inhibition was below 50%. According to their report, our result indicated that AgNPs from levan are able to display antibacterial activity without being harmful to macrophages.

Conclusions
The identification of the low-molecular weight levan confirmed the direct fermentation production of levan from B. amyloliquefaciens at a high yield, providing a safe and efficient industrial production process for levan. Moreover, AgNPs could be prepared from this levan and showed good antibacterial activity with low cytotoxicity, indicating the application potential of levan-based AgNPs in biomedical and nanotechnology industries.

Disclosure statement
No potential conflict of interest was reported by the authors.

References
[1] Tian F, Inthanavong L, Karboune S. Purification and characterization of levansucrases from Bacillus amyloliquefaciens in intra-and extracellular forms useful for  Note: Silver ion in the form of AgNO 3 was used as the negative control. Data are expressed as mean values; error bars represent ± SD (n ¼ 3). Asterisk indicates statistically significant difference between untreated cells and synthesised AgNPs (or AgNO 3 ) treated cells ( Ã P < 0.05).