Bio-based synthesis of silver nanoparticles from orange waste: effects of distinct biomolecule coatings on size, morphology, and antimicrobial activity

Abstract

Purpose: Despite the numerous reports on biological syntheses of silver nanoparticles (AgNPs), little is known about the composition of their capping agents, protein corona of plant extract-mediated synthesis, and their influence on the properties of AgNPs. Here, orange (Citrus sinensis) waste was utilized as a source of an extract for AgNP synthesis (the protein corona composition of which was elucidated), and also as a starting material for hesperidin and nanocellulose extraction, which were used for bio-based AgNP synthesis. A comparison of the results using the two methods of synthesis is presented.

Methods: AgNPs were synthesized using orange (C. sinensis) peel extract (Or-AgNPs) in a biological route, and using hesperidin (Hsd-AgNPs) and nanocellulose (extracted from oranges) in a green chemical route. Characterization of nanoparticles was carried out using zeta potential and hydrodynamic size measurements, transmission electron microscopy, and X-ray diffraction. Elucidation of proteins from protein corona was performed via ultra performance liquid chromatography-tandem mass spectrometer experiments. Antimicrobial activity was assessed via minimum inhibitory concentration assays against Xanthomonas axonopodis pv. citri (Xac), the bacterium that causes citric canker in oranges.

Results: Or-AgNPs were not completely uniform in morphology, having a size of 48.1±20.5 nm and a zeta potential of -19.0±0.4 mV. Stabilization was performed mainly by three proteins, which were identified by tandem mass spectrometry (MS/MS) experiments. Hsd-AgNPs were smaller (25.4±12.5 nm) and had uniform morphology. Nanocellulose provided a strong steric and electrostatic (-28.2±1.0 mV) stabilization to the nanoparticles. Both AgNPs presented roughly the same activity against Xac, with the minimum inhibitory concentration range between 22 and 24 μg mL^-1.

Conclusion: Despite the fact that different capping biomolecules on AgNPs had an influence on morphology, size, and stability of AgNPs, the antibacterial activity against Xac was not sensitive to this parameter. Moreover, three proteins from the protein corona of Or-AgNPs were identified.

Keywords: Citrus sinensis; Xanthomonas; hesperidin; orange peel; silver nanoparticles.

Figure 1

UV–Vis absorption spectrum of Or-AgNPs after 48 h of reaction displaying the characteristic surface plasmon resonance band at 449 nm. In detail, the photographic image of the extract (left) and the corresponding AgNPs suspension (right) (A). The kinetic progression of the Or-AgNPs reaction during the 48 h monitored setting the absorbance at 430 nm (B). UV–Vis absorption spectrum of Hsd-AgNPs after 2 h of reaction displaying the characteristic surface plasmon resonance band at 411 nm. In detail, the photographic image of the AgNPs suspension (C). The kinetic progression of the Hsd-AgNPs reaction during the 2 h monitored setting the absorbance at 411 nm (D). Abbreviations: AgNPs, silver nanoparticles; AU, arbitrary unit; Hsd, hesperidin; Or, aqueous orange; UV-vis, ultraviolet-visible.

Figure 2

X-ray diffraction pattern of orange peel extract AgNPs (A); images of orange peel extract AgNPs obtained by TEM (B and C); X-ray diffraction pattern of hesperidin/nanocellulose AgNPs (D); images of hesperidin/nanocellulose AgNPs obtained by TEM (E and F). Abbreviations: AgNPs, silver nanoparticles; AU, arbitrary unit; TEM, transmission electron microscopy.

Figure 3

Comparison of FTIR spectra of freeze-dried samples of orange peel extract and Or-AgNPs (A) and Hsd-AgNPs (B). Abbreviations: AgNPs, silver nanoparticles; AU, arbitrary unit; FTIR, Fourier-transformed infrared spectroscopy; Hsd, hesperidin; Or, aqueous orange.

Figure 4

Raman scattering spectra of Or-AgNPs suspension using 632.8 nm laser (A) and 785 nm laser (B). Abbreviation: AgNPs, silver nanoparticles; Or, aqueous orange.

Figure 5

Proteins identified as capping agents in Or-AgNPs in UPLC-MS/MS experiments, with tridimensional structures predicted by I-TASSER server. Abbreviations: AgNPs, silver nanoparticles; Or, aqueous orange; UPLC-MS/MS, ultra performance liquid chromatography-tandem mass spectrometer.