Gamma radiation mediated green synthesis of gold nanoparticles using fermented soybean-garlic aqueous extract and their antimicrobial activity
© El-Batal et al.; licensee Springer. 2013
Received: 14 October 2012
Accepted: 15 March 2013
Published: 23 March 2013
Aspergillus oryzae was used to enhance the mobilization of antioxidants of soybean matrix along with garlic as a co-substrate by modulating polyphenolic substances during solid-state fermentation. Mobilized polyphenols were used as a green tool for synthesis and stabilization of gold nanoparticles (AuNPs). The radiation-induced AuNPs synthesis is a simple, clean and inexpensive process which involves radiolysis of aqueous solution that provides an efficient method to reduce metal ions. Gamma irradiated aqueous extract of fermented soybean and garlic was used for rapid preparation of AuNPs combining both effects of radiolytic reactions by radiation and stabilization by bioactive components of fermented extract. The synthesized AuNPs were confirmed by UV-Visible spectrophotometry, dynamic light scattering (DLS), Fourier Transform infra red (FT-IR) spectrophotometry, and transmission electron microscope (TEM) analysis which revealed morphology of spherical AuNPs with size ranging from 7–12 nm. The synthesized AuNPs exhibited antimicrobial activity against both Gram positive and Gram negative bacteria, as measured by well diffusion assay.
KeywordsSolid state fermentation Soybean Garlic Gamma radiation Gold nanoparticles Antimicrobial activity
Gold is a well known biocompatible metal and colloidal gold was used as a drinkable solution that exerted curative properties for several diseases in ancient times (Daniel and Astruc 2004) and now, because of its low cytotoxicity (.,Shukla et al 2005), AuNPs have been widely used as the platform material in the field of biodiagnostics (Nam and Thaxton 2003), drug/DNA delivery (.,Paciotti et al 2004), (.,Prow et al 2006) cell imaging (.,Bielinska et al 2002), immunostaining (Roth 2003), biosensing (,Penn et al. 2003) and electron microscopy markers (Baschong and Stierhof 1998).
Solid state fermentation (SSF) of an edible plant matrix by filamentous fungi is a biotechnological strategy that may induce health beneficial naturally occurring antioxidant components including polyphenols during microbial fermentation (McCue and Shetty, 2005, Lee et al., 2008). As soybean contains phenolic and isoflavonoid compounds concentrated mainly in seed matrix, it is possible that the fungi can play role in mobilization of polyphenolic compounds during SSF period (.,McCue et al 2003;McCue and Shetty, 2005.,, Huang et al 2008.,, Bhanja et al 2009). Isoflavonoids are polyphenolic compounds acting as reducing agents (free radical terminators), metal chelators and singlet oxygen quenchers (Mathew & Abraham 2006), which suggests the reduction and stabilization of AuNPs. Garlic also is a rich source of the proteins, free amine groups and cysteine residues. These compounds contains functional groups that play a role in reducing and hence synthesis as well as stabilization of nanoparticles (Gole et al. 2001, Tortora et al. 2004) or via electrostatic attraction of negatively centerged carboxylic groups (Rastogi and Arunachalam 2011) and therefore, stabilization of these nanoparticles by "capping".
The radiation-induced synthesis is one of the most promising strategies (Mostafavi et al. 1993 ). The process is simple, clean and has harmless feature (.Li et al 2007). The formation of AuNPs can be attributed to the radiolytic reduction which generally involves radiolysis of aqueous solutions that provides an efficient method to reduce metal ions. In the radiolytic method, when aqueous solutions are exposed to gamma rays, they create solvated electrons, which reduce the metal ions and the metal atoms eventually coalesce to form aggregates (Marignier et al. 1985). The combined effect of both radiolytic reduction and presence of soybean flavonoids, and sulfur containing compounds and proteins in garlic resulted in formation of AuNPs by radiolytic reactions and stabilization by prevention of aggregates formation by "capping".
The antimicrobial activity of the synthesized AuNPs was assessed using agar well diffusion method against both Gram negative and positive bacteria and showed a good antimicrobial potential.
Materials and methods
All chemicals were purchased from Sigma-Aldrich.
Aspergillus oryzae was isolated and maintained on potato dextrose agar (PDA) plates at 4°C. Cultures were reactivated by transferring onto fresh PDA slants and cultured at 20°C-22°C for 7–10 days.
Commercial soybean seeds were obtained from the local market. The soybean seeds were ground to 30-mesh powders screen using a grinder and used along with garlic powder obtained from local market.
Media and cultivation
In an Erlenmeyer flask (250 ml), 5 gm of crushed soybean seeds and 5 gm of garlic powder were added along with 10 ml of distilled water (pH 6.5). The material was autoclaved at 121°C for 20 minutes. The spores were then harvested and suspended in 0.85% saline containing 0.1% Tween-80. The substrate was inoculated using 1 ml of spore suspension (9x106 spores/ml) for 6 days with moisture content 60% at 35°C.
Preparation of fermented extracts
The fermented product was extracted with ethanolic solution 95% (1:10, w/v) with gentle shaking 100 rpm, at room temperature for 2 hours using (LAB-Line® Orbit Environ) shaker. The filtrate was then decanted and centrifuged using (Hettich Universal 16R cooling centrifuge) at 6,000 rpm for 10 minutes at 6°C. The resulted ethanolic extract is used for further analysis. For preparation of aqueous extract, the ethanolic extracts were vacuum concentrated and dried using freeze-dryer (LyoTrap USA). The resulted powder was redispersed in equal amount of water and used for further analysis.
Preparation and centeracterization of AuNPs
AuNPs were prepared according to the method described by (Song and Kim 2009,, Noruzi et al. 2011). Briefly, to different volumes (5, 7.5, 10 and 15 ml) of fermented extract, containing total phenols 0.272 mg/ml expressed as gallic acid equivalent, different concentrations of tetrachloroauric acid (2.5, 5, 7.5 and 10 mM) were added, (purity of 49% gold metal). The reaction mixture is stirred properly using magnetic stirrer with heating at 75°C, within 1 minute the yellow colored solution started changing to pink then violet detected visually and by UV-Visible spectrophotometer indicating the formation of AuNPs.
UV/Vis spectra of AuNPs were recorded as a function of wavelength using JASCO V-560 UV/Vis spectrophotometer from 200–700 nm operated at a resolution of 1 nm.
Average particle size and size distribution were determined by PSS-NICOMP 380-ZLS particle sizing system St. Barbara, California, USA.
FT-IR measurements were carried out in order to obtain information about chemical groups present around AuNPs for their stabilization and understand the transformation of functional groups due to reduction process. The measurements were carried out using JASCO FT/IR-6300 infra-red spectrometer by employing KBr pellet technique.
The size and morphology of the synthesized nanoparticles were recorded by using TEM model JEOL electron microscope JEM-100 CX. TEM studies were prepared by drop coating Au nanoparticles onto carbon-coated TEM grids. The film on the TEM grids were allowed to dry, the extra solution was removed using a blotting paper.
X-Ray Diffraction patterns were obtained with The XRD-6000 series, including stress analysis, residual austenite quantitation, crystallite size/lattice strain, crystallinity calculation, materials analysis via overlaid X-ray diffraction patterns Shimadzu apparatus using nickel-filter and Cu-Ka target, Shimadzu Scientific Instruments (SSI) ,Kyoto, Japan.
Gamma irradiation source
The process of irradiation was carried out at the National Center for Radiation Research and Technology (NCRRT), Egypt. The facility used was Co-60 Gamma chamber 4000-A-India. Irradiation was performed using Co-60 Gamma rays at a dose rate of 10.28 kGy/hr at the time of the experiment.
Determination of total flavonoids
Total flavonoids were estimated using the method of (Ordonez et al. 2006). To 0.5 ml of sample, 0.5 ml of 2% AlCl3 ethanolic solution was added. After one hour, at room temperature, the absorbance was measured at λ 420 nm using (JASCO V-560 UV-visible spectrophotometer). 2A yellow color indicates the presence of flavonoids. Total flavonoid contents were calculated as rutin mg equivalent per gm fermented product.
Determination of total phenol
Total phenolic content was determined by the Folin Ciocalteau colorimetric method of assay by (.Singleton, et al 1999). Briefly, 50 μl of sample was mixed with 3 ml of distilled water and 250 μl of Folin reagent was added and immediately vortexed. Then, 750 μl of saturated Na2CO3 solution was added. Then, adjust the final volume to 5 ml using distilled water. Incubation for 2 hours at room temperature and then measure the absorbance at 765 nm against distilled water as blank. Total polyphenolic content is expressed as gallic acid mg equivalent.
Antimicrobial sensitivity test
The AuNPs synthesized was tested for antimicrobial activity by agar well diffusion method (,Bauer et al. 1966) against different kinds of pathogenic bacteria and yeast isolated from clinical samples; Staphylococcus aureus MRSA (Gram positive bacteria), Pseudomonas aeruginosa and Acinetobacter baumaninii/ heamolyticus (Gram negative bacteria). Standardized suspension of each tested strain 108 CFU/ml for bacteria was swabbed uniformly onto sterile Muller-Hinton Agar (MHA) (Oxoid) plates using sterile cotton swab wells of 10 mm diameter were bored into the agar medium using gel puncture. Using a micropipette, 100 μl of the AuNPs solution (7.5 mM HAuCl4 in 5 ml extract) was added into each well. After incubation at 37°C for 24 hrs, the different levels of zone of inhibition were measured and interpreted using the CLSI zone diameter interpretive standards (CLSI, 2008). Tetracycline (antibacterial agent) served as positive control for antimicrobial activity, while the filtrate alone (without AuNPs) was used a negative control. The determinations were done in triplicates and the mean values ± SD (standard deviation) were presented.
Results and discussion
Preparation and centeracterization of AuNPs
Effect of temperature on time taken for color conversion i.e. formation of AuNPs
The effect of radiation on total flavonoids
Total Flavonoids mg rutin/gm fermented product
Control (not radiated)
Peaks appeared in FT-IR spectra for fermented extract with and without tetrachloroauric acid solution
Extract + AuNPs λ (cm-1)
The broad peaks are centeracteristic to the presence of –NH2amine group and –OH stretching groups in alcoholic and phenolic compounds. Noruzi et al. 2011increased intensity which may be due to binding of gold ions to OH group. Rastogi and Arunachalam 2011
Corresponds to aliphatic C-H stretching. Noruzi et al. 2011
centeracteristic to the carbonyl group. This red shift may indicate oxidation of carbonyl group during the reaction and hence reducing gold to AuNPs. Rastogi and Arunachalam2011
May be ascribed for the presence of primary amine group C-N stretching. Kumar et al. 2012
May be attributed to SO2 absorption of sulfones present in garlic. Rastogi and Arunachalam2011
Signifies the presence of R-CH group Noruzi et al. 2011
Mean size =50 nm
Antimicrobial sensitivity test for AuNPs
Tetracycline (standard antibacterial agent)
*Diameter of inhibition zone (mm) produced by AuNPs
Staphylococcus aureus MRSA (Gm +ve)
14.67 ± 0.58
Pseudomonas aeruginosa (Gm - ve)
16.67 ± 1.15
Acinetobacter baumaninii/haemolyticus (Gm - ve)
17.33 ± 1.52
The fermented soybean-garlic aqueous extract enriched with mobilized polyphenols and proteins can be used for efficient green synthesis of stabilized AuNPs, due to the presence of functional groups, such as; carbonyl group, hydroxyl group, sulfones and amines by acting as reducing agents as well as "capping" agents for stabilization of the AuNPs.
The combined effect of both γ-radiation and mobilized polyphenolic compounds in synthesis and stabilization of AuNPs offers a highly efficient and inexpensive method which can be used in large scale production of AuNPs.
Moreover, synthesized AuNPs showed good antimicrobial activity leading to high potential uses in biological applications.
The authors would like to thank the Nanotechnology Research Unit (P.I. Prof.Dr. Ahmed El-Batal), Pharmaceutical Microbiology Lab, Drug Radiation Research Department, National Center for Research and Radiation Technology (NCRRT), Egypt, for financing and supporting this study under the project “Nutraceuticals and Functional Foods Production by using Nano/ Biotechnological and Irradiation Processes”.
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