1. INTRODUCTION:
Nanotechnology indicated a revealed field of science
that contain formation and involvement of different nanomaterials.
Nanoparticles can be determined as protest in size from 1-100 nm that due to their size may differ from
the volume. Different metallic
nanomaterials are being produced using zinc, copper, titanium, magnesium, gold,
and silver. Nanoparticles are being used for diverse purposes, from medical
treatments, using in various branches of industry production as an example,
oxide fuel batteries for energy storage(1,2).
Zinc Oxide Nanoparticles belong to a group of metallic
oxides is categorized to photo catalytic and photo oxidizing ability against
various living cells(3).
ZnO NPs have wide range of applications including,
sensors piezoelectric, transparent conductors, surface wave devices, and in biomedical
as well as chemical industry, and have wide applications in textile fibers,
energy conservation, electronics chemical gas sensing, sunscreen, paints,
catalyst and in fuel cells(4).
The anti-microbial properties of Metal oxide
nanoparticles exhibit through reactive oxygen species generation. The decline
of Gram-negative bacteria E. coli and Gram-positive bacteria S. aureus with
TiO2 was investigated in the absence and presence of irradiation(5).
The antibacterial action of ZnO NPs correlated to the construction of free
radicals on the surface, and the destruction to the lipids in the bacterial
cell membrane by free radicals,and break down cell membrane(6). ZnO
NPs have antimicrobial and antifungal effects, particularly C. albicans(7).
Zinc oxide nanoparticles are used as antimicrobial agent when incorporated into
materials such as paints, textiles, plastics and personal care products (8)
A cosmetic product is a substance, which can be
applied onto different parts of the human body e.g. (teeth, nails, face and
hair). Its role is to keep the body in a good condition, change its appearance
as well as remove body odors via perfuming, cleansing or protection. Depending
on the application area, cosmetics may be categorized as cosmetics for skin,
hair-scalp and oral care as well as fragrances. Cosmetic ingredients govern a
wide range of products ranging from oily materials, surface active agents,
polymers, ultraviolet absorbents to fragrances and vitamins(9).
Microbial spoilage can not only alter the physical properties of the product such
as color, taste, odor and viscosity, but also deactivate crucial constituents
depriving cosmetic of its features, microbiological contaminants may produce
endotoxins and metabolites causing irritation and allergic reaction of the skin(10,11).
They can be also pathogens causing hazard to the human health(12).
Powders are contaminated by fungi cause spoilage, which may lead to alteration
in organoleptic properties of the product and when a fungal organism is
pathogenic, it renders the product unfit for use and could cause serious health
risk for consumers(13).
This research is conducted to isolation and diagnosis
of fungi from cosmetic industries and estimate the antifungal special effects
of ZnO NPs as a control of fungi growth with analysis data were used HPLC
technique and Scanning electron microscopy (SEM) to show changing in fungi when
using ZnO NPs.
2. MATERIALS AND METHODS:
Ten commercial cosmetic products were randomly
delivered by consumers after approximately 6 months of usage. These included
cosmetic applied on facial skin (cream). A sterile dilution liquid (peptone 5.0
g/L, NaCl 8.5 g/L, distilled water up to 1 L, pH 7.2±0.2) was used to dilute
cream samples.
2.1 Isolation of Fungi:
Samples were placed in petri dishes containing
sabouraud dextrose agar with a width of 9cm in diameter, and incubated at
temperature 25±20C for 3 days. Fungi were sub cultured, in which
were isolated by transporting the disk from each colony and cultured in a new
petri dish. This process was repeated to obtain a pure culture.
2.2 Diagnosis of Fungi:
Diagnosis of isolated fungi was carried out
as a morphological features of
growth colony which including color, margin,texture, colony reverse and
pigments were produced and by microscopic examination, observing fungi shapes, conidia and mycelium using light microscope and scanning electron
microscope (SEM).
2.3 Preparation Method of ZnO NPs:
ZnO NPs were synthesized by hydrothermal method according
to the following: one, 0.5 of zinc acetate (Zn (CH3COO) 2.4H2O) melted in 100
ml of distelled water to make a clear solution. The mixture was put in an
ultrasonic water bath for 40 minutes and transferred to an autoclave,then kept
inside an electric oven set at 180 °C for 2 h and cooled. The quick ZnO NPs was
separated by centrifugation and washed several times with deionized water with
ethanol,after that dried at room temperature for 24h (14).
2.4 Evaluate the ability of Zinc Oxide Nanoparticles
to prevent or decrease of Fungi under study:
Sabouraud dextrose agar was prepared in 4 flasks (500
ml) which divided into four groups, flasks were sterilized in an autoclave and
left to cool then the antibiotic chloromphinicol was added. The ZnO NPs
suspension was added in a concentration (25,50,150) ppm to the culture media at
40ºC in the ratio of 1:20 for each concentration separately. The final volume
was 20 ml of media and suspension in order to ensure uniforming distribution of
ZnO NPs, the agar medium with the suspension was shocked. The last flask as control
group was left without adding of ZnO NPs.
Then these contain of flasks were poured in petri
dishes and left to harden. Followed by cultivating disk of the each fungus
separately, while C.
albicans cultured by
streaking method for two days at 37 0C and four plates per isolation.
Other plates were incubated at 25±2 0C for 7 days. Then percentage
inhibition was calculated according the following eq.(1).
Percentage of inhibition (%) = 
…………(1)
R1 = maximum radial growth of the fungus colony (control
treatment)
R2 = maximum radial growth
of the colony fungus in plates containing ZnO NPs
3. RESULTS AND DISCUSSION:
3.1 Isolation of Fungi from Cosmetics:
Table 1; figure 1 show three fungi were isolated from
ten cosmetics cream samples which are Aspergillus flavus, A. niger and Candida
albicans with the percentage (60,20,20) % respectively. A. flavus and A.
niger were grown on the sabouraud dextrose agar after 7 days, while Candida
albicans grow on chrom agar after 2days. Like in the study (15)
which isolated Aspergillus niger, Aspergillus fumigatus, Candida
albicans, Staphylococcus aureus, Clostridium tetani, Penicillium sp., Rhizopus
oligosporus and Fusarium sp.from the cosmetic samples. Contamination of the
cream may be due to poor storage, manufacturing practices. The presence of more
fungal species could be an indication that cream more susceptible to the fungi.
Aspergillus species cause Aspergillosis,
they are found all over the world. Some Asergillus sp. are harmless,
however, some species can cause a variety of diseases in humans,ranging from
simple allergic reactions to life-threatening invasive disease(16).
Composition of cosmetics varies from product to product, the specificity of
cosmetic application requires that its ingredients are nourishing and easily
incorporated, such components as proteins, minerals, vitamins and glycerine are
easily metabolized source of nitrogen, carbon, hydrogen as well as
macro-elements and micro, necessary for microbial development(17).
Table 1 The percentage of fungi isolated
from cosmetic cream samples
(A) Aspergillus flavus (B) A.
niger (C) Candida albicans
Fig. 1 Fungal genus isolated from cosmetic cream
samples viewed in petri dishes.
3.2 HPLC analysis:
A.flavus produced aflatoxin B1 by high performance liquid chromatography (HPLC)
analysis. HPLC results show that retention time of the major peak of standard
aflatoxin B1 located at 10.54 min (Figure 2) which is identical with aflatoxin
B1 produced from a filter extract of A.flavus of 10.75 min located in the
same area (Figure 3).
3.3 UV-VIS Spectroscopy Study:
Figure 4 illustrations the UV-vis of ZnO NTs, a
wide-ranging group at 365nm is the typical crowd of clean ZnO NPs. Those
optical investigations are performed to estimate the conceivably close optical
qualities of the nanoparticles. A high absorption edge for "360 nm 3. 44
eV" is observed in the sample. Thus, there may be a significant blue
change in the exciton absorption for the ZnO nanoparticles when compared with
those greater part exciton absorption for 373 nm "3.324 eV". The blue
undertaking in the exciton absorption demonstrates the quantum restriction
property from claiming nanoparticles. Generally, for tiny
area ZnO nanoparticles, those blue change will a chance to be exceptionally
substantial.
Fig.4 UV- absorption spectra of ZnO NPs.
3.4 FT-IR Study:
FT-IR spectrum of ZnO NPs (Figure5) showed significant
absorption peaks at 3457 and 1598 and 657cm-1. The absorption band
at 657 cm-1 was assigned to ZnO NPs stretching vibration. The weak
band near 1598 cm-1 is assigned to H-O-H bending vibration mode were
presented due to the absorbed water on the surface of the samples, when FTIR
sample disks were prepared in an open air atmosphere (18).
Fig.5 FT-IR spectrum of ZnO nanoparticles
3.5 Ability of Zinc Oxide Nanoparticles to prevent or
decrease the growth of Fungi:
The effect of ZnO NPs on the growth inhibition is
shown in table 2, which illustrated that ZnO NPs are proved high efficiency of decrease
growth of fungi which are Aspergillus flavus, A. niger and Candida albicans
by percentage (80,85,90) % respectively in the concentration of 50 ppm; while
150 ppm of ZnO caused complete inhibition growth of fungi under study as
compared with a control group which untreated with ZnO. This result is in
contrast with other studies about the antifungal effect of ZnO NPs(19).
The antimicrobial activity increased at concentrations increased than 5%.
Table 2: The effect of ZnO NPs concentrations in
averages of inhibition percentage of fungi by mixing with the medium (25±2)0C
after 7 days.
|
Concentrations
(ppm)
|
Average of
inhibition percentage (%)
|
|
|
Aspergillus
flavus
|
A. niger
|
Candida
albicans
|
|
0
|
0
|
0
|
0
|
|
25
|
72
|
76
|
79
|
|
50
|
80
|
85
|
90
|
|
150
|
100
|
100
|
100
|
3.6 SEM Analysis:
Figure 6;7 show changing in Aspergillus flavus and
A.niger when treated with ZnO NPs in the concentration of 50 ppm, which
decrease in the number of fungi cells, mycelium and conidia,as well as the
opening of conidia as compared with the control group which untreated with ZnO
NPs. The antimicrobial possessions of NPs are attributed to to a mixture of
elements that contain the generation of reactive oxygen species.The ROS mechanism is the use of hydroxyl radical scavengers(20). ZnO NPs as more
significant than other nanoparticles,which found that treatments with it lead
to reduce on conidia production and fungi growth(21).
The disruption of the cell membrane activity by the
direct contact between the nanoparticles and the cell membrane is also reported
to be responsible for antifungal activity of ZnO nanoparticles generation of H2O2
on the microbes(22). The proteins denatured by ZnO NPs and nucleic
acids within bacterial cells, inhibiting the replication. Its inhibitory
activity is perhaps the same in the case of fungi with moderately safe and do
not cause serious side effects (23) .
Fig.6 Scanning electron microscopy images of Aspergillus
flavus.
A, C without ZnO NPs and B,D with the treatment of ZnO
NPs.
Fig.7 Scanning electron microscopy images of
Aspergillus niger.
A, C without ZnO NPs and B,D with the treatment of ZnO
NPs.
4. CONCLUSIONS:
Fungi were isolated from cosmetic cream samples which
are Aspergillus flavus, A. niger and Candida albicans, the sabouraud dextrose agar was used to
isolate fungal contaminated cosmetics. Zinc oxide nanoparticles using an
alternative to fungicides controlling development is carried out and
investigated. UV-vis spectra of ZnO NTs,a broad band at 365nm. FT-IR spectrum
of ZnONPs showed significant absorption peaks at 3457, 1598, and 657cm-1.
It was shown that ZnO NPs as
more significant than other nanoparticles, which found that treatments with it
lead to reduce on conidia production and fungi growth.
5. ACKNOWLEDGMENTS:
I would like to thank the Faculty of Science at
University of Kufa /IRAQ is highly acknowledged.
6. CONFLICT OF INTEREST:
The Auther declared no conflict of interest
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