1. Environmental Nanotechnology, Monitoring & Management 5 (2016) 81–87
Contents lists available at ScienceDirect
Environmental Nanotechnology, Monitoring &
Management
journal homepage: www.elsevier.com/locate/enmm
Investigation of antifungal and anti-mycelium activities using
biogenic nanoparticles: An eco-friendly approach
Ashajyothi C.a
, Prabhurajeshwar C.a
, Harish K. Handralb
, Chandrakanth Kelmani R.a,∗
a
Department of Biotechnology, Gulbarga University, Gulbarga 585 106 Karnataka, India
b
Oral Sciences Disciplines, Faculty of Dentistry, National University of Singapore, 117510, Singapore, Singapore
a r t i c l e i n f o
Article history:
Received 14 November 2015
Received in revised form 7 April 2016
Accepted 8 April 2016
Keywords:
Biogenic nanoparticles
Antifungal activity
Minimum inhibitory concentration
Anti-mycelium activity
Amphotericin B
a b s t r a c t
Green chemistry approaches for synthesizing biogenic nanoparticles brings nanotechnology and micro-
bial biotechnology towards the developments in the nano-biotechnology. Nano-biotechnology is
extending diverse applications in bio-medical studies especially in the field of biosensors, electrochemical
sensors, medicine, healthcare and agriculture. In this study, we aim to evaluate antifungal and anti-
mycelium activities of biogenic Silver (Ag), Copper (Cu), Zinc oxide (ZnO) and Gold (Au) nanoparticles
(NPs) against pathogenic fungi. Selected nanoparticles except Gold have shown acceptable suscepti-
bility towards fungi including C. albicans MTCC 3017 and C. neoformans MTCC 1347. In all treatments,
Amphotericin B (AMB) and Fluconazole antifungal drugs were considered as “gold standards”. Minimum
inhibitory concentration of AgNPs, CuNPs as well as ZnONPs for non-spore forming fungi (C. albicans
MTCC 3017 and C. neoformans MTCC 1347) were recorded at ≤ = 8 ␮g/ml and for spore forming fungi (A.
niger MTCC 282 and F. oxysporum MTCC 284) at ≤16 ␮g/ml. Further, anti-mycelium studies were car-
ried using standard amended nutrient agar method. Anti-mycelium effect of biogenic NPs was recorded
towards A. niger MTCC 282 and F. oxysporum MTCC 284, showing the highest effect by AgNPs (72.8%) as
compared to Cu, ZnONPs and standard antifungal drug. Thus, highlighting the biogenic nanoparticles as
an alternative source for assessing antifungal and anti-mycelium activities.
© 2016 Elsevier B.V. All rights reserved.
1. Introduction
Pathogenic fungi have been cause for serious problems in both
plant and animal systems. Extensive use of antifungal chemicals
has severely decreased the outbreak of fungal diseases, however
with the gap of time pathogenic fungi have developed resistance
over the antifungal drugs. In addition, such antifungal chemicals
are hazardous to human and ecosystem (Vandeputte et al., 2012).
Probable adverse effects of antifungal chemicals could be one of
the major reasons for decrease in number of reports on antifungal
studies when compared to antibacterial discoveries (Doughari and
Nuya, 2008). Resistant pathogenic fungi have diversified the resis-
tance spectrum and also opportunistic fungi have raised their rate
Abbreviations: AgNP’s, silver nanoparticles; CuNP’s, copper nanoparticles;
ZnONP’s, zinc oxide nanoparticles; AuNP’s, gold nanoparticles; AMB, amphotericin
B; MTCC, microbial type culture collection; MIC, minimum inhibitory concentration;
FeSEM, field emission scanning electron Microscopy; EDX, energy-dispersive X-ray.
∗ Corresponding author.
E-mail address: ckelmani@gmail.com (C. Kelmani R.).
of infection, especially in Aspergillus and Candida species (Denning
1991; Ellis et al., 2000; Odds et al.2003). Since 30 years, Ampho-
tericin B has been used as a familiar drug for fungal infections
(Medoff et al., 1983; Baginski and Czub, 2009). However, Ampho-
tericin B has been reported to cause significant nephrotoxicity
(Safdar et al., 2010). During the last decade of 20th century, imi-
dazoles and triazoles were widely accepted for effective treatment
of systemic and opportunistic fungal infections. Fluconazole, one
of the well-known triazoles has been extensively used for treat-
ing more than 16 million patients including AIDS (Mahmoud and
Louis, 1999). Thus emergence of fluconazole resistance, toxic-
ity of Amphotericin B (Alexander and Perfect, 1997; Mukherjee
et al., 2003) and increase of drug resistant pathogenic fungi have
demanded for new research horizon for novel antifungal drugs
(Kontoyiannis et al., 2003; Prachi Kanhed et al., 2014). Due to
gradual increase in drug resistant pathogens, implementations
of metallic nanoparticle oriented antimicrobials have come up.
Hereby considering synthesis of biogenic nanomaterials a cost-
effective and eco-friendly concept, green chemistry has contributed
the synthesis of biogenic nanoparticles using microbial sources
(Kelmani et al., 2014; Ashajyothi et al., 2014a,b).
http://dx.doi.org/10.1016/j.enmm.2016.04.002
2215-1532/© 2016 Elsevier B.V. All rights reserved.

2. 82 A. C. et al. / Environmental Nanotechnology, Monitoring & Management 5 (2016) 81–87
Fig. 1. Percentage Inhibition of Mycelium growth of nanoparticles for A.niger MTCC 282.
Fig. 2. Percentage Inhibition of Mycelium growth of nanoparticles for F. oxysporum MTCC 284.
Current studies on metallic nanoparticles are being explored
and extensively investigated for potential antimicrobial
applications. The antimicrobial activity of the nanoparticles is
known to be a function of interaction between nanoparticle’s
higher surface area and microorganisms, i.e., large surface area of
the nanoparticles enhances microbes to carry out a broad range
of probable antimicrobial activities (Martinez-Gutierrez et al.,
2010).Various reports have demonstrated antimicrobial efficacy
against bacteria, viruses and eukaryotic microorganisms of various
NP materials, including silver (Adeli et al., 2013), copper (Cioffi
et al., 2005), titanium dioxide (Clarence et al., 2015), magnesium,
gold (Gu et al., 2003)and zinc oxide (Sui et al., 2013). In current
investigation, we have demonstrated the inhibitory action of sil-
ver, copper, zinc oxide and gold nanoparticles against pathogenic
fungi; Candida albicans MTCC 3017, Cryptococcus neoformans MTCC
1347, Aspergillus niger MTCC 282 and Fusarium oxysporum MTCC

3. A. C. et al. / Environmental Nanotechnology, Monitoring & Management 5 (2016) 81–87 83
284. In addition, anti-mycelium activity of all four nanoparticles
was studied against A. niger MTCC 282 and F. oxysporum MTCC 284.
2. Materials and methods
2.1. Metal nanoparticles
Four different Biogenic nanoparticles; Silver (AgNPs) (Kelmani
et al., 2014), Copper (CuNPs) (Ashajyothi et al., 2014a,b), Zinc
oxide (ZnONPs) (Ashajyothi et al., 2014a,b) and Gold nanoparticles
(AuNPs) (Ashajyothi and Kelmani, 2014), were synthesized from
Enterococcus faecalis (Nonpathogenic) by extracellular method.
Organism was procured from Medical Biotechnology and Phage
Therapy Laboratory (MBPT), Department of Biotechnology, Gul-
barga University, Gulbarga.
2.2. Fungal strains
C. albicans MTCC 3017, C. neoformans MTCC 1347, A. niger MTCC
282 and F. oxysporum MTCC 284 (Microbial Type Culture Collec-
tion and Gene Bank, Chandigarh, India) were used to evaluate the
antifungal and anti-mycelium activities of biogenic nanoparticles.
C. albicans MTCC 3017 and C. neoformans MTCC 1347 were main-
tained on Sabouraud Maltose Agar (HiMedia, Mumbai, India), A.
niger MTCC 282 and F. oxysporum MTCC 284 on Potato Dextrose
Agar (HiMedia, Mumbai, India), and subculture at least twice on
the same medium at 35 ◦C for 48–72 h prior to use in experiments
to ensure optimal growth.
2.3. Assay for antifungal activity
The in vitro antifungal activity of the nanoparticles was eval-
uated using modified disk diffusion method. Sabouraud Maltose
Agar (for C. albicans MTCC 3017 and C. neoformans MTCC 1347) and
Potato Dextrose Agar (for A. niger MTCC 282 and F. oxysporum MTCC
284) was dispensed into separate petri dishes and allowed to solid-
ify. Spores were recovered by gentle swabbing the surface of the
culture plates using a sterile cotton swab; later the swab was dipped
in 5 ml sterile saline containing 0.1% Tween 80 to suspend the
spores. Serial dilution of spore suspension was performed to adjust
the initial inoculum to 4 × 104 spores/ml using counting cham-
ber. In aseptic conditions, 0.1 ml of spore suspension (adjusted to
4 × 104 spores/ml) was pipette onto the agar plates and suspen-
sion was spread uniformly. (Doughari and Nuya, 2008). Agar wells
of 5 mm diameter were made with the help of a sterilized stain-
less steel cork borer. Aseptic conditions were maintained during
the loading of different concentrations (20, 40, 60, 80, 100 ␮g/ml)
of nanoparticle on marked agar wells using micropipette and water
was considered as control. Plates were incubated at 37 ◦C for 48 and
72 h. The zone of inhibition was measured in mm.
2.4. Assay for anti-mycelium activity
Anti-mycelium activity was performed using standard amended
nutrient agar method (Tomasino and Hamilton, 2006).Different
concentrations of each nanoparticle type samples were added
to autoclaved and cooled PDA and Sabouraud Maltose Agar. The
homogeneous mixture was poured in sterilized petri dishes. Seven
days old culture of A. niger MTCC 282 and F. oxysporum MTCC
284 was placed in the center of petri plate in two separate plates.
Three replicate plates were used per treatment. Plates containing
mycelium disc without nanoparticle and plates containing antifun-
gal drug (Amphotericin B) were considered as positive and negative
controls, respectively. All plates were incubated at 28 ± 2 ◦C for 4-
7 days. Fungal growth was measured as percent mycelia inhibition
by the formula:
Anti-mycelium activity(%) = [(Dc − Dt)/Dc] × 100
where, Dc: Diameter of colony in the control (mm), Dt: Diameter of
colony in the treatment (mm)
2.5. Determination of minimum inhibitory concentration
The MIC of nanoparticles was determined using the broth dilu-
tion method with slight modifications. Different concentrations (2,
4, 8, 16, 32, 64, and 128 ␮g/ml) of nanoparticles were added to the
10 ml of potato dextrose broth with culture and incubated at 25 ◦C
for 48–72 h. The MICs were recorded after 48 h and the absorbance
was read at 600 nm.
3. Results and discussion
3.1. Nanoparticles used
In our published reports, FeSEM (Field emission Scanning elec-
tron Microscopy) and EDX (energy-dispersive X-ray) analysis were
used to determine the morphology, shape and chemical compo-
sition of biogenic nanoparticles. FeSEM images of functionalized
silver nanoparticles synthesized from E. faecalis bacterial biomass
can be seen with core shell morphology of size 9–130 nm and
marginal variation in the particle size was observed. Negligible
amount of phosphorus and sodium elements were showed by EDX
analysis (Kelmani et al., 2014). However copper nanoparticles, were
spherical in shape with size ranging from 20 to 90 nm. In addition,
EDX spectrum gives for copper nanoparticles showed two types of
signal peaks, one was for copper atom and another for elemental
oxygen (Ashajyothi et al., 2014a,b). Biogenic ZnO nanoparticles size
was ranging from 16 to 96 nm. The EDX spectrum reports showed
strong signals of Zinc atoms, along with signals from Oxygen, Potas-
sium, Phosphorus, Sulphate and Chloride atoms. (Ashajyothi et al.,
2014a,b). Similarly, FeSEM analysis of gold nanoparticles showed
the size range from 20 to 70 nm and EDX spectrum provided
number of signals from various contaminants, viz. sodium, chlo-
rine, potassium, calcium and oxygen elements (Ashajyothi et al.,
2014a,b).
3.2. Antifungal activity of nanoparticles against different
pathogenic fungi
The antifungal activity of biogenic nanoparticles against
pathogenic fungi was investigated using standard antifungal drugs
like Amphotericin B, Fluconazole, whereas AgNPs, CuNPs, ZnONPs
and AuNPs were used as comparable drugs. Inhibition effect of all
four biogenic nanoparticles at different concentrations were esti-
mated against C. albicans MTCC 3017 C. neoformans MTCC 1347, A.
Niger MTCC 282 and F. oxysporum MTCC 284 for 72 h of incubation
as represented in Table 1.
Silver nanoparticles treated with 200 ␮g/ml concentration
showed highest fungal growth inhibition in C. albicans MTCC 3017
and C. neoformans MTCC 1347. According to Kim et al., 2007; spher-
ical shaped AgNPs were reported as potent drug against C. Albicans
when compared with commercially available antifungal drugs. In
microorganisms, Ag+ forms complexes with nucleotide base pairs
of DNA and also proven to be a potentially inhibit DNAases (Wen Ru
et al., 2010). AgNPs at 100 ␮g/ml showed 31 mm of zone inhibition
against F. oxysporum MTCC 284 and failed to inhibit the growth of A.
niger MTCC 282. According to our studies, biogenic gold nanoparti-
cles showed less effectiveness towards all four pathogenic fungi’s,
except for A. niger MTCC 282.

4. 84A.C.etal./EnvironmentalNanotechnology,Monitoring&Management5(2016)81–87
Table 1
Antifungal activity of biogenic nanoparticles and Antifungal drug used against different Pathogenic Fungi’s.
Pathogenic fungi AgNPs 50 mg/ml (stock) CuNPs 50 mg/ml (stock) ZnONPs 50 mg/ml (stock) AuNPs 50 mg/ml (stock) Antifungal drug 30mcg/ml (stock)
(␮g/ml) Zone of inhibition
(mm)
(␮g/ml) Zone of inhibition
(mm)
(␮g/ml) Zone of inhibition
(mm)
(␮g/ml) Zone of inhibition
(mm)
Amphotericin B Zone of inhibition
(mm)
Amphotericin B
Candida albicans MTCC 3017 120 25 10 19 10 17 10 NZ 10
140 26 20 23 20 20 20 NZ
160 30 30 26 30 22 30 NZ
180 32 40 28 40 24 40 NZ
200 32 50 30 50 25 50 NZ
Cr. neoformans MTCC 1347 120 NZ 10 18 10 15 10 NZ Fluconozole 11
140 22 20 21 20 22 20 NZ
160 25 30 26 30 26 30 NZ
180 27 40 29 40 28 40 NZ
200 29 50 30 50 29 50 NZ
A. niger MTCC 282 20 NZ 10 NZ 10 NZ 10 NZ Amphotericin B 14
40 NZ 20 NZ 20 NZ 20 NZ
60 NZ 30 NZ 30 NZ 30 NZ
80 NZ 40 NZ 40 NZ 40 20
100 NZ 50 NZ 50 NZ 50 31
F. oxysporum MTCC 284 20 15 10 NZ 10 NZ 10 NZ Amphotericin B NZ
40 17 20 NZ 20 NZ 20 NZ
60 20 30 NZ 30 NZ 30 NZ
80 28 40 NZ 40 13 40 NZ
100 31 50 NZ 50 16 50 NZ
(AgNPs: silver nanoparticles, CuNPs: copper nanoparticles, ZnNPs: zinc nanoparticles, AuNPs: gold nanoparticles, NZ: no Zone).

5. A. C. et al. / Environmental Nanotechnology, Monitoring & Management 5 (2016) 81–87 85
Table 2
Percentage mycelium inhibition test of biogenic nanoparticles for pathogenic fungi’s for seven days of incubation.
Pathogenic fungi AgNPs 50 mg/ml (stock) CuNPs 50 mg/ml
(stock)
ZnONPs
50 mg/ml (stock)
AuNPs 50 mg/ml
(stock)
Antifungal drug
30 mcg/ml
(stock)
(Percentage Inhibition of Mycelium growth in Pathogenic fungi’s)
A. niger MTCC 282 60 ␮g/ml 72.8% 51.6% 15.8% 5.5% 65.5%
F. oxysporum MTCC 284 60 ␮g/ml 72.8% 34% 65% NI NI
(NI: no Inhibition).
Table 3
Optical density for minimum inhibitory concentration of nanoparticles and antibiotics for pathogenic fungi’s.
Pathogenic fungi AgNPs CuNPs ZnONPs AuNPs Antifungal drug
(␮g/ml) OD at 600 nm (␮g/ml) OD at 600 nm (␮g/ml) OD at 600 nm (␮g/ml) OD at 600 nm (␮g/ml) OD at 600 nm
Candida albicans MTCC 3017 8 −0.03 8 0.190 8 0.834 8 0.192 8 0.716
16 NP 16 −0.01 16 0.86 16 −0.02 16 0.72
32 NP 32 NP 32 0.87 32 NP 32 0.64
64 NP 64 NP 64 0.87 64 NP 64 0.62
128 NP 128 NP 128 0.86 128 NP 128 0.3
Cr. neoformans MTCC 1347 8 −0.02 8 0.224 8 0.857 8 0.224 8 0.806
16 NP 16 0.02 16 0.84 16 0.02 16 0.82
32 NP 32 NP 32 0.92 32 NP 32 0.82
64 NP 64 NP 64 0.93 64 NP 64 0.86
128 NP 128 NP 128 0.94 128 NP 128 0.72
A. niger MTCC 282 8 1.02 8 1.09 8 1.02 8 1.05 8 1.04
16 0.99 16 0.96 16 1.03 16 0.99 16 0.97
32 0.96 32 0.97 32 0.98 32 0.99 32 0.95
64 0.03 64 0.98 64 1.02 64 1.02 64 0.7
128 0.024 128 0.09 128 0.62 128 0.42 128 0.06
F. oxysporum MTCC 284 8 1.26 8 1.05 8 1.07 8 1.07 8 1.06
16 0.3 16 0.87 16 1.06 16 0.97 16 0.99
32 0.04 32 0.97 32 0.98 32 0.96 32 0.78
64 −0.02 64 1.22 64 1.05 64 0.44 64 1.02
128 0.01 128 0.04 128 0.42 128 −0.002 128 0.87
(AgNPs: silver nanoparticles, CuNPs: copper nanoparticles, ZnNPs: zinc nanoparticles, AuNPs: gold nanoparticles, NP: not performed).
Table 4
MIC level of biogenic nanoparticles and antibiotics for pathogenic fungi’s.
Sl No. Pathogenic Fungi MIC level of
AgNPs (␮g/ml)
MIC level of
CuNPs (␮g/ml)
MIC level of
ZnONPs (␮g/ml)
MIC level of
AuNPs (␮g/ml)
MIC level of
Antibiotics in
(mcg/ml)
01 Candida albicans MTCC 3017 08 08 08 ≥128 ≥128
02 Cr. neoformans MTCC 1347 08 16 08 ≥128 ≥128
03 A. niger MTCC 282 64 ≥128 ≥128 ≥128 ≥128
04 F. oxysporum MTCC 284 16 ≥128 ≥128 ≥128 ≥128
Recent studies on CuNPs demonstrated that antifungal activity
of nanoparticles has received more attention because of cost-
effectiveness when compared with of other metallic nanoparticles
(Prachi Kanhed et al., 2014). Advantage of cost effective nanoparti-
cles could offer the antifungal applications in human health issues.
Different concentrations such as 10, 20, 30, 40 and 50 ␮g/ml were
checked for antifungal activity. CuNPs revealed higher antifungal
activity with inhibition zone of 32, 30 mm at 50 ␮g/ml for C. albicans
MTCC 3017 and C. neoformans MTCC 1347 respectively (Table 1).
CuNPs are not effective enough to inhibit A. niger MTCC 282 and F.
oxysporum MTCC 284.
Highly ionic nanoparticulate metal oxides such as zinc oxide
nanoparticles (ZnONPs) are unique in that they can be produced
with high surface areas and with unusual crystal structures (Lili
He et al., 2011). Reports were documented on the antibacterial
and antifungal activity of bulk ZnO powders. (Yamamoto 2001;
Sawai and Yoshikawa, 2004). In our studies, Zinc oxide nanopar-
ticles at 50 ␮g/ml showed highest inhibition zone of 25 mm and
29 mm for C. albicans MTCC 3017 and C. neoformans MTCC 1347
respectively. Amphotericin B, the standard antifungal agent has
showed the highest activity of 14 mm and 10 mm zone diameter
of inhibition against A. niger MTCC 282 and C. albicans MTCC 3017
respectively. C. neoformans MTCC 1347 showed 10 mm zone of inhi-
bition for Fluconazole antifungal agent and F. oxysporum MTCC 284
was resistant to amphotericin B.
3.3. Assay for anti-mycelium activity
Fungi used in this study are pathogenic and causative drugs of
various mycotic infections. Our results of the anti-mycelium activ-
ity assay showed, silver nanoparticles are most effective against
pathogenic fungi. Significant mycelium inhibition was recorded for
A. niger MTCC 282 and F. oxysporum MTCC 284 at 60 ␮g/ml (Per-
centage of anti-mycelium activity is 72.8%). In A. niger MTCC 282
65.5% of inhibition was recorded for standard antifungal agent at
30mcg/ml of optimum concentration (Table 2, Fig. 1) and F. oxys-
porum MTCC 284 highly resistant towards gold nanoparticles and
standard antifungal agent (Table 2, Fig. 2).

6. 86 A. C. et al. / Environmental Nanotechnology, Monitoring & Management 5 (2016) 81–87
3.4. Minimum inhibitory concentration
Tables 3 and 4 shows the results of minimum inhibitory con-
centration (MIC) along with their optical density values at 600 nm
of nanoparticles and the standard antifungal drugs. Results shows,
MIC values for the silver, copper and zinc oxide nanoparticles with
the least values of 8 ␮g/ml against C. albicans MTCC 3017 and C.
neoformans MTCC 1347, when experimental range was between
4 and 8 ␮g/ml. In addition, silver nanoparticles showed the high-
est MIC values of 64 ␮g/ml and 16 ␮g/ml against A. niger MTCC
282 and F. oxysporum MTCC 284 respectively. MIC values of stan-
dard antifungal drugs and gold nanoparticles ranged between 64
to ≥128 ␮g/ml against all four pathogenic fungi. For C. neoformans
MTCC 1347, MIC values of copper and zinc oxide nanoparticles
recorded for 16 ␮g/ml and 8 ␮g/ml respectively. Highest MIC value
for copper and zinc oxide nanoparticles ranged above 128 ␮g/ml
concentration for both A.niger MTCC 282 and F. oxysporum MTCC
284. The comparative study between biogenic nanoparticles and
standard antifungal drugs showed, silver, copper and zinc oxide
nanoparticles are more potential as compared to antifungal drugs.
Thus, highlighting the fungicidal efficiency of biogenic nanoparti-
cles against pathogenic fungi.
Our experimental reports showed all three nanoparticles
including AgNPs, CuNPs and ZnONPs exhibits acceptable antifun-
gal activity and greater mycelium inhibition action against spore
forming fungi. This observation directly states, the fungicidal action
of all three nanoparticles completely depends on their chemical
properties.
4. Conclusion
The results indicated the antifungal efficacy of bio-synthesized
silver, copper and zinc oxide nanoparticles showed promising
and most effective approach against the conventional fungicides.
In summary, all nanoparticles excluding AuNPs, are potentially
effective against pathogenic fungi when compared to standard anti-
fungal drugs. Another significance of this study, nanoparticles were
more effective in inhibiting mycelium growth in fungi at variable
concentration. MIC results showed, lower concentrations of bio-
genic NPs inhibited the tested fungal growth. These results were
confirmed by Broth dilution method. The lowest MICs of AgNPs,
CuNPs and ZnONPs for C. albicans MTCC 3017 and C. neoformans
MTCC 1347 were found to be ≤8 ␮g/ml and ≤16 ␮g/ml for A. niger
MTCC 282 and F. oxysporum MTCC 284, respectively. Experimental
outputs prove antifungal drugs from eco-friendly sources could be
the possible approach for developing cost-effective and safe anti-
fungal drugs. Also could provide applications in treating systemic
and superficial mycotic infections as well as in management of
antifungal-drug resistance pathogenic fungi.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
All authors contributed to the design and implementation of
this study. Dr. Kelmani Chandrakanth R and Ashajyothi C, con-
ducted the all experiments in Medical Biotechnology and Phage
therapy Laboratory, Department of Biotechnology, Gulbarga Uni-
versity, Gulbarga. All authors contributed to the review of study, in
discussing results, manuscript preparation, final review and data
analysis presentation.
Acknowledgment
Authors are grateful to the Department of Science and Technol-
ogy (DST-INSPIRE fellowship) New Delhi, for supporting through
funding the project and Department of Biotechnology, Gulbarga
University, Gulbarga for providing the facilities for pursuing the
research work at the Department. We would like to thank Har-
ish Handral, Faculty of Dentistry, National University of Singapore,
Singapore for their contribution in revising and rewriting the
manuscript.
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