Chemosphere 218 (2019) 477e486

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Antifungal and anti-mycotoxin efficacy of biogenic silver nanoparticles

produced by Fusarium chlamydosporum and Penicillium chrysogenum
at non-cytotoxic doses
Neveen M. Khalil a, Mohamed N. Abd El-Ghany a, Susana Rodríguez-Couto b, c, d, *
a
Botany and Microbiology Department, Faculty of Science, Cairo University, Giza, 12613, Egypt
b bal 15, 20018, San Sebastian, Spain
Ceit-IK4, Paseo Manuel de Lardiza
c
Universidad de Navarra, Tecnun, Paseo Manuel de Lardizabal 13, 20018, San Sebastian, Spain
d
IKERBASQUE, Basque Foundation for Science, Maria Diaz de Haro 3, 48013, Bilbao, Spain

h i g h l i g h t s

Silver nanoparticles were synthesized by cell-free cultures of fungi.

The synthesized nanoparticles inhibited aflatoxin production by A. flavus.

The synthesized nanoparticles inhibited ochratoxin A production by A. ochraceous.

The synthesized nanoparticles did not present cytotoxic against human melanocytes.

a r t i c l e i n f o a b s t r a c t

Article history: The cell-free culture filtrate (CFF) of the fungi Fusarium chlamydosporum NG30 and Penicillium chrys-
Received 23 July 2018 ogenum NG85 was tested to synthesize silver nanoparticles (AgNPs). The synthesized AgNPs were further
Received in revised form characterized by means of transmission electron microscopy (TEM), dynamic light scattering (DLS) and
16 November 2018
Fourier transform infra-red (FTIR) spectroscopy. TEM revealed their spherical shape, homogeneity and a
Accepted 19 November 2018
Available online 21 November 2018
size range between 6 and 26 nm for F. chlamydosporum AgNPs (FAgNPs) and from 9 to 17.5 nm for
P. chrysogenum AgNPs (PAgNPs). DLS showed that the diameter of FAgNPs was narrower than that of
Handling Editor: Tamara S. Galloway PAgNPs. FTIR spectroscopy indicated that the functional groups present in the CFF might be responsible
for the reduction of silver ions to form stabilized protein-capped AgNPs. In addition, the AgNPs showed
Keywords: notable antifungal activity and potency in thwarting mycotoxin production. Thus, using Aspergillus flavus
Fungi as a test microorganism the minimum inhibitory concentration (MIC) was 48, 45 and 50 mg/mL for
Biogenic silver nanoparticles FAgNPs, PAgNPs and the antifungal compound itraconazole, respectively. Also, when testing Aspergillus
Aflatoxins ochraceus FAgNPs, PAgNPs and itraconazole led to MIC values of 51, 47 and 49 mg/mL, respectively. The
Ochratoxin A
statistical MIC values to inhibit completely the total aflatoxin production by A. flavus were 5.9 and 5.6 mg/
Cytotoxicity
mL for FAgNPs and PAgNPs, respectively, and to inhibit the ochratoxin A production by A. ochraceus 6.3
and 6.1 mg/mL for FAgNPs and PAgNPs, respectively. The cytotoxicity assay of the AgNPs on human
normal melanocytes (HFB 4) revealed a cell survival of 80% and 75% at a concentration of 6 mg/mL for
FAgNPs and PAgNPs, respectively.
© 2018 Elsevier Ltd. All rights reserved.

1. Introduction

Nanotechnology allows the modification and development of

the main characteristics of a metal (Marcato and Duran, 2008). The
nanosize of a material is responsible for particular physicochemical
bal 15, 20018, San
* Corresponding author. Ceit-IK4, Paseo Manuel de Lardiza properties which are different from its larger bulk counterpart. This
Sebastian, Spain
E-mail addresses: srodriguez@ceit.es, susanarodriguezcouto@gmail.com
effect is chiefly due to a high surface-area-to-volume ratio which
(S. Rodríguez-Couto). results in increased reactivity. Hence, materials with nanoscale size

https://doi.org/10.1016/j.chemosphere.2018.11.129
0045-6535/© 2018 Elsevier Ltd. All rights reserved.
478 N.M. Khalil et al. / Chemosphere 218 (2019) 477e486
could be used to refine drug production, tailoring drugs at a mo-
lecular level to make them more effective and reduce side effects
than their bulk counterparts (Gu et al., 2003; Borase et al., 2015).
Due to their antimicrobial properties, nanoparticles have immense
applications in agriculture, nutrition, medicine, health and some
other sciences (Borase et al., 2015; Kathiravan et al., 2015). The
medical characteristics of silver have been known for over 2000
years. Since the nineteenth century, silver-based compounds have
been used in many antimicrobial applications. Among all the noble
metallic nanoparticles, silver nanoparticles (AgNPs) are important
candidates of choice to solve various medical problems due to their
chemical biocompatibility, inertness, oxidation resistance and wide
spectrum of antimicrobial activity against a diverse range of bac-
teria and fungi (Prabhu and Poulose, 2012).
The use of environmentally innocuous materials like plant leaf
extracts, bacteria and fungi for the synthesis of AgNPs offers
numerous benefits of eco-friendliness and compatibility for phar-
maceutical and biomedical applications as they do not use toxic
chemicals in the synthesis protocols. Frequently, chemical synthe-
sis methods cause some chemically toxic compounds which, when
adsorbed on the nano-structured surface, can obstruct their utili-
zation in medical applications (Parashar et al., 2009). Biomimetic
technologies are environmentally friendly and economically
feasible for material synthesis (Kalishwaralal et al., 2008). Bacteria,
fungi and plant extracts are the three main sources used for the
biological synthesis of AgNPs. They produce microbial enzymes or
phytochemicals with antioxidant or reducing characteristics acting
on metal ions to produce the desired nanoparticles. Three major
constituents are involved in the preparation of nanoparticles using
biological methods: the solvent medium for synthesis, the reducing
agent, which is environmentally friendly, and a stabilizing agent
that is nontoxic (Prabhu and Poulose, 2012).
Researchers are involved in the control of contamination caused
by fungi to diverse crops since the nineteenth century (Backhaus,
2009). Most of the current research is related to the control of
mycotoxins produced by Aspergilli fungi (Pitt and Hocking, 2006;
Reddy et al., 2008). These mycotoxins are toxic at low concentra-
tions to higher vertebrates and other animals (Bennett, 1987). The
FDA limits aflatoxin (AF) in corn grain according to its intended use:
no more than 200 ppb for breeding cattle, 300 ppb for finishing
beef cattle and 20 ppb for lactating dairy cattle (Van Egmond, 1989).
The most important mycotoxins are aflatoxins (AFs), ochratoxin A
(OTA), fumonisins, trichothecenes and zearalenones. The widely
distributed mycotoxigenic fungi, which cause risk on human
health, are Aspergillus ochraceus, Aspergillus niger, Aspergillus flavus,
Aspergillus carbonarius and Aspergillus parasiticus (Gonzalez et al.,
2005).
Fungal metabolism results in the production of AFs as well as
several other mycotoxins. Aflatoxin B1 (AFB1) is produced by
several Aspergilli and is considered one of the most potent hep-
atocarcinogenic agents in humans and animals. Thus, the presence
of Aspergilli in food raises high concern and, hence, it is monitored
by the health authorities in many countries (Van Egmond and
Jonker, 2004).
OTA is a mycotoxin produced by some fungal species belonging
to the Aspergillus and Penicillium genera (Milicevic et al., 2010). OTA
was firstly extracted and identified in South Africa from a culture of
A. ochraceus (Van der Merwe et al., 1965). OTA is considered
carcinogenic, nephrotoxic, immunotoxic, teratogenic and hepato-
toxic for domestic and laboratory animals. It was also described as a
probable agent causing development of urothelial tumors and ne-
phropathies in humans (O'Brien and Dietrich, 2005).
In this study AgNPs were biologically produced using Fusarium
chlamydosporum NG30 and Penicillium chrysogenum NG85. The
efficacy of the produced AgNPs to control the mycotoxigenic
species A. flavus and A. ochraceous was evaluated. In addition, the
potency of AgNPs to diminish the production of mycotoxins by
A. flavus and A. ochraceous was investigated. Finally, the safety of
the prepared AgNPs was assessed against human normal melano-
cytes (HFB 4).
2. Materials and methods
2.1. Fungal strains, media and culture conditions
In the present study, fungal species were isolated from an
agricultural soil in Giza, Egypt, using the soil dilution plate method
(Johnson et al., 1960). Soil samples were used as inoculum on
Czapek-Dox agar (CZA) medium which contained (g/L): sucrose 20;
NaNO3 2; K2HPO4 1; KCl 0.5; MgSO4 7H2O 0.5; FeSO4 7H2O 0.01 and
agar 15. Streptomycin (30 mg/mL), previously sterilized by filtration
(0.2 mm), was added to the above medium after sterilization and
cooling. The obtained fungal colonies were purified by the streak
plate method. Then, they were identified based on their morpho-
logical and microscopic characteristics including color, mycelia
texture, spore formation pattern, etc. according to Gilman (1957),
Raper and Fennel (1965), Moubasher (1993) and Watanabe (2002).
The fungal species were grown on slants containing CZA at 28  C for
4 days. Spores of each strain were harvested and stored at 4  C in a
sterilized spore suspension buffer containing 0.9% (w/v) NaCl and
1% (v/v) Tween 80.
2.2. Molecular identification of fungal isolates
The identification of Fusarium chlamydosporum and Penicillium
chrysogenum was further confirmed using nuclear ribosomal DNA
internal transcribed spacer (ITS) sequencing. The genomic DNA was
obtained using the protocol of GeneJet Plant genomic DNA purifi-
cation Kit (Thermo) #K0791 (http://www.thermoscientificbio.com/
). Internal transcribed spacer (ITS) region of 5.8S rRNA was ampli-
fied using the primers ITS1 (50 TCC GTA GGT GAA CCT TGC GG 30 )
and ITS4 (50 TCC TCC GCT TAT TGA TAT GC 30 ). PCR sequencing of the
amplified product was carried out at the GATC Company (Ger-
many). The resulting sequence was entered into the BLAST algo-
rithm of National Center of Biological Information (NCBI) database
to obtain the closely related phylogenetic sequences. A phyloge-
netic tree was constructed using the MEGA 6 software. The ob-
tained sequence was then submitted to the GenBank of the
National Center for Biotechnological Information (NCBI) database.
A strain identifier and an accession number were obtained for each
isolate.
2.3. Synthesis and detection of the AgNPs
The stock spore suspensions of the fungal species
F. chlamydosporum NG30 and P. chrysogenum NG85 were used to
inoculate 100 mL of malt extract glucose yeast extract peptone
(MGYP) medium (0.3% malt extract, 1.0% glucose, 0.3% yeast extract,
0.5% peptone; pH 6.8) in 250-mL cotton-plugged Erlenmeyer flasks.
The flasks were incubated for 24 h at 28  C inside a shaking incu-
bator (150 rpm). This starter culture was used as an inoculum (10%
v/v) for the same medium and the same culture conditions except
the incubation period was three days. The fungal mycelium was
removed from the culture medium by centrifugation (5000 rpm,
15 min and 4  C) and washed thrice with sterilized deionized water.
The biomass was then autolyzed to release the intracellular en-
zymes and other constituents into an aqueous solution. This was
performed by suspending 10 g of fresh biomass in 100 mL of ster-
ilized deionized water and incubated for 72 h at 28  C under
 N.M. Khalil et al. / Chemosphere 218 (2019) 477e486 479

shaking (150 rpm). The flask contents were filtered through
Whatman filter paper no. 1 to obtain the cell-free filtrate (CFF). For
the biosynthesis of the AgNPs, 50 mL of CFF were mixed with 10 mL
of 10 mM AgNO3 and incubated at 28  C under shaking at 150 rpm
in the dark.
2.4. Characterization of the AgNPs
The reduction of silver ions to AgNPs by F. chlamydosporum
NG30 and P. chrysogenum NG85 was determined at 420 nm by using
a UVevisible spectrophotometer (model Perkin-Elmer Hitachi
2000). The particle size of the prepared samples was measured by
the dynamic light scattering (DLS) technique, using a PSSNICOMP
Zeta Potential/Particle Sizer 380ZLS (PSS-NICOMP, Santa Barbara,
CA, USA) at room temperature. Prior to the measurement, the
samples were diluted in freshly prepared deionized water. The
morphology of the synthesized AgNPs was detected by a Jeol JEM-
1400 transmission electron microscope (TEM).
2.5. Fourier-transform infrared spectroscopy
Fourier-transform infrared (FTIR) spectra of freeze-dried AgNPs
powder were obtained by a Fourier Transform Infrared spectrom-
eter (FTIR 6100).
2.6. Antifungal activity
The spore germination inhibitory effect of AgNPs was tested
against the mycotoxigenic fungi A. flavus NRRL 3145 and A. ochra-
ceus ATCC 22947, which were kindly obtained from the National
Research Center (NRC), Dokki, Giza (Egypt). The minimum inhibi-
tory concentration (MIC) test was performed. Spore suspension

Fig. 1. Phylogenetic tree of (a) Fusarium chlamydosporum NG30 and (b) Penicillium chrysogenum NG85.
480 N.M. Khalil et al. / Chemosphere 218 (2019) 477e486
concentrations were determined using a hemocytometer and
adjusted to 2  106 spores/mL. Fifty mL of spore suspension were
transferred to each well of a microtiter plate containing 100 mL of
Czapek-Dox medium with different concentrations of AgNPs. Ex-
periments containing double distilled water and itraconazole
instead of AgNPs were considered as negative and positive controls,
respectively. The plate was incubated for 16 h at 30  C until spore
germination. Spores were regarded as germinated when the length
of the germ tube was at least twice the length of the spore itself
(Griffin, 1994). A hemocytometer was used to count the germinated
spores. About 100 spores per replicate were observed to detect
spore germination. MIC was determined as the lowest AgNPs
concentration that completely inhibited spore germination.
2.7. Leakage of proteins and DNA
To study the probable mechanism of AgNPs affecting cells, the
leakage of proteins and DNA from A. flavus and A. ochraceous cells
was studied. Spore suspension solutions of A. flavus and
A. ochraceous (106 spore cells/mL) were prepared in saline solution
(NaCl, 0.9% w/v). The fungal spore suspensions were incubated for
24 h at 30  C with FAgNPs and PAgNPs at their respective MIC
values for spore germination inhibition. Control spore suspension
sets (without AgNPs) were used as blanks. Spore suspension in each
case was centrifuged for 15 min at 4000 rpm at 4  C. The optical
density (OD) of the supernatant was read at 280 and 260 nm to
estimate the proteins and DNA leaked from the cells.
2.8. Effect of AgNPs on fungal production of mycotoxins
Total aflatoxins (B1, B2, G1, G2) and ochratoxin A (OTA) pro-
duced by the mycotoxigenic fungi A. flavus NRRL 3145 and A.
ochraceus ATCC 22947, respectively, were monitored. Different
concentrations of AgNPs synthesized by F. chlamydosporum NG30
and P. chrysogenum NG85 were added into 250-mL cotton-plugged
Erlenmeyer flasks containing 100 mL of YES liquid medium. The
medium contained 2% (w/v) yeast extract and 15% (w/v) sucrose
(Davis et al., 1966) and was autoclaved at 121  C for 20 min. Spores
of A. flavus NRRL 3145 and A. ochraceus ATCC 22947 were collected
from 10-day old PDA slants and used to inoculate the respective
flasks. They were incubated for 21 days at 30  C in an incubator.
Fig. 2. Mechanism of the synthesis of silver nanoparticles (AgNPs) by the fungal Cell Free Filtrate (CFF).
2.9. Extraction and measurement of mycotoxins
The following procedures were performed at the Regional
Center for Food and Feed (RCFF), Giza (Egypt). The cultures ob-
tained after incubation were filtered through Whatman number 1
filter paper. The culture filtrates were extracted in the presence of
chloroform (1:2 v/v). The chloroform extract was evaporated until
obtaining a dry film in a rotary evaporator. The dry films containing
total AFs and OTA were reconstituted with DMSO (dimethyl sulf-
oxide). The concentrations of total AFs and OTA were quantitatively
measured using an HPLC (Agilent 1200 series USA with a C18 col-
umn, Lichrospher 100 RP-18.5 mm  25 cm). The isocratic mobile
phase was composed of water-methanol-acetonitrile (54:29:17, v/
v/v) for AFs and water-acetonitrile-acetic acid (121:90:1, v/v/v) for
OTA at a flow rate of 1 mL/min. The wavelengths of excitation and
emission were 362 and 460 nm for AFs and 333 and 465 nm for
OTA.
2.10. Cytotoxicity assay
The cytotoxicity of the prepared FAgNPs and PAgNPs was tested
against the normal human cell line: human normal melanocytes
(HFB 4) according to the Skehan et al. (1990) method. Cells were
plated in a 6-multiwell plate (104 cells/well) for 24 h before the
treatment with AgNPs to allow the attachment of the cells to the
well of the plate. Different concentrations (0, 1, 2, 3, 4, 5 and 6 mg/
mL) were added to the cell monolayer, triplicate wells composed for
each dose. Monolayer cells were incubated with FAgNPs and
PAgNPs at 37  C for 48 h in an atmosphere of 5% CO2. Cells were
fixed, washed and stained with Sulfo-Rhodamine-B after incuba-
tion. Acetic acid was used to wash the excess of stain. The attached
stain was recovered using Tris-EDTA buffer. ELISA reader (Meter
tech. S 960, USA) was used to measure the color intensity. The
relation between cell survival (as a percentage of the control) and
AgNPs concentration was plotted to get the survival curve of the
normal human melanocytes (HFB 4 cell line) after subjecting to
AgNPs. Cell survival (%) was calculated as follows:
Survivalð%Þ ¼ ðIt=IcÞx100
where It is the color intensity of the treated cells and Ic is the color
 N.M. Khalil et al. / Chemosphere 218 (2019) 477e486 481

Table 1 3. Results and discussion

FTIR spectra characteristics and functional groups.

Assignment Peak no., wavelength (cm1) 3.1. Isolation of fungal strains

P. chrysogenum NG85 F. oxysporum NG30
In this study, the two fungal species Fusarium chlamydosporum
-OH 3, 3431.71 2, 3426.89
NG30 and Penicillium chrysogenum NG85 were isolated and puri-
C-H stretching 4, 2925.48 3, 2975.62
C-O- stretching 6, 1636.3 7, 1625.7 fied. The identification of the isolates was further confirmed using
COO- symmetrical stretching 8, 1383.68 9, 1382.71 nuclear ribosomal DNA internal transcribed spacer (ITS)
-N-H stretching 10, 1049.09 10, 1033.66 sequencing. The traditional Sanger technology and the new 454
technology were combined for sequencing the PCR products. The
obtained nucleotide sequence was deposited at the NCBI GenBank
intensity of the control cells. and a strain identifier was given to each isolate. Thus, they were
identified as Fusarium chlamydosporum NG30 and Penicillium
chrysogenum NG85 with accession numbers MH141316 and
2.11. Statistical analysis MH119131, respectively. The dendrogram was established (Fig, 1) to
show sequence alignments with available sequences from the NCBI
The data presented in each experiment are the mean of tripli- data bank. F. chlamydosporum NG30 and P. chrysogenum NG85 were
cate assays. The SPSS 20.0 software was used for the determination utilized as cell factories for the AgNPs production.
of the standard error (SE) and for the regression analysis.

Fig. 3. Left: Transmission electron microscopy (TEM) photographs of silver nanoparticles (AgNPs) synthesized by (a) Fusarium chlamydosporum NG30 and (b) Penicillium chrys-
ogenum NG85. Scale bar: 100 nm. Right: Measurements of Dynamic light-scattering (DLS) for particle size distribution analysis of AgNPs.
482 N.M. Khalil et al. / Chemosphere 218 (2019) 477e486

3.2. Biosynthesis and characterization of the AgNPs
AgNPs were formed without any mycelial materials using the
Cell Free Filtrate (CFF) obtained from the autolyzed biomass.
Consequently, a clear solution was obtained and the complications
due to nanoparticle/cell interactions were avoided. The CFF con-
tained reducing enzymes as well as proteins. The reducing enzymes
were responsible for reducing the silver ions (Agþ) to silver atoms
(Ag), while the proteins acted as capping materials for the silver
atoms which stabilized them. The mechanism by which AgNPs
were synthesized by the fungal CFF is shown in Fig. 2.
For each 50 mL of CFF, a 10-mL solution of 1.699 mg/mL AgNO3
was added to be transformed to AgNPs.
The extracellular synthesis using CFF is, thus, more advanta-
geous for the isolation of AgNPs from the solution and a less
number of steps is involved in both synthesizing and purifying
AgNPs (Kalpana and Lee, 2013).
In the current work, the CFF color of F. chlamydosporum NG30
and P. chrysogenum NG85 changed to brown after mixing with
AgNO3. This brown color indicated the AgNPs formation. To confirm
the AgNPs synthesis in the reaction mixture, UVevisible spectro-
photometry was utilized at 420 nm (Jain et al., 2011; Alani et al.,
2012). The increase in absorbance with the incubation time is
shown in Supplementary Fig. 1. The brown color development
resulted from surface plasmon vibration excitation in the metal
nanoparticles, which is typical for AgNPs (Gurunathan et al., 2009).
The maximum amount of AgNPs for F. chlamydosporum NG30 was
obtained at 48 h and there was almost no rise in absorbance from
there onwards. As for P. chrysogenum NG85, the highest amount of
AgNPs was obtained at 72 h with a slight increase at 96 h. The final
reduction in activity could be due to a decrease of nitrate reductase
activity or cofactor availability.
3.3. FTIR analysis
The produced AgNPs were freeze-dried to carry out FTIR spec-
troscopy. This was performed to clarify the probable interactions
between silver and bioactive molecules. These molecules could be
regarded as the capping material responsible for AgNPs synthesis
and stabilization. The amide linkages found between residues of
amino acids in proteins are responsible for well-known signatures
in the infrared region of the electromagnetic spectrum (Jain et al.,
2011). The FTIR spectra are shown in Supplementary Fig. 2, while
in Table 1 the characteristics of the FTIR spectra and the functional
groups are elucidated.
The peaks at 3426.89 and 3431.71 cm1 for F. chlamydosporum
NG30 and P. chrysogenum NG85, respectively, could have resulted
from the strong stretching vibrations of hydroxyl functional groups
(Priyadarshini et al., 2013) which may belong to proteins. The C-H
stretching of protein methylene groups could be denoted by the
bands at 2975.62 and 2925.48 cm1 (Ghaseminezhad et al., 2012).
The bands at 1033.66 and 1049.09 cm1 and the bands at 1625.7

Fig. 4. Linear regression model fit of spore germination of i: Aspergillus flavus, ii: Aspergillus ochraceus, as a function of, a: Fusarium chlamydosporum NG30 silver nanoparticles
(FAgNPs), b: Penicillium chrysogenum NG85 silver nanoparticles (PAgNPs) and c: Itraconazole.
 N.M. Khalil et al. / Chemosphere 218 (2019) 477e486
Fig. 5. Effect of silver nanoparticles (AgNPs) in the spore suspension on the leakage of
proteins (a) and DNA (b). Error Bars show Mean ± SE.
and 1636.3 cm1 can be ascribed to the -N-H and the carbonyl C-O-
stretching vibrations, respectively, in the amide linkages (amide I
and amide II) of the proteins (Suresh et al., 2011).
On the other hand, the peaks observed at 1382.71 and
1049.09 cm1 may correspond to the COO- symmetrical stretch
from the carboxyl groups of residues of the amino acids (Gajbhiye
et al., 2009). Xie et al. (2007) stated that the carboxyl groups of
Asp and Glu residues and the hydroxyl groups of Tyr residues are
considered the most active functional groups for Agþ reduction and
the anisotropic growth of AgNPs.
3.4. TEM and DLS analysis
The morphology and shape of the produced AgNPs were
analyzed by TEM. The well-dispersion, homogeneity and spherical
structure of both FAgNPs and PAgNPs are depicted in Fig. 3. Their
size appeared to be falling in the submicron range, ranging from 6
to 26 nm in the case of the FAgNPs and from 9 to 17.5 nm for the
PAgNPs. To further verify the particle size of the produced AgNPs,
DLS was performed (Fig. 3). DLS confirmed that FAgNPs had a
narrower distribution of diameters than those of PAgNPs. The
average particle size of FAgNPs was 16.1 nm while that of PAgNPs
was 11.1 nm. Various reports revealed that the size of the AgNPs
depended on the utilized biological system to synthesize them.
Also, diverse shapes were found although the spherical particles
were the predominant ones (Alani et al., 2012; Chan and Don, 2013;
Ammar and El-Desouky, 2016; Xue et al., 2016).
3.5. Antifungal activity
The antifungal activity of the synthesized AgNPs was assessed
483
against the mycotoxigenic fungi A. flavus and A. ochraceus. The re-
sults proved the ability of the synthesized FAgNPs and PAgNPs to
inhibit fungal spore germination. The MIC which caused zero spore
germination of A. flavus and A. ochraceus was statistically deduced
from the linear regression model fit (Fig. 4). In the case of A. flavus,
the deduced MIC values were 48, 45 and 50 mg/mL for FAgNPs,
PAgNPs and itraconazole, respectively. A. ochraceus caused MIC
values of 51, 47 and 49 mg/mL for FAgNPs, PAgNPs and itraconazole,
respectively. The slight antifungal superiority of PAgNPs over
FAgNPs could be attributed to the smaller size of the PAgNPs
(11.1 nm) compared to that of FAgNPs (16.1 nm). It was previously
reported that particles with smaller sizes exhibited faster pene-
tration to microbial cells, hence more antimicrobial activity
(Martınez-Castanon et al., 2008).
The AgNPs antimicrobial potency was previously demonstrated.
Thus, Jogee et al. (2017) evaluated the antifungal potential of
AgNPs, synthesized from ten different plants, against the test fungi
A. flavus, A. tamarii, A. niger, A. versicolor, Macrophomina phaseolina
and Penicillium spp. Their results showed that all the synthesized
AgNPs inhibited the fungal growth. Kasithevar et al. (2017) found
that AgNPs formed by Alysicarpus monilifer aqueous leaf extract
showed antibacterial efficacy against methicillin-resistant Staphy-
lococcus aureus (MRSA) and coagulase-negative Staphylococci
(CoNS) isolates from HIV patients.
To gain a further insight into the process of antimicrobial ac-
tivity of AgNPs, the probable leakage of proteins and DNA was
studied. The results in Fig. 5 show that the presence of AgNPs in the
spore suspension caused leakage of proteins as well as DNA.
Generally, PAgNPs were more effective than FAgNPs in causing
cellular membrane damage (Fig. 5). Moreover, A. flavus was more
susceptible to the leakage of proteins and DNA than A. ochraceous.
The OD of the proteins leaked from A. flavus spore cells incubated
with FAgNPs and PAgNPs was 2.05 and 3, respectively, and in the
case of DNA leakage, the OD was 1.91 for FAgNPs and 2.27 for
PAgNPs but it was the same trend for the proteins. The leakage of
proteins from A. ochraceous spore cells was higher in the case of
PAgNPs (OD 2.86) than for FAgNPs (OD 1.94). The respective OD
values for the DNA leakage caused by FAgNPs and PAgNPs were 1.67
and 1.89.
AgNPs primarily anchor the cell membrane at different sites and
penetrate it readily, causing perforations resulting in the leakage of
substances from the interior of the cell (Dizaj et al., 2014). Hence,
the leakage of proteins and DNA from the tested fungal cells was
observed. The higher capability of PAgNPs compared with FAgNPs
in causing protein and DNA leakage is concomitant with the pre-
vious results of more antimicrobial activity against the tested
fungal cells. This was attributed to the smaller size of PAgNPs than
those of FAgNPs.
It is noteworthy to mention that Cao et al. (2017) found that high
concentrations of AgNPs can cause deterioration of the mutual
interaction between plants and arbuscular mycorrhizal fungi and
have a negative effect on the rhizospheric soil phosphorus cycle,
both of which decrease soil fertility and plant growth. Accordingly,
in the current study, it was thought highly advisable to determine
the MIC of the produced AgNPs to inhibit mycotoxin production.
The results in Fig. 6 show that the statistical MIC values for com-
plete inhibition of the total AFs production by A. flavus were 5.9 and
5.6 for FAgNPs and PAgNPs, respectively. The MIC values inhibiting
the production of OTA by A. ochraceus were 6.3 and 6.1 for FAgNPs
and PAgNPs, respectively. It is, thus, obvious that these MIC values
are much lower than those determined for the total inhibition of
fungal spore germination. This proves the importance of con-
ducting this experiment in order to reduce the harmful effect that
may be caused when using high concentrations of AgNPs. In this
sense, Ammar and El-Desouky (2016) indicated that the AgNPs
484 N.M. Khalil et al. / Chemosphere 218 (2019) 477e486

Fig. 6. Linear regression model fit of mycotoxin production of i: Aspergillus flavus, ii: Aspergillus ochraceus, as a function of, a: Fusarium chlamydosporum NG30 silver nanoparticles
(FAgNPs), b: Penicillium chrysogenum NG85 silver nanoparticles (PAgNPs) and c: Itraconazole.

biosynthesized by A. terreus and P. expansum at a concentration of
220 mg/100 mL of media exhibited the highest OTA reduction, with
respective percentages of 58.87 and 52.18%. Yehia and Ahmed
(2013) found that, in a concentration dependent manner, zinc ox-
ide nanoparticles reduced the ability of Penicillium expansum and
Fusarium oxysporum to produce the mycotoxins fusaric acid and
patulin, respectively.
raised up to 6 mg/mL (Fig. 7). Thus, concentrations equal or lower
than 6 mg/mL can be considered as non-cytotoxic. However, Sur
et al. (2012) did not find a toxic effect of AgNPs on normal human
dermal fibroblasts (NHDF) up to a concentration of 50 mg/mL. Also,
in another study, AgNPs were found non-toxic to NHDF and normal
human epidermal keratinocytes (NHEK) in the tested range be-
tween 0 and 25 mg/mL (Galandakova et al., 2016).

3.6. Cytotoxicity assay 4. Conclusions

Recent studies have shown evidence of AgNPs cytotoxicity,
causing safety concerns. The current experiment was conducted to
test the effect of prepared AgNPs on cell survival of human normal
melanocytes (HFB 4). The MIC values to inhibit mycotoxin pro-
duction obtained from the previous experiment were taken as a
guide to select the test concentrations in this experiment. The
cytotoxicity assays revealed that the cell survival of HFB 4 reached
80% and 75% when the concentration of FAgNPs and PAgNPs was
AgNPs were successfully prepared and characterized from
F. chlamydosporum NG30 and P. chrysogenum NG85. The antimi-
crobial activity of FAgNPs and PAgNPs were studied against A. flavus
and A. ochraceous. The prepared FAgNPs and PAgNPs proved their
effectiveness in reducing AFs and OTA production by A. flavus and
A. ochraceous, respectively, at non-cytotoxic doses as shown by the
cytotoxic assay against human normal melanocytes (HFB 4). Under
food safety regulations, if AgNPs are used in polymeric matrices
 N.M. Khalil et al. / Chemosphere 218 (2019) 477e486
Fig. 7. Cytotoxicity assay of cell survival of human normal melanocytes (HFB 4) as a
function of a: Fusarium chlamydosporum silver nanoparticles (FAgNPs) and b: Penicil-
lium chrysogenum silver nanoparticles (PAgNPs). Error Bars show Mean ± SE.
intended for packaging of fresh food, the authors recommend
further in vivo AgNPs cytotoxicity tests using laboratory animals are
conducted.
Competing financial interests
The authors declare that they have no competing financial
interests.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.chemosphere.2018.11.129.
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