A rapid synthesis and antibacterial property of selenium nanoparticles using egg white lysozyme as a stabilizing agent
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A simple method for the rapid synthesis of selenium nanoparticles (SeNPs) by the reduction of selenium dioxide using an aqueous egg white lysozyme solution (stabilizing agent) and ascorbic acid solution (reducing agent) has been described. The formation of SeNPs is assured by characterization with UV–Vis, FT-IR, XRD and morphological characters that were observed using TEM analyses. The absorbance of the SeNPs is found at 250 nm. The TEM images show that the SeNPs are uniform and in spherical shape of size 40–60 nm. The crystalline nature of SeNPs is assured by XRD analysis. The antibacterial property of synthesized SeNPs was tested against the pathogenic bacteria, i.e., Bacillus subtilis, Bacillus cereus, Escherichia coli, Staphylococcus pneumoniae, Proteus mirabilis and Klebsiella pneumoniae. SeNPs showed more antibacterial activity against B. subtilis (19 mm) and Streptococcus pneumoniae (15 mm) as compared with commercially available antibiotics such as streptomycin and erythromycin. The SeNPs synthesized via this process has proficient antimicrobial activity against pathogenic bacteria. SeNPs could play a significant role in pharmaceutical industries for the development of efficient antibiotic agents.
KeywordsSelenium nanoparticles Egg white lysozyme Antibacterial activity Pathogenic bacteria TEM analyses
Nanotechnology is a futuristic and emerging discipline in science concerning the creation or manipulation of particles and materials in nanoscale sizes [1, 2, 3]. The most significant characteristic of nanoparticles is that the chemical, physical and biological properties of nanoparticles are quite different from the properties of the same materials on a larger scale [4, 5]. These materials are obtained from the structured arrangement of groups of atoms and molecules or by minimizing macroscopic materials into a nanometer scale [6, 7]. In the present years, various novel nanoparticles have received much attention due to their great potential in biomedical sciences [8, 9], engineering [10, 11], agriculture [12, 13] and food safety [14, 15]. Among them, selenium nanoparticles (SeNPs) are emerging as promising nanoparticles that can potentially be used for a wide range of applications [16, 17]. This element can be considered as an essential trace element micronutrient for living creatures at low concentrations, but it becomes toxic and harmful at higher doses. Normal selenium levels in adults are around 81 mg, and the dietary requirement is ~ 55 mg per day, with an upper limit of ~ 400 mg [18, 19]. SeNPs have been sold on the market as a food additive in a tea product that claims to possess several health benefits [17, 20]. Some studies have shown that SeNPs have unique antibacterial activities [21, 22] and antifungal activities [23, 24]. SeNPs can be used for food safety applications which includes as antibacterial nanocoatings [25, 26], in food packaging [27, 28] and in functional foods [29, 30].
Recently, many efforts have been made in the fabrication of bioactive and biocompatible nanoparticles for a variety of applications [31, 32]. For example, DNA [33, 34], peptides [35, 36] and proteins  have been bioconjugated to nanoparticles with the use of the intermediate protecting agents of the nanoparticles or the linking agents . Nowadays, research on direct conjugation of biocompatible agents and biomolecules to nanoparticles is accomplishable . For example, SeNPs capped with biocompatible agents, such as lipid bilayers [39, 40] and dendrimers , have been prepared with water solubility and potential biocompatibility. Biomolecules, such as amino acid, have also been used as protective agents for the formation of SeNPs. For example, l-cysteine-capped SeNPs were used for biomedical applications that have been reported by Prasanth et al. .
Lysozyme is a commercially valuable small enzyme with an antibacterial function. It kills bacteria by breaking down the cell walls of bacteria and makes the bacteria burst under their internal pressure . Furthermore, evidence has recently shown that the lysozyme is also an active agent for killing human immunodeficiency virus (HIV) in vitro . Lysozyme is readily available, and its structure and properties are well known. The previous reports showed that the lysozyme-coated silver nanoparticles for differentiating bacterial strains based on antibacterial activity . Lysozyme is present in nature as part of the innate immune system in higher organisms and primarily acts against bacteria through enzymatic hydrolysis of the peptidoglycan layer that surrounds the cell membrane . To further explore the mechanisms of nanoparticle formation and expand the repertoire of antimicrobial materials, we investigated methods to integrate lysozyme with SeNPs to form multifunctional antimicrobial agents. Using lysozyme as the catalyst, preparations of selenium nanoparticles were synthesized, and the physical and chemical properties were assessed to characterize the functionality of these hybrid bioinorganic composites. The material was also incubated with a variety of bacterial strains to determine whether the antimicrobial properties of lysozyme, as well as the biocidal effects of selenium, were investigated to develop new bio-nanocomposites.
The synthesized selenium nanoparticles were characterized by UV–Vis, FT-IR, XRD and TEM analyses. This study reports for the first time, the preparation and synthesis mechanism of SeNPs using ascorbic acid and egg white lysozyme (EWL) as reducing and stabilizing agent, respectively. This work has been carried out to produce a cost-effective synthesis of spherical SeNPs at an appropriate temperature with shorter reaction times. Further, we have described in detail the formation of SeNPs, which were stabilized with EWL molecules. Moreover, the synthesized SeNPs was also investigated for its antibacterial activity against different pathogenic bacteria. Our result signifies that the synthesized SeNPs, along with EWL, are suitable for the formulation of new types of bactericidal agents.
2 Materials and methods
The purified egg white lysozyme was obtained from Sigma-Aldrich (St. Louis, USA). Selenium dioxide (SeO2) was purchased from Bio-Rad (California, USA). Ascorbic acid was procured from Qualigens Fine Chemicals Co. Ltd (Mumbai, India). Bacteriological media components were purchased from Hi-Media (Mumbai, India). All the other chemicals utilized in this study were of analytical grade procured from Sisco Research Laboratories (Mumbai, India).
2.2 Preparation and synthesize of SeNPs stabilized with EWL
A stock solution of 0.01% EWL and 0.04 M SeO2 was subsequently prepared in double-distilled water. 0.04 M of the solution of ascorbic acid was added to prepare SeO2 solution. Color change from honey yellow to dark red was observed, indicating the presence of SeNPs. In the following, the synthesis of SeNPs was carried out according to the method described by Xia .
2.3 Characterization of SeNPs stabilized with EWL
The synthesized SeNPs stabilized with EWL were characterized by UV–Vis, FT-IR, TEM and XRD analysis.
2.3.1 UV–visible spectral analysis
Hitachi 2900 absorption spectrophotometer was used for the absorption spectral studies. The nanosolution was filtered through a 0.2 micron sterilized filter to set apart impurities, if any, before the UV–visible spectral studies.
2.3.2 FT-IR spectral analysis
FT-IR spectroscopy was performed to study the chemical interactions in SeNPs formulations. A small quantity of SeNPs was mixed with KBr, and then, the mixture was pelleted out with a pressure of 150 kg cm−2. The pellet was studied using EQUINOX (Bruker) FT-IR spectrophotometer in the range of 4000 to 450 cm−1.
2.3.3 X-ray diffraction analysis
The crystallographic phase of the SeNPs stabilized with EWL was determined on Philips X-ray diffractometer over the 2θ range from 10 to 70° at a rate of 4°/min using Cu Kα radiation (λ = 1.54060 Å). Philips PW 1830 (USA) X-ray generator was operated at a voltage of 40 kV.
2.3.4 TEM analysis
The shape and size of SeNPs stabilized with EWL were characterized by TEM (JEOL, JEM-2100, Japan). The samples were prepared by placing 3.0 µl of the nanosolution filtrate on the carbon-coated copper grid as a thin film.
2.4 Antibacterial study of SeNPs stabilized with EWL
The antibacterial activity of SeNPs stabilized with EWL was studied by the agar diffusion method. Different pathogenic bacterial strains such as Bacillus subtilis, Streptococcus pneumoniae, Bacillus cereus, Escherichia coli, Proteus mirabilis and Klebsiella pneumoniae were used for this analysis. The primary cultures of the organisms were subcultured in Luria–Bertani (LB) broth at 35 °C and kept on a rotary shaker at 200 rpm for 24 h. The Mueller–Hinton agar (MHA) plates were swabbed with 24-h-old bacterial cells to produce a lawn culture (1 × 106 cfu ml−1). Paper disks of 6 mm (Hi-Media, India) were impregnated with SeNPs (10.0 µg ml−1). The disks were gently pressed to get better contact with MHA. The plates were incubated upside down for 24 h at 37 °C. After incubation, the different levels of the zones of inhibitions were measured. After incubation, the susceptibility of the test organisms was calculated by measuring the diameter of the zone of inhibition.
2.5 Statistical analysis
Statistical analyses were carried out using GraphPad Prism 6.0 software (San Diego, CA, USA). Data values were presented as mean ± SEM. Differences between two groups were assessed by using Student’s t test. Statistical significance is reported as highly significant using ** (p < 0.002).
3.1 Synthesis of SeNPs stabilized with EWL
Uniform shaped SeNPs stabilized with EWL were synthesized via reduction of selenium dioxide in the presence of ascorbic acid as a reducing agent and EWL as a stabilizing agent. The color intensity increased as the incubation time increased. The change of color was observed at two different time intervals (i.e., 1 h (Fig. 1Bb) and 2 h (Fig. 1Bc)). The maximum color intensity was seen in the solution incubated for 2 h (Fig. 1Bc).
3.2 Characterization of SeNPs stabilized with EWL
3.2.1 UV–vis spectral analysis
The formation of SeNPs stabilized with EWL via the reduction of selenium dioxide was monitored in the UV–visible spectrophotometer. A strong characteristic absorption peak at 250 nm was indicating the presence of the SeNPs (Fig. 1A).
3.2.2 FT-IR spectral analysis
3.2.3 XRD analysis
3.2.4 TEM analysis
3.2.5 Effect of stability of SeNPs stabilized with EWL visualized under TEM analysis
3.3 Antimicrobial studies SeNPs stabilized with EWL
3.3.1 MIC and antibacterial activity under TEM analysis
Antibacterial activity of SeNPs stabilized with EWL against different pathogenic bacteria
Zone of inhibition (mm)
SeNPs + EWL
24 ± 0.41
19 ± 0.77
23 ± 0.50
13 ± 0.46
21 ± 0.54
14 ± 0.45
22 ± 0.60
15 ± 0.44
21 ± 0.33
11 ± 0.55
20 ± 0.49
0 ± 0.0
In the past years, there seems to be an increased interest in the application of SeNPs as they exhibit antioxidant, anticancer and antimicrobial activities [21, 48]. Therefore, the present study deals with the synthesis of SeNPs using a natural protein, lysozyme obtained from egg white lysozyme. Previous research reveals the photoinduced synthesis of silver nanoparticles stabilized with lysozyme . The nanoparticle synthesis occurred within 2 h, much lesser duration compared to the earlier report where the synthesis process had taken 4 h using fibroin . In the synthesis process, the aqueous solution of SeO2 was reduced by ascorbic acid. The ascorbic acid solution was used as a precursor to initiating the reaction with SeO2, and the SeNPs were synthesized. In the current study, SeNPs were synthesized with ascorbic acid as reducing agent, and EWL as a stabilizing agent. Previous reports state that employed fibroin protein obtained from silkworm was used as the stabilizing agent for the synthesis of SeNPs . The formation of SeNPs via the reduction of SeO2 was monitored in the UV–visible spectrophotometer. A strong characteristic absorption peak observed at 250 nm suggests the synthesis of SeNPs (Fig. 1A). Functional groups in the lyophilized SeNPs were examined using FT-IR spectroscopy by scanning in through 4000–450 cm−1. The FT-IR spectrum of SeNPs stabilized with EWL showed bands at 3301 and 2961 cm−1 that were characteristic of hydroxyl groups. The bands at 1308 and 1239 cm−1 correspond to the aromatic primary amine C–N stretching vibration, while the bands at 1171 and 1105 cm−1 were characteristic of tertiary and primary C–N stretching vibrations. The absorbance band at 1648 cm−1 was characteristic of C=C stretching. The absorbance bands at 668, 743, 897 and 932 cm−1 were due to aromatic skeleton. The FT-IR peaks of hydroxyl, C-N and aromatic groups were observed and they were similar to the bacterial Comamonas testosteroni S44 synthesized SeNPs stabilized with protein . The peak at 1536 cm−1 was due to aromatic C–H stretching vibration. These data suggest that the stabilization of the nanoparticles could be due to the aromatic and hydroxyl group of EWL (Fig. 2; blue line).
X-ray diffraction was analysis done to substantiate the crystallite structure of SeNPs and the XRD pattern of SeNPs matches the pattern of SeNPs listed in the ASTM XRD standard card NO.NP-194A, confirming the crystalline structure of SeNPs. A comparison of our XRD spectrum against the standard verified that the synthesized SeNPs formed in this study is nanocrystalline indicated by the peaks at 2θ corresponding to 2451, 725, 591, 477 and 281, respectively, and was compared with the results of Shin (Fig. 3). From the previous study, the X-RD diffraction peaks of green synthesized pure crystalline SeNPs from Withania somnifera was congruent to our SeNPs . The average crystallite size of the synthesized SeNPs was 40–60 nm, following the results obtained in TEM studies (Fig. 4). As shown in Fig. 4, the TEM image of the nanoparticles appeared to be well-dispersed spherical particles, with a diameter of 40–60 nm. Khurana had synthesized SeNPs, which were spherical of size ranging from 20 to 60 nm . Long-term stability was analyzed to check for aggregation, apart from other parameters like the average diameter and shape, which is a prerequisite for the application of nanomaterial. An excellent stabilization efficacy of EWL on SeNPs formulation was exhibited the stability analysis UV–visible spectrophotometer (Fig. 5). The absorption spectra of SeNPs revealed the changes in the diameter of the nanoparticles, e.g., changes caused due to aggregation of the SeNPs (Fig. 6). Therefore, all stability studies performed in this work were evaluated using UV–visible spectroscopy. The previous study state that the long-term stability of lipid nanoparticles using for biomedical applications . In the systems where ascorbic acid was employed as the reducing agent, the long-term stability was not prone to the changes in concentrations of the gelatin nanoformulation. Nguyen et al.  observed the same results, and the synthesis SeNPs was followed at 250 nm by UV–visible spectrometer. There were not many changes in the UV–visible spectra recorded at varying intervals (Fig. 2a). Dispersions were found to be stable against aggregation . The previous study state that the SeNPs showed antibacterial activity against P. aeruginosa (9.25 mm) and S. aureus (10.75 mm) . In the present study, the synthesized SeNPs stabilized with EWL exhibited stronger antibacterial effect against pathogenic bacteria. 10.0 µg ml−1 of SeNPs stabilized with EWL was potent enough to inhibit the growth of B. subtilis, S. pneumoniae, B. cereus, E. coli, P. mirabilis and K. pneumoniae. The highest zone of inhibition was recorded for B. subtilis (19 mm) followed by S. pneumoniae (15 mm), B. cereus (14 mm), E. coli (13 mm) and P. mirabilis (11 mm). Though SeNPs stabilized with EWL can inhibit both the gram-positive and gram-negative bacteria, more pronounced antibacterial activity was observed with gram-positive bacteria. The SeNPs stabilized with EWL were not effective against K. pneumoniae (Fig. 7B; Table 1). Nguyen et al.  also reported similar study; 10 μg concentration of SeNPs inhibits the food-borne pathogens. The reason why SeNPs stabilized with EWL failed to curb the growth of K. pneumoniae remains to be investigated. TEM image reveals the antibacterial potential of SeNPs stabilized with EWL on B. subtilis and E. coli, causing bacterial damage to the peptidoglycan layer. SeNPs stabilized with EWL mainly target the peptidoglycan layer of the bacterial cell membrane, causing the leakage of cytosol content resulting in cell death, as observed by Narayanan (Fig. 7B).
This study deals with the synthesis of SeNPs using EWL and ascorbic acid for the generation of metal nanoparticles. The solvents and reducing agents determine the physical and morphological characteristics of SeNPs. The size, conformation and morphological characteristics of the newly synthesized nanoparticles were studied by UV–Vis, FT-IR, XRD and TEM analyses. The EWL stability dispersions of SeNPs were observed to exhibit prolonged stability minimum 12 months, which was confirmed by the TEM image. The antibacterial efficacies of the reported SeNPs stabilized with EWL have been proved to be promising against different pathogenic bacteria. Hence, further studies are to be conducted in this research area to be employed in the pharmaceutical industry, and this would be a cost-effective alternative to other approaches.
The authors thank the University Grant Commission-Basic Science Research (UGC-BSR) Science Meritorious Fellowship (GCCO/A2/UGC/PT/CASB/MER FELLOW/2009/1484), New Delhi, India, for financially supporting this work.
Compliance with ethical standards
Conflict of interest
The authors report no conflict of interest. The authors are the only responsible person for the content and drafting of this article.
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