Evaluation of anti-fungal activity of Fe2O3 and Fe3O4 nano particles on Aspergillus flavus

ABSTRACT

Nanoparticle is a new innovation of using particles in
a new size which ranged from1- 100 nanometers that equal 1 X10-6
meter. In this study we evaluated the antifungal effect of Fe2O3
and Fe3O4 nanoparticles on Aspergillus flavus (KP137700)
selected for the study isolated from broiler feed was isolated from broiler
feed by Mycology Department of the Animal Health Research Institute, Giza,
Egypt. Fe2O3 and Fe3O4 nanoparticles
synthesized by co-precipitate method. The size of synthesized nanoparticles was
45 nanometer for Fe2O3 and 9 nanometer for Fe3O4.
The antifungal effect Fe2O3 and Fe3O4
nanoparticles against Aspergillus flavus measured by MIC method and evaluated
by scanning electron microscope. The results revealed that Fe2O3
nanoparticles have antifungal effect more than and Fe3O4 nanoparticles.

Keywords: Nanopaticles,
Fe2O3, Fe3O4, Antifungal,
Aspergillus flavus

 

 

 

 

 

1.
INTRODUCTION

Nanotechnology has offered great possibilities in
various fields of science and technology (Adibkia et al., 2007).
Pharmaceutical nanotechnology with numerous advantages has growingly attracted
the attention of many researchers. Nanoscale, Size range from approximately
from 1 nm to 100 nm. This definition is accompanied by two notes: Note 1: Properties
that are not extrapolations from a larger size will typically, but not
exclusively, be exhibited in this size range. For such properties the size
limits are considered approximate. Note 2: The lower limit in this definition
(approximately 1 nm) is introduced to avoid single and small groups of atoms
from being designated as nano-objects or elements of nanostructures, which
might be implied by the absence of a lower limit (ASTM 2006).

Nanoparticles
and nanocomposites prepared chemically by precipitation or in situ formation in
a given matrix through the sol–gel processes (Sanchez et al., 1996).
The conditions determinants of nanoparticle growth are changed in the
dependence on method preparation of nanoparticles. Materials scientists and
engineers have made significant developments in the improvement of methods of
synthesis of nanomaterial solids (Hansany et al., 2012).

Aspergillus
flavus is one of the most important pathogenic fungi that contaminate grains or
feed mixtures and cause serious problems in such as mycotoxicosis due to its
aflatoxins production (Hassan et al., 2014). Development of new
and effective antimicrobial agents seems to be of paramount importance, through
the antimicrobial activity of metals having various properties, potencies and
spectra of activity, has been known and applied for centuries especially in nano-size
(Malarkodi et al., 2014).The aim of this study is the founding of
a new effective antifungal compounds through the nanotechnology against
Aspergillus flavus which is the most dangerous fungi on animal productions.

 

 

 

 

2. MATERIAL AND
METHODS

 

2.1.Synthesis of
nanoparticles

Fe2O3 synthesised by co-precipitation
technique method by Using titra-hydrate ferrous chloride FeCl2.4H2O
?(> 99:9%) dissolved in distilled water. The
ammonium ?hydroxide NH4OH were added
drop-wise to the mixture with stirring under strong ?ultrasonic
agitation was added to the solution to adjust the pH value at 9 or10 till
precipitation occurred. Dark brown precipitate is formed and washed using
distilled water for several times to remove excess ammonia (10 times) the
precipitate was dried at 400°C for 4 hours (Kandpal et al., 2014).
Synthesis of Fe3O4 nanoparticles, Fe3O4
nanoparticles were synthesized by co-precipitation technique method ?of titra-hydrate ferrous chloride FeCl2.4H2O
?(> 99:9%) ,? about 1.9881g,
and hexa-hydrate ferric ?chloride FeCl3 .6H2O
?(> 97:9%) ?, about 5.406gm  (Ozkaya et al., 2009).

 

2.2. Characterization of
synthesized nanoparticles:

X-ray diffraction analyses were carried out to identify the
previously prepared nanoparticles in pure single phase. Also it confirms the
successful formation of these nanoparticles according to (Cullity et al.,
2001)

Infrared Spectra were recorded on a Perkin – Elmer (FT-IR):

FT-IR were carried out to identify the synthesized nanoparticles
and to determine its size according to (Guan et al., 2003).

Evaluation of Fe2O3 and Fe3O4
nanoparticles effect on the growth of A. flavus.

 

2.3.
Selection of test pathogen:

Pathogenic Aspergillus flavus (KP137700) selected for
the study isolated from broiler feed was obtained from the Mycology Department
of the Animal Health Research Institute, Giza, Egypt. Preparation of dilutions
of synthesized compounds 10 mg of the each nanoparticle (Fe2O3
and Fe3O4) were weighed accurately and dissolved in 10 ml
dimethyl sulfoxide giving a solution of 1mg/ml concentration. 1 ml of the above
solution was again diluted to 10 ml with dimethyl sulfoxide giving a solution
of 100 µg/ml concentration. Determination of minimum inhibitory concentration
and minimum fungicidal concentration Minimum inhibitory concentration (MIC) was
determined for Fe2O3 and Fe3O4
nanoparticles showing antifungal activity against test pathogen by serial
dilution method. Broth microdilution method was followed for determination of
MIC values. Sterilized loop wire was used to transfer A. flavus  to sabouraud dextrose agar and incubated at
25 °C for 5 days. From the A. flavus strain, small portion was transferred to
3ml of sabouraud dextrose broth media separately and incubated at 25°C for 24
hrs.0.1 ml of the above five medias were transferred to five different
stoppered conical flasks containing 0.9% NaCl solution.1ml of media was taken
in a test tube, to which, 1ml of test solution (100 µg/ml) was added.
Thereafter, 0.1ml of the microbial strain (A. flavus) prepared in 0.9% NaCl was
added to the test tube containing media and test solution. Serial dilution were
done five times giving concentrations of 50, 25, 12.5, 6.25, 3.75, 1.5 µg/ml.
The test tube were stoppered with cotton and incubated at 25°C for one week. The
MIC values were taken as the lowest concentration of the particles in the test
tube that showed no turbidity after incubation. 
The turbidity of the contents in the test tube was interpreted as
visible growth of microorganisms.

 

2.4.
Minimum fungicidal concentration

The minimum fungicidal concentration (MFC) was
determined by sub culturing 50µl from each test tube showing no apparent
growth. Least concentration of test substance showing no visible growth on sub
culturing was taken as MFC.

 

 

 

2.5.
Scanning electron microscope examination of A. flavus after the exposure to
Fe2O3 and Fe3O4:

In conical flasks one liter containing 200 ml of yeast
extract broth (YEB) than the flasks were autoclaved for 15 minutes at 121°C
cooled at room temperature. The Fe2O3 and Fe3O4 nanoparticles were taken
separately at different concentrations of 1.5, 3, 4.5 and 6 mg/100 ml, each
concentration dissolved in sterilized distilled water using an ultrasound bath.
 Preparation of spore suspensions of A.
flavus was standardized to 106 spores/ml then adding one millimeter of spore
suspension of A. flavus and different concentrations of Fe2O3 and Fe3O4 nanoparticles
were add to Yeast extract broth (YEB). All the inoculated flasks were incubated
at 25°C for 21 days. After the end of incubation period, the content of each
flask was filtrated. The treated fungi mycelia sections were collected, fixed
with formaldehyde, washed with
phosphate buffer solution and dehydrated with alcohol solution (30, 60, 80, 90
and 100%, maintaining the mycelia at 100%) and then submitted to critical point
drying according to (Al-othman et al., 2014).
Aspergillus flavus became ready for scanning electron microscopy (SEM) using JEOL
(JSM-
6380 LA) instrument.

 

3. RESULTS

Phase identification and structural analysis were
carried out by XRD and FTIR spectra at room temperature for Fe2O3
and Fe3O4 nanoparticles showed that the investigated
sample crystallized in a single phase 45nm size of Fe2O3
nanoparticles and 9nm size of Fe3O4 nanoparticles as
shown in Fig 1, 2, 3 and 4.

Minimum inhibitory concentration (MIC) of Fe2O3,
and Fe3O4 nanoparticles was 25mg and 50mg respectively
against Aspergillus flavus.

SEM examination confirmed the anti-fungal effect of Fe2O3
and Fe3O4 nanoparticles through different degree of fungi
cell membrane rupture fungal spores were observed damage such as reduce in
spores number, malformations and hypertrophy these effects lead to destroyed
and damaged of spores resulting in possible reduction of multiplication and the
enzymatic activity of the micro-organism. This
aspect is great important because Aspergillus reproduction involves mainly
formation of spores. The severity of damage and malformation of Aspergillus flavus
is high with Fe2O3 nanoparticles and low with Fe3O4
nanoparticles as in Fig 5 and 6.  

 

4. DISCUSSION

(Gehan et al., 2014) supported results of this study through evaluation of the
antifungal potential Fe2O3 nanoparticles in inhibiting
the growth of Aspergillus flavus.  There
was antifungal potential of prepared Fe2O3 nanoparticles
in disc diffusion test. When the treated fungi with Fe2O3
nanoparticles were subjected to SEM, the damage and rupture of their cell wall
were detected in the area surrounding growth. The normal conidial cell of
Aspergillus flavus has a spherical shape and smooth cell wall and intact cell
membrane. The effect of high concentration of Fe2O3
nanoparticles on the treated fungi was observed as membrane damage of cells and
some pits that have been caused in intercellular components, leading to leakage
and finally cell death.

The antifungal effect of Fe3O4 nanoparticles
that evaluated in our study is agreed with (Bhupendra et al., 2013)
who studied the minimum inhibitory concentration (MIC) values of Fe3O4–Ag
nanoparticles is less than Ag nanoparticles against Aspergillus glaucus. The
fungal growth declines as the concentration of Fe3O4–Ag
is raised and when the latter reaches MIC, no growth is observed. Presence of
Fe3O4 in composite with Ag gives the Ag more antifungal
effect. 

 

5.Conclusion

In conclusion, the current study shows that Fe2O3 nanoparticles have a
strong effect on Aspergillus flavus as antifungal if compared with Fe3O4
nanoparticles which
have a little antifungal effect on Aspergillus
flavus