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Enhancement of antioxidant activity of levan through the formation of nanoparticle systems with metal ions

Levan, a natural polymer, is widely used in biomedical applications, such as antioxidants, anti-inflammatory, and anti-tumor. The present study aimed to enhance the antioxidant activity of levan by combining it with various metal ions in the nanoparticle (NP) system. Levansucrase encoding gene from Bacillus licheniformis BK1 has been inserted into an expression vector and the obtained recombinant was labeled as Lsbl-bk1 (accession number MF774877.1). That enzyme was used for in vitro levan synthesis in 12% (w/v) sucrose as a substrate and about 4.28 mg/mL of levan was obtained. Levan-based metal ion NPs were synthesized using the coprecipitation method. In the production of NPs, levan acts as a reducing and stabilizing agent. Four types of levan-based metal ion NPs were synthesized, namely, levan–Fe2+ NPs, levan–Cu+ NPs, levan–Co2+ NPs, and levan–Zn2+ NPs. The transmission electron microscopy (TEM) technique was applied to visualize the size and shape of the synthesized levan–metal NPs. All levan-based metal ion NPs have a particle size of less than 100 nm, and even levan–Cu+ and levan–Zn2+ have particle sizes less than 50 nm. Levan–Fe2+ NPs and levan–Cu+ NPs exhibited prominent antioxidant activity with an inhibition level of up to 88% and 95%, respectively. And the inhibition level of two metal ion NPs had about 33%–40% higher antioxidant activity compared with the inhibition level of levan only. The two levan–metal ion NPs, therefore, have future prospects to be developed as the new formulation for the antioxidant drugs.


Introduction
Free radicals, which are unstable compounds that have unpaired electrons, are very reactive because they always try to form electron pair by reacting with other substances (proteins, fats, and DNA) in the body. In certain amounts, free radicals are needed for health to fight inflammation, kill bacteria, and regulate smooth muscle tone in organs and blood vessels. Phagocytes, such as neutrophils, macrophages, and monocytes, release free radicals to destroy invading pathogenic microbes as part of the body's defense mechanism against diseases (Pham-Huy et al., 2008). However, the amount of excessive free radicals can be damaging and very dangerous. Free radicals are formed through normal cell metabolism, inflammation, lack of nutrients, gamma radiation, ultraviolet (UV), environmental pollution, and cigarette smoke. Naturally, the body's antioxidant system as a mechanism of protection against free radical attacks is already present in the body. There are two kinds of antioxidants: internal and external antioxidants. Internal antioxidants are those produced by the body itself. Naturally, the body is able to produce its own antioxidants, but this ability has some limitations. The body's ability to produce natural antioxidants will decrease because of age (Buehler, 2012). Oxidative damage from free radicals in the body can basically be overcome by endogenous antioxidants, such as enzyme catalase, glutathione peroxidase, superoxide dismutase, and glutathione S-transferase (Peng et al., 2014). But if there are excess free radical compounds in the body or if they exceed the limits of cellular antioxidant protection capabilities, additional antioxidants from the outside or exogenous antioxidants are needed to neutralize the formed radicals. The ideal exogenous antioxidant should be easily absorbed and sent to the intracellular location needed to reduce pathological oxidative damage. To develop effective antioxidants, the best strategy is to create a new nanoscale drug delivery system because of its size. The nanosized particles which is in the range of 10-100 nm can facilitate the penetration to the tissue system thereby improving the uptake of the drug by the targeted cells (Mirza and Siddiqui, 2014).
Exopolysaccharide is a polysaccharide produced and secreted by microbial cells into the medium (extracellular) (Rehm, 2009). Several types of bacteria, fungi, yeast, and microalgae have been investigated to have the ability to produce exopolysaccharides. The presence of exopolysaccharides is characterized by the growth of mucus in the growth medium of microorganisms (Nwodo and Okoh, 2012). In general, exopolysaccharides produced by these microorganisms have significant biological functions which are as cell protector and preventing cell aggregation (Nicolaus et al., 2010). Levan is a branched exopolysaccharide composed of fructose units with β-(2,6)-glycosidic bonds in the main chain and β-(2,1)-glycosidic bonds in its branching (Liu et al., 2012). Levan has been widely applied in the food industry as a low-calorie prebiotic and sweetener, as well as in the health sector as antidiabetic, antiviral, anti-inflammatory, antitumor, and antioxidant (Kang et al., 2009). Currently levan is also used as material for the manufacture of nanoparticles to encapsulate drugs, catalysts and metal ions (Carlos et al., 2018).
Levan synthesis requires the help of levansucrase biocatalyst (E.C 2.4.1.10), with sucrose as its substrate (Freitas et al., 2011). This enzyme synthesizes levan in three reaction stages, including hydrolysis of sucrose to glucose and fructose, then transfer fructose to sucrose (transfructosylation), and the fructose chain extension stage (polymerization) (Badel et al., 2011). Levans can be obtained by direct production of original bacteria or by recombinant techniques (Jathore et al., 2012). However, problems exist in the use of bacteria originating from levan production, such as the level of acquisition of levan which is on a low scale. Isolation and characterization of levansucrase-encoding genes from Bacillus licheniformis BK1 were performed and named as Lsbl-bk1 (accession number MF774877.1). The gene was successfully cloned into the pGEM-T Easy Vector in the host E. coli TOP10 cell and was also subcloned to the pET-30A expression vector in the E. coli BL21 (DE3) plysS host cell. Therefore, the production of recombinant levansucrase from host cell E. coli BL21 (DE3) plysS-Lsbl-bk1 was the first step in obtaining biocatalysts to synthesize levan in vitro. It is hoped that the production of recombinant levansucrase in optimum conditions can produce a greater amount of levan.
Many researchers have conducted studies of the potential of levan as an antioxidant (Srikanth et al., 2015;Zhao et al., 2018). Further studies on increasing antioxidant activity have been conducted by modifying antioxidant material in terms of particle size. Nano-sized particles have more reactive properties, large surface area, small diffusion distances, and greater adhesion styles (Sarilmiser and Oner, 2014). In addition, levan nanoparticle has been also well-studied by Gonz alez-Garcinuño et al. (2020) which performed a comprehensive study about levan nanoparticle formation by developing a kinetic model for a self-assembly. The model predicted well the required critical aggregation concentration (CAC) to form nanoparticles. Gonz alez-Garcinuño et al. (2019) also have studied silver-coated nanoparticles with bactericidal effect and found that levan nanoparticle did not interfere the killing effect of the silver to kill the bacterial targets.
This present study presents that nanotechnology provides new information on the use of levan as an encapsulation material for metal ions which has antioxidant activity to measure NPs to increase those activities. It also demonstrates that the antioxidant activity of metal ioncoated nanoparticles did not interfere by the levan nanoparticle system instead, it was enhanced significantly.

Bacterial strains
The bacterial strains Escherichia coli BL21(DE3)plysS carrying pET-Lsbl-bk1 used was from the stock culture maintained in our laboratory on LB medium which already contains 50 μg/mL kanamycin antibiotics (selective medium). This recombinant clone contains the levansucrase gene from Bacillus licheniformis BK1 (deposited at GenBank NCBI with the accession number of MF774877.1).

Production and characterization of levan in vitro
Production of levan in vitro was following the procedure by Srikanth et al. (2015) with modification. The transformant E. coli BL21(DE3), carrying pET-lsbl-bk1, was inoculated into 100 mL LB medium containing 50 μg kanamycin and incubated at 37 C. Enzyme expression was induced by adding 1 mM IPTG when the optical cell density (OD 600 ) of the culture reached 0.6-1.0. The expressed enzyme excreted extracellularly and separated from the cell by centrifugation at 8000 rpm for 15 min. Levan production in vitro was performed by mixing 50 mL of the crude recombinant levansucrase Lsbl-bk1 30 μg/mL with 50 mL sucrose solutions 12% (w/v) dissolved in 50 mM phosphate buffer pH 7 and incubated at 37 C for 18 h. That reaction was stopped by heating at 100 C for 5 min and then cooled down to room temperature. The precipitated impurities were discarded by centrifugation at 8,000 rpm for 15 min. Levan was obtained by precipitating the supernatant with 95% cold ethanol in a ratio of 1:3. Then, the pellet was freeze-dried for 4 h to obtain dry levan. The concentration of levan was determined by the phenol-sulfuric method at 490 nm using fructose as the standard. Levan was characterized using FTIR. And then the FTIR spectra of levan were compared with the FTIR spectra of pure levan from Erwinia herbicola.

Synthesis of levan-based metal ion nanoparticles
Levan-based metal ion nanoparticles (levan-metal NPs) were synthesized using coprecipitation methods, following the procedure by Pudovkin et al. (2018). In the production of nanoparticles, levan acts as a reducing and stabilizing agent. The metal ions which were used to be coated with levan in this study were Fe 2þ , Cu þ , Co 2þ , and Zn þ . Therefore, to obtain these metal ions, the metal solutions with a higher oxidation number were chosen, such as FeCl 3 , Cu(NO 3 ) 2 , Co(NO 3 ) 3 , and Zn(CH 3 COO) 2 , respectively. These metal ions were chosen because they are easily oxidized and has been applied as an antibacterial in the form of nanoparticles (Valgimigli et al., 2018). For each metal, 25 mg levan was dissolved in 25 mL NaOH 0.2% (w/v) in a nutshell. Then, levan solutions were mixed with 25 mL metal solutions of 100 mM, at room temperature, 500 rpm, for 10 min. The mixture then slowly heated at 100 C for 4 h or until a color change occurs. The color product obtained was centrifuged at 8000 rpm for 15 min and washed three times with distilled water. The precipitate was collected and freeze-dried for 4 h for further analysis.

Characterization of levan-based metal ion nanoparticles
All of the levan-based metal NPs was characterized using UV-Vis spectroscopy to observe the maximum wavelength from metal NP solution colors and FTIR (Fourier transform infrared) to observe the changes in levan wavelength before and after binding metal ions. The morphology and individual size of levan-metal NPs were analyzed using TEM (transition electron microscopy). The composition of metal ions from levanbased metal NPs was analyzed using EDS (energy-dispersive spectroscopy).

UV-Vis spectrophotometric analysis
UV-Vis spectra of the levan-based metal NPs were analyzed by Per-kinElmer UV-Vis double beam spectrophotometer. Deionized water was used as blank. The scanning range for each samples was 250-600 nm, operated at a resolution of 1 nm.

Fourier transform infrared (FTIR) spectroscopy
FTIR spectrum of levan, levan-Fe 2þ NPs, levan-Zn 2þ NPs, levan-Cu þ NPs, and levan-Co 2þ NPs was recorded using PerkinElmer Spectrum BX, FT-IR (PerkinElmer, Akron, OH, USA) at room temperature through potassium bromide pellet method. A total of 10 mg of NPs, metal salt, and levan were mixed with 10 mg of solid KBr, then it mixed well and pelletized using a vacuum pump. The pellet was kept in IR path and spectrum was measured in the 400-4,000 cm-1 range. FTIR spectroscopy measurements were carried out to recognize the biogroups that are attached on the surface of each samples.

Transmission electron microscopy (TEM) analysis
The morphology and particle size of levan-based metal ion nanoparticles were analyzed using TEM (Hitachi Model H-7500, Ltd., Tokyo, Japan) with an acceleration voltage of 100 kV. Firstly, the sample solution was sonicated for 10 min. Then, drop on a small of the sample solution was insulated in plate. The morphology of the particles was observed at 100,000x magnification.

Energy-dispersive spectroscopy (EDS) analysis
The composition of metal ions from levan-based metal NPs was analyzed using EDS (Hitachi Model SU3500, format EDS, manufactured by EDAX from AMETEK, Inc). Analysis using EDS was carried out to see the composition of metal elements contained in levan-based metal ion nanoparticles. A little solid of levan-based metal ion nanoparticles is taken and placed on a carbon adhesive. Then the sample was coated in gold (Au) with a power of 10 mA for 20 min. The composition of metal elements were analysed with an acceleration voltage of 10 kV at 500 magnification.

Analysis of levan-based metal ion nanoparticle stability
Stability analysis on these NPs was determined using the AAS method. The stability analysis of levan-based metal ion NPs was determined by calculating the levels of metal ions that were successfully encapsulated in the levan-based metal ion NP system and then calculating the levels of loose metal ions in levan-based metal ion NP solutions. Solids NPs were dissolved in demineralized water and stirred then left for a few minutes. Then, the solution of the NPs measured the concentration of metal ions by observing the standard curves of each metal ion.

Antioxidant activity
The antioxidant activity was measured using the 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) assay. The main standard compound used was ascorbic acid, which has high antioxidant activity. Briefly, 750 μL ascorbic acid, levan, all of the levan-based metal NPs, and all of the metal ions at different concentrations were added into 250 μL of the DPPH solution (0.4 mM, dissolved in 95% ethanol, v/v) in microtubes 1.5 mL. The mixture was stirred for 10 s to dissolve all of the components. The solution was incubated at room temperature for 30 min in a dark room. The changes in color from purple to yellow were measured at 518 nm using spectrophotometer UV-Vis. The antioxidant activity was measured based on inhibition percentage as follows: where A control is the absorbance in the absence of the DPPH solution, and A sample is the absorbance in the presence of the sample. Then, a curve was made based on percent inhibition with variations in concentration and the IC 50 value was calculated. The IC 50 is defined as the concentration of antioxidants needed to reduce radical DPPH by 50%.

Statistical analysis
All data were expressed as mean AE standard error of mean (n ¼ 5) and analyzed using one-way analysis of variance (ANOVA), followed by Dunnett's test.

Production and characterization of levan
The levan mass obtained from a production medium of 100 mL using 12% (w/v) sucrose substrate with 30 μg/mL crude recombinant levansucrase Lsbl-bk1 was 4.28 mg/mL. In the same condition, the levan mass obtained in vitro using recombinant Lsbl-bk1 levansucrase was still smaller compared to the levan obtained using recombinant levansucrase L8-37-0-1, which was 6.5 mg/mL. It may be caused by Lsbl-bk1 used in synthesizing levan in vitro has not been purified. The FTIR analysis could prove and confirm that the product obtained was levan, as can be seen from the possible functional groups responsible. The levan structure has several distinctive bonds that appeared on the IR spectrum. A significant peak typical for levan is the OH vibration peak at wavenumber  3600-3200 cm À1 , -C-H vibration peak at wavenumber 3000-2800 cm À1 , vibration peak C ¼ O at wavenumber 1641-1600 cm À1 , and absorption peak for glycosidic bonds at wavenumber 2900 cm À1 .
Comparing the IR spectrum of levan production results with the standard levan, it can be seen that there are similarities in the spectrum at significant peaks (see Table 1). The levan structure has several distinctive bonds that can appear on the IR spectrum so that it becomes a qualitative analysis to show the real levan product obtained. The FTIR spectra ( Figure 1) (Zhao et al., 2005). The wavelength region 939.74-1, 031.73/cm is reported to be fingerprint of molecule because it allows the identification of major chemical groups in polysaccharide (Fellah et al., 2009). The phenolic sulfuric method for determining the total fructant in the levan obtained.

Synthesis and characterization of levan-based metal ion nanoparticles
Levan-based metal ion NPs have been successfully synthesized, namely, levan-Fe 2þ NPs, levan-Zn 2þ NPs, levan-Cu þ NPs, and levan-Co 2þ NPs. All of levan-based metal ion NPs have been confirmed qualitatively from the appearance of color precipitate at the end of the reaction. The colors of levan-Fe 2þ , levan-Zn 2þ , levan-Cu þ , and levan-Co 2þ NPs were brownish red, white, light blue, and green, respectively. Solids of levan-metal NPs can be seen from Figure 2. Then, colloidal solutions were characterized by UV-Vis spectroscopic analysis. Levan-Fe 2þ NPs had a brownish red color and had an absorption peak at 301 nm, levan-Zn 2þ NPs had a white color (288 nm), levan-Cu þ NPs had a light blue color (298 nm), and levan-Co 2þ NPs had a green color (303 nm). Sundaram et al. (2012) has reported that exopolysaccharide-based Fe 3 O 4 NPs had a maximum wavelength in the range of 250-350 nm. Murali et al. (2017) also conducted research on ZnO NPs, which had a maximum wavelength of 320 nm. Dobrucka (2018) has also reported that biosynthesized CuO NPs using the extract of Galeopsidis herba had a maximum wavelength of 296 nm.  The levan modification of metal ions was confirmed using FTIR. The IR spectrum profile in Figure 3 presents the difference in absorption at wavenumbers that are typical for each metal. The FTIR of levan-Fe 2þ observed in wavenumbers around 3300 cm À1 , there is still an O-H vibration from levan, as well as after the formation of elevating NPs with metal. However, at a wavenumber of below 700 cm À1 , there is a vibration of Fe metal in the levan-Fe 2þ spectrum, and the fingerprint of the wavenumber refers to the furanose ring in levan. It can be stated that in the formation of levan-based Fe 2þ NPs, there is an interaction between the part of the furanose ring and the Fe 2þ metal ion, which is a similar explanation to that for levan-Co 2þ and levan-Cu þ . However, in levan-Zn 2þ , there are visible spectrum IR differences in the wavenumbers around 1700-1600 cm À1 , which show the vibrations of carbonyl or C-O-C in levan. So, it can be said that for levan-Zn 2þ NPs, levan interacts with metal ions in the glycosidic bond section. The number of metal ion components bound to levan-based metal ion NPs can be analyzed using EDS.
The EDS results from levan-based metal ion NPs can be seen from Figure 4. Based on the analysis of the EDS results, the readable elements on the sample surface for all levan-based metal ion NPs are carbon (C), oxygen (O), and the metal itself. This shows that the levan system has been formed (the element of carbon and oxygen), as well as the metal ions. In addition, in Figure 3, there is also a difference in the color of each readable element, and it can be observed that in levan-Fe 2þ , the metal content of Fe was only 14%, whereas the oxygen content was 49%. From the image of the elemental position in the EDS levan-Fe 2þ NPs result, it can also be seen that Fe was in the inside of the sample, which is marked with orange (showing the Fe element) covered by green (oxygen component) and purple (carbon component). The explanation for levan-Zn 2þ NPs, levan-Cu þ NPs, and levan-Co 2þ NPs resembles the analysis of levan-Fe 2þ NPs.
The morphology and size of levan-based metal ion NPs were analyzed using TEM (see Figure 5). Before the analysis, the sample was first modified to ensure that the particles were properly separated. So, the results obtained can determine the morphology and particle size of the sample. Levan-Fe 2þ NPs, levan-Zn 2þ NPs, and levan-Cu þ NPs have a spherical morphology compared with irregular levan-Co 2þ NPs. This indicates that there are different molecular geometries between levanbased metal ion NPs. Thus, the ability of levan to encapsulate different types of metal ions affects the geometry of the NPs system formed. Based on the results of this TEM, levan is sufficient to encapsulate Zn 2þ and Fe 2þ ions, as evidenced by the neatly rounded surface of the NPs. However, all levan-based metal ion NPs have a particle size of less than 100 nm, and even levan-Cu þ and levan-Zn 2þ have particle sizes of less than 50 nm. Thus, levan is quite promising to be used as a material for creating NP systems.

Stability of levan-based metal ion nanoparticles
The results of metal ion levels and the stability of levan-based metal ion NPs can be seen from Figure 6. Based on these data, it can be seen that all metal ions have about 80% bound to levans. The stability of levan- Figure 5. TEM image of levan-based metal ion nanoparticles, a) levan-Cu þ NPs, b) levan-Zn 2þ NPs, c) levan-Co 2þ NPs, and d) levan-Fe 2þ NPs. Figure 6. The percent curve of the metal ions in levan-based metal ion nanoparticles (indicated by the stripe motif box) and the stability of levan-based metal ion nanoparticles (indicated by black spots on white spots). Figure 7. Reaction of DPPH with ascorbic acid.
based metal ion NPs is seen from the percentage of release measured from the concentration of metal ions after dissolving in aqua and a few minutes. According to the results, all levan-based metal ion NPs are quite stable with a percentage of metal ion release of less than 5%.

Antioxidant activity
The antioxidant activity of levan-based metal ion NPs was determined using DPPH compounds. Basically, the general reaction that occurs in the determination of antioxidant activity using DPPH, reaction with ascorbic acid, for example, can be seen from Figure 7. DPPH radical compounds that are purple when oxidized will experience a change in color to yellow. Then, it is used to determine the antioxidant activity of each sample. In the formation of levan-based metal ion NPs, levan acts as a reducing and stabilizing agent. An illustration of the formation of levan-based metal ion NPs can be seen from Figure 8. Levan has a -OH functional group that can interact with metal ions. In this context, functional groups in their structure that can serve as reductive and stabilizing agents in the synthesis of metal nanoparticles by a chelating and capping process (Escarcega-Gonzales et al., 2018). This interaction results in a reduction in metal ions. The metal ions used in this study were Fe 3þ , Cu 2þ , Co 3þ , and Zn 2þ . Based on the reduction potential; The reduced from of each metal ion encapsulated with levan has the potential to increase antioxidant activity. This phenomenon is explained by the value of the potential reduction energy of each metal ion. Co 2þ , Fe 2þ , and Cu þ have negative potential energy values, so that they are spontaneous enough to experience oxidation to Co 3þ , Fe 3þ , and Cu 2þ , respectively. It is observed that levan acts as a reducing agent in the manufacture of levan-based metal ion NPs. Based on the data analysis of  one-way ANOVA for the antioxidant activity of only levan, metal ions only, and levan-metal NPs exhibited a significant difference in each of the metal ions, except for Zn 2þ metal ions which were not significantly different.
The results of the testing of the antioxidant activity between standard compounds, only levan, only metals, and levan-based metal ion NPs, can be seen from Figure 9. Without levan, the antioxidant activity of metal ions were lower. So, the interaction with levan could increase the antioxidant activity of those metal ions. Red curves with round symbols show the antioxidant results of ascorbic acid. It can be seen that at small concentrations (<0.1 mg/mL), ascorbic acid has been able to act as an antioxidant until, finally, it reaches an inhibition of 85% of free radical compounds. In addition, other new samples provide antioxidant properties at a concentration of 0.5 mg/mL. Levan itself has antioxidant properties that can inhibit radical compounds up to 58%. Kim and Chung (2016) hypothesized that levan has antioxidant activity because it contains many hydroxyl groups. The hydroxyl functional groups, which can react with free radicals and terminate radical chain reactions, are highly related to the antioxidant activity. Moreover, several previous studies reported that levan and its derivatives have antioxidant activity (Liu et al., 2012). Metal ions (Fe 2þ , Cu þ , Co 2þ , and Zn 2þ ) also provide antioxidant properties that can inhibit radical compounds up to 22%. So, a combination of levan and some metal ions which have antioxidant activity is used. The combination of levans with these metal ions that form levan-based metal ion NPs can increase antioxidant activity by up to 95% in levan-based Cu þ NPs (especially for Cu þ NPs after 5h), followed by levan-based Fe 2þ NPs which have a percent inhibition of radical compounds by 85%. So, combining levans with metal ions can increase antioxidant activity by 33%-40%. However, negative results are given on the incorporation of levan with Zn 2þ ions. A decrease in antioxidant activity due to Zn 2þ has stabilized, so that oxidation does not easily occur, which results in decreased antioxidant activity after being combined with levan. Efrekhari et al. (2018) mentioned that the reduction of Cu(II) to Cu(I) occurs in the presence of GSH and ascorbic acid, which induces the decomposition of H 2 O 2 through Fenton reaction and OH À * generation, leading ultimately to membrane lipid peroxidation.

Conclusions
This study focuses on the synthesis of levan using levansucrase resulting from heterologous expression. The synthesis of levan-based metal ion nanoparticles (NPs) aimed to obtain the possibility of increasing the antioxidant activity of the levan itself. Levan has been successfully produced in vitro using the Lsbl-bk1 recombinant levansucrase biocatalyst. Characterization of functional groups constitute the levan structure has also been performed through FTIR analysis. Based on the IR levan spectrum profile, the production results obtained a significant IR spectrum profile with a standard pure levan from Erwinia herbicola. The use of levan in various fields has been widely reported, but this study focuses on the use of levan as a reducing agent and stabilizer in the manufacture of metal ion NPs for antioxidant activity. Four types of levan-based metal ion NPs have been synthesized, namely, levan-Fe 2þ NPs, levan-Cu þ NPs, levan-Co 2þ NPs, and levan-Zn 2þ NPs based on analysis via UV-Vis, FTIR, EDS, and TEM. Levan-based metal ion NPs also have high stability and can be measured using the AAS method. Four levan-based metal ion NPs have been synthesized, which have the potential to be used as antioxidants, namely, levan-Fe 2þ NPs and levan-Cu þ NPs, with a percentage of inhibition of radical compounds in the amount of 85% and 95%, respectively. Consequently, the two NPs have the potential to be used as new formulation in antioxidant drugs. In addition, levan-Cu þ NPs after 5 h were quite stable and gave a percentage of inhibition of radical compounds greater than that of ascorbic acid, although it takes longer time than ascorbic acid.

Suggestions
Research related to the kinetic and thermodynamic characteristics of levan-based metal ion nanoparticles which have the potential as antioxidant.

Author contribution statement
Rukman Hertadi: Conceived and designed the experiments; Performed the experiments; Contributed reagents, materials, analysis tools or data.
Muzayana Mahbuba Saleha Amari: Performed the experiments; Analyzed and interpreted the data; Wrote the paper.
Enny Ratnaningsih: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper.