A novel chitosan agarose nanocopper composite film (Cs/Agr/Cu-Cu2O NPs) using Aspergillus niger K Y 401431: preparation, characterization and evaluation of their antibacterial activity

Abstract

Aspergillus niger K Y 401431 was used in the present study for biosynthesis of copper/copper oxide nanoparticles. A niger K Y 401431 was checked for the production of the carcinogenic mycotoxin 'ochratoxin A' to prove the safety of the strain and the culture filtrate was free from ochratoxin A. For the optimized production of Cu/Cu₂O NPs, the influence of some parameters was investigated. 10 mM of CuSO₄ was optimal for nanoparticle production, well-defined Cu/ Cu₂O NPs formation occurred after 240 min of incubation using culture filtrate of A. niger K Y 401431developed under submerged culture conditions for 3 days culture age. The biosynthesized copper nanoparticles were characterized by UV-visible spectroscopy, transmission electron microscopy (TEM), energy dispersive X-ray (EDX), Fourier-transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS), X-ray diffraction (XRD). The characterization of Cu/Cu₂O NPs revealed the formation of crystalline spherical shaped (Cu/Cu₂O NPs) with 4-10 nm size. The cytotoxic efficacy was investigated against three cancer cell lines MCF7 (Human Caucasian breast adenocarcinoma), HEPG2 (human hepatocellular carcinoma cell line) and A549 (Lung carcinoma cell line) using MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. The cytotoxic effect of the biosynthesized Cu/ Cu₂O NPs on A549, HEPG-2, and MCF-7 showed IC₅₀ of 20.3, 56.9, and 43.3 µg / ml, respectively. In this study, the biosynthesized Cu/ Cu₂O NPs were used for the preparation of chitosan/agarose/copper bionanocomposite film by casting method. The Cs/Agr/ Cu-Cu₂O bionanocomposite films were investigated using SEM, XRD, and FT-IR. The prepared bionanocomposite film displayed enhanced antibacterial activity against pathogenic bacteria.

Keywords: Antibacterial activity; Aspergillus niger K Y 401431; Characterization; Cs/Agr/Cu-Cu2O bionanocomposite film; Cu/Cu2O NPs.

Fig. 1

a HPLC chromatogram of Ochratoxin A standard b: HPLC chromatogram of A. niger K Y 401431 extract

Fig. 2

Effect of one variable at time in the synthesis of nanoparticles. A Effect of different concentration of copper sulphate (CuSO₄) on the production of Cu/Cu₂O NPs. B Effect of submerged and solid- state culture of A. niger on the production of Cu/Cu₂O NPs. C Effect of different incubation periods of A. niger (culture age) on the production of Cu/Cu₂O NPs under shaking culture conditions. D Effect of Reaction Time on the production of Cu/Cu₂O NPs

Fig. 3

A Characterization of Cu/Cu₂O NPs synthesis A: CuSO₄ salt solution; B: culture filtrate; C: reaction at 0 time; D: reaction after 2 h; E: reaction after 3 h, F: reaction after 4 h. B UV–visible spectrum of culture filtrate of A. niger K Y 401431, green reaction medium at zero time and biosynthesized CuNPs. C HR- TEM image of biosynthesized Cu/Cu2O NPs

Fig. 4

A EDX analysis of biosynthesized Cu/Cu₂O NPs. B FTIR spectra of biosynthesized Cu/Cu₂O NPs. C Particle size analysis of biosynthesized Cu/Cu₂O NPs on the basis of DLS. D XRD of biosynthesized Cu/Cu₂O NPs

Fig. 5

SEM images of A Cs/Agr blend and Cs/Agr/Cu-Cu₂O bionanocomposite films containing different concentrations of Cu-Cu₂O NPs, B 1%, C 5% and D 10%

Fig. 6

XRD patterns for: A chitosan/agarose and Cs/Agr/Cu-Cu₂O bionanocomposite films loaded with B 1%, C 5% and D 10 wt% Cu-Cu₂O NPs

Fig. 7

FTIR spectra for A chitosan agarose and Cs/Agr/Cu-Cu₂O bionanocomposite films loaded with B 1%, C 5% and D 10 wt% Cu-Cu₂O NPs

Fig. 8

Antibacterial activity of biosynthesized Cu/Cu₂O NPs and chitosan agarose nanocomposite films loaded with different concentrations of biosynthesized Cu/Cu₂O NPs. BL: Cs/Agr composite, Cu: biosynthesized Cu/Cu₂O NPs and Cs/Agr/Cu- Cu₂O bionanocomposite films loaded with; 1c: 1%, 5c: 5% and 10c: 10% wt% Cu- Cu₂O NPs