Characterization and Antibacterial Efficacy of Mycosynthesized Silver Nanoparticles from the Desert Truffle Tirmania nivea
Authors: Muhsin Tawfik Muhammed , Hachim Ahmad K. 
Published: July 2019

Desert truffles are an obligate hypogenous ectomycorrhizal fungi in association with host plant roots Helianthemum sp. and are of socioeconomically important and naturally grown in the Middle East, North Africa, Southern Europe, Mediterranean countries including Arab Gulf countries. Truffles are edible and a rich source of protein and various chemical compounds and traditionally have been used as folk medicine in the Arabian countries. The last decade has witnessed an increase research interest focused on the biosynthesis of metal nanoparticles using fungi as natural sources and as a good tool in nanobiotechnology. Nevertheless, recently metal nanoparticles have been widely applied in multidisciplinary fields including medical and pharmaceutical applications. Among nanometals, silver nanoparticles are of great significance to be used in pharmaceutical aspect as antimicrobial agent. According to our knowledge little information so far is available regarding biosynthesis of silver nanoparticles by the truffles and to search for new antimicrobial alternatives, therefore, the objective of this study was to explore the desert truffle (Tirmania nivea) for its potentiality to biosynthesize silver nanoparticles (AgNPs) and to examine their efficacy against five strains of human pathogenic bacteria namely; Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi and Staphylococcus aureus. Fruiting bodies (Ascocarps) of the truffle T. nivea were collected from the sandy desert of Iraq and brought to the laboratory, washed thoroughly with distilled water and dried at room temperature. Twenty gram of dried fruit bodies of truffle were grounded and dissolved in boiled water and filtered using Whatman filter paper No 1. For synthesis process of silver nanoparticles, 100 mL of truffle extract filtrate was treated with 1 mM of AgNO3 solution and kept for 24 hr at dark condition and synthesis of silver nanoparticles (AgNPs) was checked by visual observation of color changes from pale yellow to dark brown and was further confirmed by UV – Vis spectrum. Fungal filtrate without AgNO3 was maintained as control. The potentiality of silver nanoparticles was examined for their antibacterial efficiency using agar well diffusion method against the selected strains of pathogenic bacteria. Wells (5 mm diam) were made in Muller-Hinton agar (MHA) plates streaked with swabs of each bacterial strain. The wells were loaded with two concentrations (50 and 100 ul) of synthesized silver nanoparticles solutions. Incubated at 37°C for 24 hr and examined for the appearance of inhibition zones around the wells and their diameters were measured. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) assay was carried out using the micro dilution method with serial dilutions (100, 50, 25, 12, 6.5, 3.13, 1.56, 0.78, 0.39, 0.2, 0.1, 0.05, 0.025 μg/L) of the truffle extract filtrate against two strains of bacteria E. coli (ATCC 25922) and S. aureus (NCTC 6571). Disc diffusion method was used to assay the synergistic effect of synthesized AgNPs with commonly used antibiotic Gentamycin. Cytotoxicity of the truffle extract was examined against human blood. Characterization of the biosynthesized silver nanoparticles from truffle extract was carried out by using UV-Vis spectrophotometer analysis, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM). The results showed that the biosynthesized silver nanoparticles exhibited a high growth inhibition activity at 50 ul/ml concentration (12–25.5 mm inhibition zones dim) and at 100 ul/ml (14.5–28 mm inhibition zones diam) against the tested pathogenic bacterial strains. Among the tested bacteria, highest growth inhibition was noticed against P. aeruginosa (25.5 and 228 mm diam) at the two concentrations of AgNPs, respectively. However, a remarkable increase of bacterial growth inhibition zones (23–37 mm diam) was observed for a combination of silver nanoparticles and Gentamycin compared with Gentamycin alone (20–30 mm diam). MIC values were very low (0.312 and 0.0097 ug/ml) against the two tested bacterial strains E. coli and S. aureus, respectively. The truffle extract did not show any toxicity against human blood. UV-Vis spectrophotometer analysis revealed a peak at 420 nm indicating the biosynthesis of silver nanoparticles, FTIR analysis verified the detection of protein capping of biosynthesized AgNPs while SEM images showed that the synthesized silver nanoparticles are dispersed or aggregated and mostly spherical shape and their size ranging between 3–41 nm. It can be concluded that the biosynthesized silver nanoparticles by the desert truffle T. nivea are a promising for future medical and pharmaceutical applications as antibacterial agent and a further investigation to examine their efficacy in vivo is recommended.