Antibacterial action of insect chitosan/gum Arabic nanocomposites encapsulating eugenol and selenium nanoparticles

2.1 Materials

Spray-dried Acacia gum (Gum Arabic; GA, Merck Millipore, Germany); eugenol (Eug), acetic acid, Tween 80, hydrochloric acid (HCl, 37%), sodium selenite (Na₂SeO₃-5H₂O), ethanol (≥99.8%), triphenyl tetrazolium chloride (TTC), L-ascorbic acid (99%), methanol (99.9%, HPLC-grade), chloroform (HPLC-grade, 99.5+%), sodium hydroxide (NaOH, anhydrous, ≥98%), and microbiological media (obtained from Sigma-Aldrich, MO), were used throughout current study.

2.2 Insect chitosan extraction

Insect CT was extracted from BSF “Hermetia illucens larvae”, which were attained from the “Experimental insect farm, University of King Abdulaziz, Jeddah, KSA”, at their 5th instar. The insects’ proteins and oils were mostly eliminated via oil-press approach, and BSF residues were rinsed with double-distilled water (DW), and then lyophilized. The CT extraction from BSF residual materials involved (Marei et al., 2016; Al-saggaf, 2021):

1. Defatting: by treatment with 12 folds (v/w) of chloroform–methanol mix (7:3, respectively) under stirring for 5 h at room temperature (RT; 25 ± 2 °C).

2. Demineralization: by treatment of resulted materials from (1) with 12 folds (v/w) of 2% HCl solution for 2 h at RT.

3. Deproteinization: by treatment of resulted materials from (2) with 10 folds (v/w) of 1.0 M NaOH solution at 48 ± 2 °C for 3 h.

4. Deacetylation: by treatment of resulted materials from (3) with 20 folds (v/w) of concentrated NaOH (58%, w/v) solution, at RT for 1 h then at 115 °C for 2 h. The extensive DW washing and air drying was performed after every step, and the final resulted CT was then lyophilized. For the lyophilization of CT and subsequent composites, the vials of lab-scale benchtop lyophilizer (VirTis adVantage Plus; SP Scientific, Gardiner, NY) were filled with composites’ solution, stoppered and positioned into the instrument tray. The lyophilization was performed at − 40 °C for 22 h with 0.111 kPa chamber pressure.

2.3 Preparation of biopolymers nanocomposites

For preparing polymers nanocomposite from inset NCT, GA, Eug and SeNPs, the GA and extracted CT were firstly dissolved (at 0.1%,w/v) in DW and acetic acid 0.1%, respectively. The pH of dissolute CT solution was modified to 4.8 by adding few drops (when necessary) from 0.2 M NaOH solution, and then Eug and Tween 80 were added to this solution (at 0.1%, v/v each) while stirring at 520g at RT. After that, GA solution was dropped slowly into this solution while stirred. The stirring continued for further 2 h, and then the mixing solution was centrifuged at 11500g for 22 min (Sigma 2-16P-10350, GmbH, Germany). The supernatant was collected, recurrently washed with DW, re-centrifuged and lyophilized (Rajabi et al., 2019).

For NCT/GA/Eug/SeNPs nano-compositing, 100 µM of sodium selenite was dissolved in GA solution before dropping and the chitosan/Eug solution was supplemented with 0.1% (w/v) of ascorbic acid, as precursor for SeNPs reduction, and the step were conducted as above. The formed nanocomposites were purified via repeated washing by DW and ethanol, followed by centrifugation after each wash and pellets’ harvesting, to eliminate the excess free molecules.

2.4 Analysis of materials physiognomies

2.5 The encapsulation efficiency (EE) and loading capacity (LC) of Eug-loaded nanopolymers

The EE and LC of Eug encapsulated into NCT/GA nanocomposite were analyzed according to Woranuch and Yoksan (2013). The dispersed sample in DW was combined with 5% (v/v) of 1 M HCl and heated to 96 °C for 32 min. Ethanol (1%, v/v) was introduced to the mixture, then it was centrifuged at 7500g for 1 min at RT. The gathered supernatant absorbance was measured via UV–vis spectrophotometry at 283.5 nm wavelength. The EE and LC of Eug in nanopolymers were calculated as follow: $$EE\left(\%\right)=\left[\mathit{weight}ofloadedEug/weightofinitialEug\right]\times 100$$ $$LC\left(\%\right)=\left[\mathit{weight}ofloadedEug/weightofsample\right]\times 100$$

2.6 Antibacterial assessment of nanopolymers-based composites

The antibacterial assessment of NCT/GA, NCT/GA/Eug and NCT/GA/Eug/SeNPs involved diverse techniques for qualitative, quantitative and imaging examination.

2.7 Statistical analysis

Experiments in triplicates were performed, the SPSS package (V 11.5, Chicago, USA) was used for computing means ± SD (standard deviation) and the differences’ significances at p ≤ 0.05 were computed using one-way ANOVA and t-test.

3.1 Chitosan extraction from BSF

The CT was efficaciously extracted from larval biomass of BSF; the yielded purified CT reached 1.62%, with 88.2% deacetylation degree, molecular weight of 95.7 kDa and solubility of 97.1% in diluted acetic acid (1%, v/v). The attained physiochemical characteristics of BSF-CT here are comparable to the formerly obtained from recent investigations (Marei et al., 2016; Al-saggaf, 2021).

3.2 Infra-red analysis of produced molecules

The biochemical bonding and interactions of employed molecules (NCT, GA, Eug and their composites) were analyzed spectroscopically via FTIR (Fig. 1).

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Fig. 1

FTIR spectra of fabricated biomolecules/composites, including insect chitosan nanoparticles (NCT), gum Arabic (GA) and eugenol (Eug).

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For NCT (Fig. 1-NCT), the spectral analysis indicated the occurrence of most distinctive bonding in extracted CT from insects, which were illustrated in earlier investigations (Marei et al., 2016; Luo et al., 2019; Hahn et al., 2020). The main distinctive bands in NCT were positioned at 3414 cm⁻¹ (pertained to overlapped O–H and N–H extensional vibrations), 1568 cm⁻¹ (N–H bending vibration), 1415 cm⁻¹ (C–H extensional vibration) Additionally, the following characteristic bands were appeared an derived from native CT, at 2913 cm⁻¹ (C–H stretching), 1642 cm⁻¹ (amide I), 1538 cm⁻¹ (amide II), 1079 cm⁻¹ (C-O-C), and at 904 cm⁻¹ (pyranose ring) (Luo et al., 2019; Hahn et al., 2020; Al-saggaf, 2021). The specified peak for CT-TPP binding and forming of P⚌O linkage was detected at 1251 cm⁻¹, confirming NCT formation (Anand et al., 2021).

The GA spectrum (Fig. 1-GA) displayed the distinguishing band for this biopolymer; including the sharp bands at 1622 and 1413 cm⁻¹ (assigning the asymmetrical and symmetrical vibrations of carboxyl groups, respectively), at 2911 cm⁻¹ (C–H extension), and around 3400 cm⁻¹ (O–H extensional vibrations) (Espinosa-Andrews et al., 2010; Tan et al., 2016; Rajabi et al., 2019).

The band at 2911 cm⁻¹ could signify the existence of sugars, e.g. arabinose, galactose, and rhamnose. The 1622 cm⁻¹ band indicates occurrence of amino acids and aromatic/aliphatic galactoproteins, along with the 1376 cm⁻¹ band. The glucuronic acids indicative band (at 1413 cm⁻¹) is attributed to symmetric C⚌O stretching. Additionally, the 1252 cm⁻¹ band could represent C-O alcohol stretch, alkane CH₃, stretching ether C-O-C or stretching amines C-N, whereas the 1024 cm⁻¹ band represents bending alkene C–H from gum polysaccharides (Rajabi et al., 2019).

The conjugated NCT/GA nanoparticles spectrum (Fig. 1-NCT/GA) has many distinctive peaks from both biopolymers, and shifting of other specified peaks after conjugations (e.g. the band at 1622 cm⁻¹ in GA spectrum shifted to 1578 cm⁻¹ in NCT/GA spectrum), which indicate physical/chemical interactions after polymers’ conjugation (Avadi et al., 2010; Espinosa-Andrews et al., 2010; Rajabi et al., 2019).

The FTIR spectrum of loaded Eug into NCT/GA nanocomposite reflected the main distinctive peaks of Eug in addition to the biopolymers characteristic peaks (Fig. 1-NCT/GA/Eug). The Eug distinctive bands were clearly detected at 3077 and 2999 cm⁻¹ (⚌C–H vibrated stretching), at 1508 and 1607 cm⁻¹ (C⚌C vibrated stretching in aromatic rings), at 1261 and 1203 cm⁻¹ (C-O bending vibration), at 891 cm⁻¹ (–C⚌CH₂ bending), at 741 cm⁻¹ (–CH2 bending), and at 1358 cm⁻¹ (–CH3 bending) (Woranuch and Yoksan, 2013; Anand et al., 2021; Das et al., 2021). the detection of bands at 1231 and 1120 cm⁻¹ that appointed ionic-crosslink formation between TPP and NH³⁺ groups in NCT, in addition to Eug distinctive bands in NCT/GA/Eug composite, strongly validated the physiochemical conjugation between the composite materials of chitosan and anionic groups conjugation (Bonilla et al., 2018; Anand et al., 2021; Das et al., 2021).

3.3 Structural analysis of fabricated nanoparticles/nanocomposites

The structural physiognomies of fabricated nanocomposites are demonstrated in Table 1 and Fig. 2. The NCT/GA nanocomposite have homogenous and well-dispersed characteristics, with mean Ps diameter of 124.8 nm, whereas the NCT/GA/Eug composite were had slightly larger Ps mean diameter (132.6 nm) and this mean additionally increased (134.2 nm) with the incorporation of SeNPs to form NCT/GA/Eug/SeNPs composite. The entire nanocomposites (NCT/GA, NCT/GA/Eug, and NCT/GA/Eug/SeNPs) carried strong positive charges on their surface (e.g. zeta potential > 30 mV); the highest positivity was recorded for NCT/GA (+38.7 mV) and slightly reduced with the incorporation of further molecules to nanocomposites. The used ratio and pH for NCT/GA composite fabrication were should be 1:1 (w/w) and 4.0, respectively, which was formerly illustrated as the optimal conditions for this nanocomposite construction (Tan et al., 2016); the resulted nanoparticles with this technique/condition have high affinity for encapsulating hydrophobic molecules. The illustrated mechanism for producing NCT/GA nanocomposites, which are highly stable, is the interaction between positively charged amine groups in NCT with negative charges in GA surface; the excess positive charges in NCT groups give the composite its positive values of zeta potential (Avadi et al., 2010; Pant and Negi, 2018).

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Fig. 2

Electron microscopy imaging of produced nanomaterials including the SEM of insect chitosan/gum Arabic nanocomposite (A), their loaded particles with eugenol (B), and the TEM imaging of eugenol-mediated SeNPs (C).

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The slight increase in Ps of NCT/GA/Eug and NCT/GA/Eug/SeNPs composites than the Ps of plain NCT/GA, and the decrease of the nanocomposites’ zeta potential after incorporation of Eug and SeNPs, both indicate that NCT/GA could entrap/uphold the other molecules within their matrix. This feature generally leads to Ps enlargement and occupation of some free charges with these added molecules, which cause zeta potential reductions (Tan et al., 2016; Rajabi et al., 2019).

The calculated EE and LC of NCT/GA toward Eug were 71.94 ± 0.42% and 17.45 ± 0.34%, respectively. The LC represented the quantity of loaded Eug per weight unit of biopolymer matrix, while EE denoted the efficacy of biopolymers to protect the loaded Eug (Das et al., 2021); the EE is economically very important for nanocapsules’ fabrication (Woranuch and Yoksan, 2013; Rajabi et al., 2019). The encapsulation into nanopolymers (e.g. NCT) was validated to augment Eug thermal stability, and the used ratios from Eug to nanopolymer were reported as the optimum to have stronger and thicker layer around bioactive molecules and gave higher EE (Woranuch and Yoksan, 2013; Rajabi et al., 2019). Additionally, the employment of Tween-80 emulsifier in Eug encapsulation in core of NCT led to elevated percentages for EE and LC (Das et al., 2021).

3.4 Antibacterial assessment

The bacterial inhibitory actions of fabricated polymers nanocomposites (NCT/GA) and their conjugates with Eug and SeNPs were assessed qualitatively (via ZOI measurement), quantitatively (via MIC and MBC determination) and confirmed through SEM imaging for the Gram- (E. coli) and Gram⁺ (S. aureus) bacteria (Table 2 and Fig. 3, respectively).

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Fig. 3

Scanning imaging of exposed Escherichia coli (E) and Staphylococcus aureus (S) cells to nanocomposite of chitosan/gum Arabic/eugenol/SeNPs for 0, 5 and 10 h. Arrows indicate examples of attached nanoparticles to microbial cells.

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