Phyto-fabrication of selenium nanorods using extract of pomegranate rind wastes and their potentialities for inhibiting fish-borne pathogens


 The invasion of fish/seafoods by zoonotic pathogens causes health threats to humans. Plant derivatives and phytosynthesized nanometals could promisingly overcome bacterial infections/contaminations. The extract of pomegranate rinds (PRE) was innovatively employed for biosynthesizing selenium nanorods (Se-NRs). These agents were assessed as antibacterial candidates against diverse fish-borne pathogenic species (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium, and Sphingomonas paucimobilis). The PRE-synthesized Se-NRs, within 60 min of contact, were negatively charged (‒32 mV) and had mean diameter of 62.31 nm and length range of 443.5–1236.9 nm. The designated infrared spectra for PRE and PRE/Se-NRs composite validated the biosynthesis, bonding, and interactions of the nanocomposite. The antibacterial potentialities of PRE, phytosynthesized Se-NRs, and PRE/Se-NRs composite was confirmed toward the entire challenged pathogens; S. aureus had the highest resistance (with inhibitory concentrations of 72.5, 60.0, and 55.0 mg/L, respectively) and S. paucimobilis was the most sensitive (with inhibitory concentrations of 55.0, 45.0, and 42.5 mg/L, respectively). The ultrastructure of the treated S. paucimobilis with PRE/Se-NRs emphasized the composite potentiality for deforming/distorting cells within 4 h and causing full cells’ destruction and deformation within 8 h of exposure. The PRE-phytosynthesized Se-NRs are advocated as potent antibacterial products against fish-borne pathogens for decontaminating fisheries farms and products.


Introduction
Nanotechnology is the fast-growing discipline of science and technology, which targets the production, characterization, and applications of novel materials in nanoforms, e.g., with particles' diameter in nanometer scale [1]. The nanoparticles' (NPs') applications and features effectually served in numerous fields, including chemical, mechanical, optical, and biomedical applications [2]. NPs represent entirely new and augmented features than bulk particles based on their particular properties, e.g., greater surface area, diminished size, distribution, and morphology. Ordinarily, NPs were synthesized via chemical/physical approaches; the physical methods' disadvantages include their high requirements of energy and cost and the low NPs yield, whereas chemical methods frequently cause ecological and toxicological consequences because of the involvement of hazardous chemicals in synthesis and their residues' effects [3,4].
The bio (green) synthesis of NPs could overcome most of the above disadvantages by providing facile, ecofriendly, and economical approaches that apply microorganisms, algae, biopolymers, plant materials, or their derivatives for NPs synthesis [2,5].
Selenium (Se), the essential element for higher organisms' life, is required at 40-300 µg in daily intakes for human. Se at these concentrations is necessary for conventional maintenance of organisms' functions, but it may cause toxicity at high dosage intakes (≥3,200 µg/day) [6,7]. However, the Se nanostructures (e.g., spheres, nanorods, nanowires, hexagonal prism, amorphous, nanotubes, nanoribbons, trigonal, and nanoplates) could diminish the Se risk and toxicity, which enable their effectual application in biomedical/pharmaceutical agents [8,9]. The transformation of Se to its nanostructures was achieved using different protocols, but the biosynthesis using plant derivatives (i.e., phytosynthesis) recently gained more successfulness, because of its elevated efficiency and safety for reducing/stabilizing Se nanoforms [10][11][12][13][14][15]. The phytosynthesis of nano-Se was also reported to augment their particles' stability, biocompatibility, and bioactivity as antimicrobial, anticancer, and antioxidant agents.
The pomegranate fruits (Punica granatum L.) grow in warm climates and their rinds were historically applied as herbaceous remedies for treating and managing numerous disorders, including diarrhea, dysentery, inflammation, cancers, parasitic, and microbial infections [16,17].
Accordingly, the biosynthesis of Se-nanorods (Se-NRs) using PRE and their characterization were investigated in the present study; the antibacterial actions of biosynthesized nano-Se with PRE were also elucidated toward various fishborne bacterial pathogens.

Pomegranate rind extract (PRE) preparation
The pomegranate fruits were organically farmed and harvested at KFS research farm, Kafrelsheikh University, Egypt. Fruits were cleansed accurately with chlorinated water and their rinds were manually peeled, washed with deionized water (DIW), and dried (using hot air at ∼43°C for 60 h). The dried rinds were pulverized mechanically, and their powder (∼100 g) was soaked in 1,000 mL of 70% of ethanol, agitated with stirrer magnet (at 25 ± 2°C for 72 h, 125×g), and filtered to eliminate rind's residues. The PRE was vacuum dried at a temperature of 42°C and pressure of 13.0 kPa, weighed, and re-dissolved in stirred DIW to reach 10% concentration.

Phytosynthesis of Se-NRs
First, 10 mM solution of sodium selenite (Na 2 SeO 3 , molar mass: 172.94 g/mol, Sigma-Aldrich, MO) in DIW was prepared. Afterward, equal vol. of PRE (1% concentration) and Na 2 SeO 3 solution (10 mL each) were mixed and stirred (540×g) with magnetic stirrer for 60 min at 25 ± 2°C. The development of brownish-orange color indicated Se-NRs biosynthesis using PRE. The formed PRE/Se-NRs in reaction solution was precipitated via centrifugation at 12,500×g for 35 min (SIGMA 2-16KL centrifuge; Sigma Lab. GmbH, Germany). Then, the portions from the precipitates were washed first by DIW four times and then with ethanol and recentrifuged after each washing to obtain plain Se-NRs [33]. The plain Se-NRs and the PRE/Se-NRs composite were subsequently freeze-dried and characterized.

FTIR spectroscopic analysis
For detecting the distinctive biochemical bonding and interactions in the produced materials as well as their potential compositions, the PRE and PRE/Se-NRs spectra were spectrophotometrically investigated operating Fourier transform infrared spectroscopy (FTIR) (JASCO FT-IR-360, Tokyo, Japan). The transmission was appraised at 450-4,000/cm wavenumber range.

Se-NRs' optical analysis
For validating the formation of metal NPs, via detecting their surface plasmon resonance associated with free electrons on formed NPs' surfaces, the Se-NRs' spectrum were analyzed by UV-Vis spectrophotometer (model UV-2450, Shimadzu, Japan) at 300-1,000 nm wavelength range.

NRs' size and charge
The appraising of Se-NRs' size (Ps) and zeta potential (ζ) were performed via dynamic light scattering technique, applying zetasizer (Zeta plus, Brookhaven, USA).

Antibacterial assay
The generated agents (PRE, Se-NRs, and PRE/Se-NRs composite) were assessed as antibacterials against fish-borne pathogens including Gram positive (

Qualitative assay
The "zones of inhibition (ZOI) by disc diffusion assay" was appraised as indicator for bacterial inhibitory action of inspected agents [34]. Concisely, solutions (10% in DIW) were made from each agent and sterile paper discs (Whatman No. 2, 6 mm diameter) were impregnated with solutions to carry 50 μg/disc. Bacteria were spread onto the NA plates and the assay discs were positioned onto the inoculated plates' surface. Then the plates were incubated aerobically at 37°C for 18-24 h until the appearance of growth-free zones around the discs. The mean values of the appeared ZOIs' diameters were appraised from triplicated trials.

Quantitative assay
The minimal inhibitory concentrations (MICs, mg/L), assessment, of each inspected agent toward each pathogen, were implemented thru microdilution technique [22,34], validating results with triphenyl tetrazolium chloride (TTC) staining (Sigma-Aldrich, MO., 5 mg/mL in DIW). Gradual concentrations of PRE, Se-NRs, or PRE/Se-NRs composite (10-100 μg/mL) in NB were prepared in microtiter plates (96 well) and each well was inoculated with ∼2 × 10 6 cells/mL of each individual pathogen. The plates were incubated for 24 h and the free wells from cells' growth (with no obvious turbidity) were treated with 100 μL of TTC solution. 100 μL from the color-free wells (without red formazan formation) were streaked onto the NA plates and the growth-free plates confirmed the agents' inhibitory actions.

Antibacterial visualization via electron microscopy
The SEM photo capturing, of S. paucimobilis exposed to PRE/Se-NRs composite, was employed for perceiving structural and morphological alterations/distortions in cells, after exposure to composite for 0, 4, and 8 h, to elucidate its potential action mode. The SEM bacterial imaging applied standardized procedure [35]. Grown bacteria in NB (24 h old) were exposed to 100 μg/mL of composite and incubated at 37°C. Bacterial samples were centrifuged 4,500×g for 30 min, washed with 0.9 % NaCl solution, re-centrifuged and then subjected to SEM preparing and imaging.

Statistical analysis
The mean values ± standard deviation (SD) of triplicated experiments were calculated using SPSS package (SPSS V-11.5, Chicago, IL, USA),. Results' significances at p ≤ 0.05 were computed using t-test and one-way ANOVA.
3 Results and discussion 3.1 Phytosynthesis and characterization of biosynthesized Se-NRs using PRE

Visual and optical observation
By using PRE, Na 2 SeO 3 was bioreduced to Se-NRs, which was visually evidenced by gradual change in solution color from bale yellow to the appearance of deep brownishorange color after 60 min (1 in Figure 1), signifying the Se-NRs biosynthesis [8]. The UV analysis of PRE-synthesized Se-NRs shows two strong peaks positioned at 464 and 581 nm and one weak peak at 349 nm (2 in Figure 1). These peaks are adjacent to the absorption peaks at 346, 450, and 570 nm for t-Se nanowires found in C12EO10 micelles and at 347, 462, and 586 nm which were reported for Se-NRs spectrum [36]. As formerly reported, the different properties of Se nanomaterials are according to the size and microstructure of the materials and thus could be altered by changing the synthetic conditions [37]. The peaks above 530 nm can be solely ascribed to inter-chain interactions vertical to the c axis within a given t-Se crystal. Therefore, the location of the low-energy peak at high wavelength may provide useful information for the inter-chain interactions in addition to the degree of crystallinity [14,15,33].

Size and charge of PRE-synthesized Se-NRs
The Ps analysis of phytosynthesized Se-NRs, mediated by PRE, revealed that their size ranged from 28.41 to 92.61 nm, with mean and median diameters of 62.31 and 64.53 nm, respectively. The mean diameter was the ζaverage hydrodynamic diameter. The resulted green phytosynthesis of Se-NRs is simple, cost effective, and eco-friendly; the resultant NPs have non-toxic and high stability attributes [12]. Additionally, the ζ potential of Se-NRs was computed to be −32 mV, which indicates high stability of NRs in aqueous solution and advocates the PRE stabilization potentiality [38]. ζ potential can provide a definite measurement of specific molecular surface charges, and additionally, it provides indications of the produced electric double layer by the contiguous ions in solution. Characteristically, the NPs with greater ζ values than +30 mV or lesser than −30 mV display higher degrees of stability due to their elevated inter-particle electrostatic repulsion [14,15,33,37].

FTIR analysis
The biochemical bonding and reactions of PRE and PRE/Se-NRs were detectable from their FTIR spectra ( Figure 2). The IR pattern of PRE (upper curve in Figure 2) reflected the key attributes of the extract structure. The wide absorption band at 3376.41/cm distinguishes the O-H group in PRE polyphenols, flavonoids, and terpenes, whereas the clear band at 2912.15/cm corresponds to C-H vibrated stretching of alkyl [19,39]. The PRE absorption peaks at 1442.88/cm (aromatic rings) and 1311.21/cm (N-O stretches of nitro compound) were clearly detected in extracted IR spectrum [18,40].
The detectable sharp peaks at 1632.23 and 1741.74/cm are assumingly referring to N-H vibration (in primary amines) and C]O (carbonyls) stretching, respectively [25,41]. The peak at 1042.11/cm, assigning the variable C-O covalent stretching in PRE, was shifted after conjugation with Se-NRs, indicating their combined interactions [24,42].
After conjugation of Se-NRs and their reduction with PRE (lower curve in Figure 2), the O-H wide band shifted and the C-H band (at 2912/cm) disappeared, which indicated their roles in Se-NRs reduction/conjugation [19]. Also, the disappearance of C]O stretching band (at 1742/cm), in PRE/Se-NRs spectrum, strongly indicates the involvement of this biochemical bonding in Se-NRs' biosynthesis and the interaction between Se and this bond in PRE [25,41]. The alteration in PRE aromatic rings and N-O designative bands' intensities (after conjugation with Se-NRs) indicated their potential interactions with biosynthesized NPs [19,40]. The strong band, appearing at 723.12/cm in PRE/Se-NRs spectrum, designates the vibrated bending of Se-O, which clearly evidenced the Se-NRs' interaction and stabilization by the PRE [23,26]. The reductions/stabilizations of various Se-NP forms (e.g., nanospheres, nanowires, or nanorods) principally depend on the nature of the stabilizer's capability to interact with Se ions [43,44]. Thus, PRE could be proposed as ideal reducer/stabilizer for biosynthesis of Se-NRs.

SEM imaging
The SEM technique was employed for visualizing the size and shape of the phytosynthesized Se-NRs using PRE (Figure 3). The bioformation of Se-NRs and their morphological dimensions appeared as rod-shape clusters with average rods' diameter of ∼64.42 nm and length range from 443.51 to 1236.86 nm, which harmonized the obtained figures from Ps analysis. These Se-NRs' diameter and length are lesser than those obtained in previous study [44], in which the rods' diameter range was 100-200 nm and length was 1,000-3,000 nm, indicating the high reducing efficiency of PRE to biosynthesize Se-NRs. Furthermore, the complete synthesis of Se-NRs within 6 h of bioreaction with PRE indicates the efficacy of the extract as the bioformation of Se-NRs normally needs long time (up to 48 h) for giving that structure [8].

Qualitative and quantitative assays
The assessment of PRE, phytosynthesized Se-NRs, and PRE/Se-NRs composites as antibacterial agents (qualitatively using ZOI and quantitatively using MIC) verified the bacterial inhibitory action of the entire agents (Table 1). Generally, the Gram + strain (S. aureus) had higher resistance than Gramspecies (P. aeruginosa, E. coli, S. typhimurium, and S. paucimobilis) toward all examined agents/ composite. Oppositely, S. paucimobilis showed the highest sensitivity to antibacterial agents, which were proved by the widest ZOIs and least MICs within the challenged strains. The PRE/Se-NRs composites' action were significantly the most forceful, whereas the actions of PRE and Se-NRs were comparable. The PRE/Se-NRs exhibited powerful antibacterial activities, which were insignificantly different from the standard antibiotic (ampicillin), toward the entire challenged strains.
The Gram + strain (S. aureus) was illustrated to have more resistance to biosynthesized Se-NPs than Gramspecies [13,33,45], which harmonizes the current obtained findings for PRE-synthesized Se-NRs.
The Gram + bacteria are assumed to possess higher resistance to antimicrobial nanometals than Gramspecies due to the presence of Gram + thick protective  peptidoglycan layer that contains teichoic/lypoteichoic acids. While the Grambacteria have specific proteins (porines) that selectively permit molecules' penetration into cells, thus the generated reactive oxygen species (ROS) from Se-NPs can diffuse more easily to interior Gramcells and destruct/inactivate their vital components [33,46,47].

Antibacterial elucidation via SEM
The consequences of S. paucimobilis exposure to PRE/Se-NRs composite on cellular structure, morphology, and deformation are indicated via SEM imaging (Figure 4).
The choice of bacterial strain (S. paucimobilis) was based on its greatest sensitivity to challenging nanocomposites and its novelty as challenged pathogen; thus, it was assumed to offer more useful explanations/evidences for composite action. In the experiment initiation (C in Figure 4), cells had normal, healthy, and contracted appearance with smooth surface and uniformed cell walls. Manifested morphological distortions in the bacterial cells were noticed after the 4th h of exposure to PRE/Se-NRs composite. Bacterial walls were expanded, became puffy, and lots of Se-NRs were attached to the outer cell membranes (4H in Figure 4). With PRE/Se-NRs exposure prolongation to 8 h, cells' deformation, distortion, and lyses became very noticeable (8H in Figure 4). The challenged cells were largely exploded/lysed at this stage. The damaged cell wall residues and interior cellular components were mostly intermingled with Se-NRs. The PRE/Se-NRs composite bactericidal actions are assumed to involve synergistic mechanisms from PRE and Se-NRs [48,49].
The ROS generation and elevated intracellular ROS levels were reported after cells' treatment with biosynthesized Se-NPs [51]. These high ROS levels are frequently associated with mitochondrial dysfunction and DNA damage, leading to cellular death [8]. The PRE-mediated nanometals could have influential antibacterial mechanisms, involving the anchoring/penetration through cell walls and restraining  cellular signals via diverse peptides' dephosphorylation [19,24]. Despite their forceful antibacterial actions, biosynthesized Se-NRs were recommended to possess fascinating attributes for drug delivery because of their diminished side-effects, elevated biocompatibility, and low cytotoxicity [52]. The antibacterial capacities of PRE-mediated metal NPs were reported to depend on the PRE antibacterial action added to NPs' potentialities [26]. Metal NPs with miniature Ps can interact with cellular membranes (as evidently shown in Figure 4), pass into interior cells, and prohibit DNA and protein functions, thereby triggering cells' apoptosis/death, because bacteria cannot habitually replicate. The metallic NPs' (including Se-NPs) antibacterial actions are principally based on generated ROS by NPs, their electrochemical binding to microorganisms' cells, cellular ATP and biomolecule depletion, and cations release [1,7,13]. Additionally, the Se-NPs' bactericidal action was assumed to be associated with osmotic imbalance after interaction among bacterial cells and Se-NPs. This leads to cellular biochemical bonds' interruption in membrane structure and deformation of cell-walls' permeability and functionality [6,15]. The Se-NPs were additionally suggested to interact with thiol and sulfhydryl groups in cellular membrane proteins (e.g., porines), denaturing them, and subsequently causing membranes' deformation and distortion [33].

Conclusion
In the presented investigation, PRE was extracted from P. granatum rinds and effectively employed for phytosynthesis of Se-NRs. The synthesized NRs were negatively charged and had mean diameter of 62.31 nm. The antibacterial actions of PRE, plain Se-NRs, and PRE/Se-NRs composite were proved toward various fish-borne bacterial pathogens, i.e., S. aureus, P. aeruginosa, E. coli, S. typhimurium, and S. paucimobilis, which advocate their applicability for decontaminating fish farms and products.
Acknowledgement: The authors are greatly thankful for the mercy help and guidance from ALLAH. The appreciation is indebted for The Central Lap for Aquatic Health and Safety -Kafrelsheikh University (CLAHS-KFS), for providing the required facilities to conduct the study.
Funding information: Authors state no funding involved.