Biosynthesis MgO and ZnO nanoparticles using chitosan extracted from Pimelia Payraudi Latreille for antibacterial applications

Chitosan (CS) is one of the most abundant biopolymers in nature with superior properties such as biocompatibility, biodegradability, lack of toxicity, antimicrobial activity, acceleration of wound healing, and stimulation of the immune system. In this study, chitosan was extracted from the exoskeletons of beetles (Pimelia payraudi latreille) and then used for the biosynthesis of highly pure MgO NPs and ZnO NPs by a facile greener route. The extracted chitosan exhibited excellent physicochemical properties, including high extraction yield (39%), high degree of deacetylation (90%), low ash content (1%), high fat-binding capacity (366%), and unusual crystallinity index (51%). The MgO NPs and ZnO NPs exhibited a spherical morphology with crystallite sizes of 17 nm and 29 nm, particle sizes of about 20–70 nm and 30–60 nm, and band gap energies of 4.43 and 3.34 eV, respectively. Antibacterial assays showed that the extracted chitosan exhibited high antibacterial activity against Gram-positive and -negative bacteria, while ZnO NPs showed much stronger antibacterial activity against Gram-positive bacteria than against Gram-negative bacteria. For MgO NPs, the antibacterial activity against Gram-positive bacteria was lower than against Gram-negative bacteria. The results suggest that the synthesized MgO NPs and ZnO NPs are excellent antibacterial agents for therapeutic applications.


Introduction
Antimicrobial nanoparticles are potential broad-spectrum antibiotics that can overcome resistance to conventional antibiotics (Laouini et al. 2021). The adhesion of nanoparticles to microbial cells and reactive oxygen species and their penetration into cells have been identified as the main antimicrobial mechanisms of action . Depending on particle size, shape, and concentration, nanoparticles can be either bactericidal or bacteriostatic (Harish et al. 2022;Cunha et al. 2018;Salem et al. 2022a;Elakraa et al. 2022;Al-Zahrani et al. 2022b). In addition, the development of resistance to nanoparticles is less likely, but not impossible. Physicochemical approaches such as sol-gel and solvothermal methods are the most common methods for preparing metals and metal oxide nanoparticles (Salama et al. 2021). However, these methods are usually expensive and can pollute the environment. Therefore, an alternative green biosynthesis method was used to synthesize nanoparticles (Abdelaziz et al. 2022;Shehabeldine et al. 2022a;Saied et al. 2021;Al-Zahrani et al. 2022a). Plant extracts or biopolymer molecules can be used as reducing agents to produce nanoparticles (Belaiche et al. 2021;Hamimed et al. 2022). The biosynthesis methods using plant extracts or biopolymer molecules are very important in the preparation of Ag, Au, Cu, ZnO and MgO NPs (Said et al. 2021;Djamila et al. 2022;Al-Rajhi et al. 2022;Shehabeldine et al. 2022b;Hashem et al. 2022;Salem et al. 2022b).
Chitosan a natural polycationic polymer, it is used as a reducing agent for nanoparticle synthesis and an antimicrobial agent for biomedical applications. Chitosan composites with nanoparticles have significant antibacterial activity against a variety of bacterial pathogens. It is mainly available in the exoskeletons of arthropods, in Saccharomyces cerevisiae and in the cell walls of fungi (Iber et al. 2022). It is also found in the cuticle of insects and crustaceans, which contains 15-20% chitin along with proteins, minerals and pigments (Said et al. 2021). Chitosan is commercially produced by deacetylation of the chitin extracted from the exoskeleton of crustaceans. The degree of deacetylation ranges from 60 to 100% for commercial chitosans. During extraction, the proteins, minerals and pigments of the cuticle are separated from the chitin matrix using chemicals under mild conditions. Commercially produced chitosan has an average molecular weight (Mwt) of 4000-20,000 Daltons and a pKa of ~ 6.5 (Rinaudo 2006). The amino groups make chitosan water-soluble and bind readily to negatively charged functional groups. chitosan is a non-toxic, biocompatible and biodegradable biopolymer with antitumor, antioxidant and antimicrobial properties (Jiménez-Gómez and Cecilia 2020).
Chitosan is gaining importance as biopolymers used as reducing agents, shapers, or size adjusters in the production of metal and metal oxide nanoparticles (Ottonelli et al. 2020). Coating the surfaces of nanoparticles with chitosan makes them more biocompatible with normal cells. Chitosan has been shown to regulate drug release, promote mucosal adhesion and tissue penetration (Ahmed and Aljaeid 2016), support cell interaction (Zubareva and Svirshchevskaya 2016) and enhance antimicrobial activity (Phan et al. 2021). The amino groups of chitosan are rapidly protonated in the acidic state and can combine with anionic functional groups. Large amounts of amino groups on chitosan lead to easy binding of metal oxide nanoparticles with many bioactive molecules, including drugs, enzymes, protons, etc. (Hojnik Podrepšek et al. 1913). Chitosan-coated nanoparticles possess antibacterial and antioxidant activity, as well as antituberculosis activity and antifungal activity against Candida (Sharifiaghdam et al. 2022). The antibacterial activity of chitosan is due to its electric charge and high adsorption capacity, as well as chemical processes that enable them to effectively destroy the bacterial cell membrane and inhibit the growth of bacterial cells (Yilmaz Atay 2019).
Given the growing concern about biofilm-associated infections and drug-resistant bacteria, the development of new antibacterial agents is a pressing concern (Youssef et al. 2017;Forster et al. 2022). The present work aims to isolate chitosan from beetle (Pimelia payraudi latreille) and then use the extracted chitosan to prepare MgO NPs and ZnO NPs. Various physical characterization methods such as extraction yield, deacetylation degree (DD), ash content (AC), moisture content measurement (MC), water binding capacity (WBC) and fat binding capacity (FBC) were performed for the synthesized chitosan. Chitosan, ZnO NPs and MgO NPs were characterized by UV-Vis, FTIR, XRD and SEM. The antibacterial activity of chitosan, MgO NPs and ZnO NPs was evaluated against Staphylococcus aureus (ATCC6538), Bacillus subtiliis (ATCC6633), Listeria innocua (CLIP74915), Pseudomonas aeruginosa (ATCC9027) and Salmonella typhimuruim (ATCC14028). Chitosan is enriched with amino functional groups which bind with ZnO NPs and MgO NPs, improving their properties and preventing nanoparticle agglomeration. Therefore, we believe that the prepared chitosan, ZnO NPs and MgO NPs could be used as antimicrobial nanomaterials in the cosmetics, biomedical, and pharmaceutical industries.

Materials
Zinc chloride (ZnCl 2 , 99%), magnesium chloride hydrates (MgCl 2 ,6H 2 O, 98%), sodium hydroxide (NaOH, 97%), hydrochloric acid (HCl, 99%), acetic acid (CH 3 COOH, 99.5%), hydrogen peroxide (H 2 O 2 , 98%) and dimethyl sulfoxide (DMSO, 99%) were purchased from Biochem Chemophara. Mueller-Hinton agar was purchased from Bioscan Industrie, Algeria. The beetles (Pimelia payraudi latreille) were the primary supplier for chitosan extraction. The beetles were collected in the city of El-oued, Algeria (33° 22′ 06″ N and 6° 52′ 03″ E) and the exoskeleton was selected for chitosan extraction. The insects used in this study are widely distributed worldwide and in Algeria and were not endangered or protected species in the field study. The specimens (insects) were acquired in a dead and dry state, and no special permit was required for access to the site. Bacteria were provided by the Algerian Microorganisms Culture Center. Image J software was used to calculate the particle size distribution of the nanoparticles.

Extraction of chitosan
Beetles were starved in the laboratory for 3 days to remove the gut contents. Then, the beetles were washed with distilled water, dried, ground, and then stored at − 20 °C. As shown in Fig. 1, the exoskeleton of the beetles was thoroughly freed from loose tissue, cleaned, dried, and crushed to fit through a 250 μm mesh sieve (Kaya et al. 2014a). Briefly, 30 g of the sample (beetle exoskeleton) was treated with 1 M HCl (demineralization) at a solid-to-liquid ratio of 1:15 for 1 h at 40 °C. After demineralization, the resulting solid fraction was rinsed with distilled water until the pH returned to pH 7. The extracted chitin was deproteinization with 1 M NaOH at 80 °C for 2 h. The chitin was then filtered, washed, and then decolorized by treatment with 10% H 2 O 2 at 50 °C for 30 min. The chitin was then neutralized by rinsing with distilled water until pH 7 was reached. The decolorized chitin was subjected to a deacetylation procedure in which it was treated with 50 wt% NaOH at 100 °C for 4 h. The precipitate was washed with distilled water and the resulting chitosan was dried under a vacuum at 50 °C for 24 h.

Biosynthesis of MgO NPs and ZnO NPs
To prepare MgO NPs and ZnO NPs from the chitosan extract, a solution with a concentration of 1% w/v chitosan in an acetic acid solution was prepared. After completion of the dissolution process, 5 mL of the previously prepared chitosan was mixed with 50 mL of an aqueous solution containing MgCl 2 ·6H 2 O or ZnCl 2 at a concentration of 0.1 M for 4 h. The pH of each mixture was adjusted by adding 0.1 M NaOH with constant stirring until the pH became basic (pH 9) at 60 °C. The precipitate was then rinsed several times with deionized water, centrifuged at a speed of 2500 rpm, and then heated at 500 °C for 4 h to remove all organic residues.

Physicochemical characterization
After drying the chitosan extract, the dried chitosan was weighed and the chitosan content was calculated as follows: Yield (%) = (weight of chitosan, g)/(weight of beetle exoskeleton, g) × 100 (Failed 2017). The ash content of chitosan was determined gravimetrically by combustion in an air atmosphere using a constant weight crucible at 550 °C for 3 h. The degree of deacetylation (DD) of chitosan was determined by FTIR spectroscopy using the following equation: DD (%) = 100−[100*(A 1655 /A 3450 )/1.33]. Where: A 3450 and A 1655 are the heights of the absorption bands of the hydroxyl and amide groups, respectively. The factor of 1.33 indicates the value of the A 1655 /A 3450 ratio for fully N-acetylated chitosan (Hao et al. 2021). The ash content of chitosan was determined according to the method (Rødde et al. 2008). The following equation was used to determine the ash content: Ash % = (W 2 /W 1 ) × 100, where W 1 and W 2 are the initial weight of the chitosan sample and the weight of the residue (grams), respectively. The moisture content of the prepared chitosan was determined gravimetrically by vacuum drying at 110 °C for 24 h. The moisture content (MC, %) = (W 1 −W 2 /W 1 ) × 100, where W 1 and W 2 are the weights of the wet and oven-dried samples, respectively (Klute 1986). WBC and FBC were determined according to a modified method by Wang and Kinsella (Samar et al. 2013). Briefly, a centrifuge tube containing 0.5 g of chitosan was weighed first. Then, 10 mL of water for the WBC test and soybean oil for the FBC test were added to the chitosan and mixed in the centrifuge tube for 1 min using a vortexer. The mixture was allowed to stand at 25 °C for 30 min, shaken every 10 min for 5 s, and centrifuged at 3200 rpm for 25 min. After decanting the supernatant, the tube was reweighed. WBC and FBC were calculated as follows: WBC% = (water-bound, g/initial weight chitosan, g) × 100 and FBC% = (fat-bound, g/initial weight chitosan, g) × 100 (Marei et al. 2016).
The crystalline structure of chitosan, MgO NPs and ZnO NPs was studied by using X-ray diffraction (XRD, Rigaku Miniflex 600). A UV-Vis spectrophotometer (Jasco V160 UV-Vis) was used to record the absorption spectra of chitosan, MgO NPs, and ZnO NPs. The UV-Vis spectra of ZnO NPs and MgO NPs were studied by dispersing 0.1 mg each of ZnO NPs and MgO NPs in 2 mL of distilled water. The optical properties of chitosan were investigated by dissolving 0.1 mg of chitosan in 2 mL of acetic acid. The band gap energy (Eg) was estimated using the Tauc relationship (αhν) = A (hν−Eg) n (Barhoum et al. 2021). Here α is the absorption coefficient, h is Planck's constant, A is a constant, Eg is the energy bandgap, and n is a constant equal to ½ for the direct band gap (Bouafia et al. 2021). The IR spectra were characterized using a Nicolet iS50 FT-IR spectrometer with the KBr technique (Barhoum and García-Betancourt 2018). Particle size, morphology, and elemental composition were studied with a scanning electron microscope (FESEM, Leo Supra 55-Zeiss Inc., Germany).

Antibacterial bioassay
Agar well diffusion method was used to test the antibacterial activity of chitosan, MgO NPs and ZnO NPs against a variety of bacterial strains, particularly Staphylococcus aureus (ATCC6538), Bacillus subtiliis (ATCC6633), Listeria innocua (CLIP74915), Pseudomonas aeruginosa (ATCC9027) and Salmonella typhimuruim (ATCC14028). Culture plates are prepared and streaked with 100 µL of a 24 h matured broth culture of bacterial strains using a sterile glass rod. Wells are made with a sterile cork borer; 6-mm wells are made in each Petri plate. Different concentrations of MgO NPs and ZnO NPs (2 mg/mL, 4 mg/mL, 6 mg/mL in DMSO) and chitosan (1%, 4%, and 8% in acetic acid), acetic acid is used to dissolve chitosan because the latter is insoluble (Santoso et al. 2020). The antibacterial assay of the samples tested against ciprofloxacin (CIP-5) as a reference. The plates were incubated for 24 h at 37 °C. All experiment was done in triplicate, the diameters of the zone of inhibition after the incubation period are summarized in Table 1.

Statistical analysis
The statistical calculations (standard deviation) are based on three measurements at least.

Characteristics of chitosan
Chitosan is a deacetylated derivative of chitin, both are biocompatible, biodegradable, and non-toxic. Previous studies reported that the yield of chitosan from other insects ranges from 2 to 79% (Zainol Abidin et al. 2020). This study showed a chitosan yield of 39%. The lower limit of yield (39%) of chitosan is due to the removal of the proteins and impurities during the deacetylation and precipitation process. In contrast to chitin, chitosan has a large proportion  Figure 2A shows the UV-vis absorption spectra of chitosan, ZnO NPs, and MgO NPs. The chitosan has two main absorbance bands, a weak peak at 270 nm and a strong peak at 220 nm. The band at 270 nm is due to the n-π* transition of the amino groups. The spectral absorption at 320 nm is due to the n-π* transition for the carboxyl or carbonyl groups

FTIR spectroscopy
FTIR spectra of chitosan (Fig. 3a, b) showed a broader band at 3100-3500 cm −1 corresponding to the -OH and the -NH stretching vibrations (Kumirska et al. 2010). The bands at 2862 cm −1 correspond to the stretching of polysaccharide bonds CH. The band at 1650 cm −1 is attributed to amide I resulting from the removal of the acetyl group (deacetylated chitin) (Ben Seghir and Benhamza 2017). The band at 1580 cm −1 corresponds to an amide II (-NH 2 bending). The degree of acetylation of the chitosan extract was calculated from the FTIR spectra and was found to be 90% (

X-ray diffraction
The crystallinity and crystalline structure of chitosan strongly depend on the degree of deacetylation of chitin.
The XRD pattern of chitosan (Fig. 4a) shows three diffraction peaks occurring at 10.4°, 20.0°, and 30°, corresponding to the (020), (110), and (100) planes of the crystal lattice, respectively. The intensity of the peak at 10.4° is lower than that at 20.0°. This is due to the intramolecular hydrogen bonds formed after deacetylation (Kaya et al. 2015). The crystallinity of chitosan was determined using the following equation: CI = (I 110 −I am )/I 110 . Where I am is the intensity of amorphous diffraction centered at 2θ = 10° and I 110 is the maximum diffraction intensity at centered 2θ = 20° (Kumirska et al. 2010;Kaya et al. 2014b). The determined value of CI is 51%. Figure 4b shows Where d is the average crystallite size, λ is the wavelength of X-ray (0.15406 nm), K is a constant assumed to be 0.9 and β is the FWHM of the intensity for the peak centered at 20°; and θ is the Bragg angle. The average crystallite size of the MgO NPs is 17 nm, while the crystallite size of the ZnO NPs is 29 nm.

Scanning electron microscopy
SEM was used to analyze the morphology of the extracted chitosan, ZnO NPs, and MgO NPs. Chitosan extracted from Pimelia payraudi latreille has irrigualr (semicrystalline) structure with a rough surface similar to the structure of shrimp and crab (Fig. 5a, b). The morphology and particle size of ZnO NPs and MgO NPs are affected by several factors, such as salt concentration, chitosan concentration, solution pH and temperature (Hassan et al. 2021). SEM images show that the ZnO and MgO NPs have a spherical morphology (Fig. 5c, e). The particle size distribution histograms (Fig. 5d, f) show indicated that the average size distribution of the MgO NPs around 20-70 nm and 30-60 nm for ZnO NPs. SEM-EDS elemental analysis (Fig. 5g, h and  and Gram-negative bacteria (Pseudomonas aeruginosa, Salmonella typhimuruim). Table 3 and Fig. 6a shows that the zone of inhibition of chitosan increases with increasing chitosan concentration (Ardean et al. 2021).Chitosan is most active on the surface of bacterial cells and leads  Table 3 shows the strong antibacterial activity of MgO NPs against Gram-negative bacteria (Salmonella typhimurium), but shows no significant effect on Gram-positive bacteria. The mechanism of action of MgO NPs with bacterial cells depends on the cell wall structure and the binding sites of MgO NPs to the cell surface, subsequently the interaction of Mg NPs with internal cell components. In contrast to MgO NPs, ZnO NPs have a much stronger antibacterial effect on Gram-positive bacteria compared to Gram-negative bacteria (Fig. 6c). The difference between Gram-positive bacteria and Gram-negative bacteria in the outer membrane is probably the main reason for this behavior (Tan et al. 2018). The cell wall of Gram-negative bacteria consists of two cell membranes, an outer membrane and a plasma membrane with a thin peptidoglycan layer. On the opposite side, the MgO NPs and ZnO NPs have also different crystal structure and surface charges, which interacts differently with Gram-positive bacteria and with Gram-negative bacteria (Wang et al. 2017). The main problem in evaluating the antimicrobial activity of chitosan has been to find the concentration at which acetic acid ceases to have an effect and the inhibitory activity can be attributed exclusively to chitosan. Recent studies have shown that the antibacterial activity of acetic acid is related to concentration. At low concentrations (below 200 ppm), acetic acid had no antibacterial activity. At medium concentration (200 ppm), the antibacterial effect was evident. At high concentrations (above 200 ppm), it was able to kill all E. coli (Ardean et al. 2021;Mendoza et al. 2017). Recent studies on the antibacterial efficacy of chitosan, MgO NPs and ZnO NPs against bacterial strains were compared to results of this work and summarised in Table 4.

Conclusion
Chitosan was extracted from the exoskeletons of the beetle (Pimelia payraudi latreille) and then used for the biosynthesis of antibacterial MgO NPs and ZnO NPs. After precipitation, the yield of pure chitosan from the beetle Pimelia payraudi latreille was 39%. The extracted chitosan exhibited excellent physicochemical properties compared to previously published studies: Extraction yield (39%), DD (90%), AC (1%), MC (9%), WBC (577%), FBC (366%), CI (51%). The ZnO NPs and MgO NPs prepared from chitosan exhibited a spherical morphology with an average particle size of the MgO NPs around 20-70 nm and 30-60 nm for ZnO NPs.The antibacterial properties of chitosan, MgO NPs and ZnO NPs were investigated against different types of Gram-negative and Gram-positive bacteria by the agar well diffusion method. Chitosan showed good antibacterial activity against both Gram-positive and Gram-negative bacteria. The ZnO NPs showed much stronger antibacterial activity against Gram-positive bacteria than against Gram-negative bacteria. While the MgO NPs showed strong antibacterial activity against Gram-negative bacteria and low activity against Gram-positive bacteria. The results suggest that the chitosan extracted from the exoskeletons of the beetle and the biosynthesized MgO NPs and ZnO NPs are excellent antibacterial agents for biomedical applications.