International Journal of Innovative Pharmaceutical ...C. Topographical analysis by SEM Scanning...

RESEARCH ARTICLE Moharekar et.al / IJIPSR / 2 (9), 2014, 2106-2118

Department of Biotechnology ISSN (online) 2347-2154

Available online: www.ijipsr.com September Issue 2106

EXPLOITATION OF ASPERGILLUS NIGER FOR SYNTHESIS

OF SILVER NANOPARTICLES AND THEIR USE TO IMPROVE

SHELF LIFE OF FRUITS AND TOXIC DYE DEGRADATION

1Shubhangi Moharekar,

2Pradnya Bora,

3 Varsha Kapre,

4Mahadev Uplane,

5Vishakha Daithankar,

6Bapusaheb Patil,

7Sanjay Moharekar*

1,2,3,5,6, 7

Department of Biotechnology, New Arts, Commerce and Science College, Ahmednagar,

INDIA 4Department of Instrumentation Science, University of Pune, Ganeshkhind, Pune-411007,

INDIA

Corresponding Author:

Dr. Sanjay T. Moharekar

Department of Biotechnology,

New Arts, Commerce and Science College,

Ahmednagar, INDIA

Email: [email protected],

Mobile: +91 9970544366

International Journal of Innovative

Pharmaceutical Sciences and Research www.ijipsr.com

Abstract

Extracellular biosynthesis of silver nanoparticles (AgNPs) by Aspergillus niger isolated from spoiled bread

was reported in the present study. The biosynthesis of AgNPs was monitored by ultraviolet-visible

spectroscopy, and the AgNPs obtained were characterized by Scanning electron microscopy and X-ray

diffraction. The synthesized AgNPs were spherical particles having size of 7 nm. The AgNPs showed

remarkable antibacterial activity against four major human pathogens, Staphylococcus aureus,

Pseudomonas aeruginosa, Salmonella typhi and Escherichia coli. The efficacy of test was performed with

thin film of silver nanoparticles incorporated with sodium alginate. Futher this film was found to increase

the shelf life of grapes and chikku compared to control with respect to weight loss and soluble protein

content. In addition, the silver nanoparticles were also able to decolorize Congo red dye up to 51% within

48 hrs. Based on these findings, it was concluded that the present study provides potential of fungal-

mediated biosynthesis of AgNPs having effective antibacterial activity, efficient decolorizing property and

can be used in combination with sodium alginate for preservation of fruits.

Keyword: Silver nanoparticles; Aspergillus niger; Antibacterial activity; Fruit shelf life.

mailto:[email protected]




INTRODUCTION

Nanotechnology is an emerging field which deals with synthesis of nanoparticles and also with

their applications. Such nanomaterials displaying novel properties have effective and wide

applications in biophysical and biomedical fields like diagnostics, therapeutics, medicine, drug

delivery system, agriculture, consumer goods, and cosmetics. Noble metal nanoparticles such as

gold, silver, platinum and palladium have been most effectively studied [1, 2]. Nanoparticles can

be synthesized through physical, chemical and biological approach. Existing physical and

chemical methods poses certain disadvantages in terms of toxic chemicals and harsh conditions

employed. In comparison to the physical and chemical methods, the biological route of

nanoparticle synthesis is being paid increasing attention due to its eco-friendly approach, cost-

effectiveness, and it do not involve the use of any toxic chemicals for the synthesis of NPs [3]. A

biological method employs use of either microorganisms (bacteria and fungi) or plant extract for

nanoparticles production. It has been reported that fungi are extremely good candidates in the

synthesis of metal nanoparticles [4].

A number of different genera of fungi have been investigated for production of AgNPs like

Aspergillus fumigatus [5], Penicillium sp. [6], Coriolus versicolor [7], Alternaria alternate [8],

Trichoderma reesei [9], Penicillium purpurogenum [10], Aspergillus flavus [11].

India is major agriculture-based country, where preservation of perishable vegetable is the main

problem. Different microorganisms are responsible for the spoilage of fruits and vegetables, thus

decreasing their quality and shelf life [12]. The emerging multi drug resistance in microbes is a

matter of great concern as these pathogens are reported to be the leading cause of death

worldwide [13, 14]. Nanoparticles can serve as potent alternative to the antibiotics, because NPs

have antibacterial and antifungal activity [13]. Use of metallic nanoparticles entrapped in

varieties of coatings for enhancement of shelf-life in different food products is topic of interest in

food nanotechnology branch [15-17].

In present work, we investigated the biosynthesis of AgNPs using Aspergillus niger. The

properties of obtained AgNPs were characterized by ultraviolet-visible spectroscopy, Scanning

electron microscopy (SEM) and X-ray diffraction (XRD) techniques. This work provided a

potent application of AgNPs to serve as an alternative to antibiotics against human pathogens

and in preservation of vegetables to increase shelf life. A preliminary study was also conducted

to find out the potential of nanoparticles as dye degraders.




MATERIALS AND METHODS

MATERIALS

Aspergillus niger was isolated from spoiled bread, and maintained on nutrient Agar slant at -

20°C in refrigerator till further use. The isolated fungus was identified using morphological

characteristics and Lactophenol Cotton Blue Staining. Four kinds of bacteria were tested for their

susceptibility for AgNPs: Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi,

Escherichia coli and antibiotic – Gentamycin (10 µg/ml) as positive control.

PRODUCTION OF BIOMASS

Aspergillus niger was grown in 250 ml culture flask containing 100 ml potato dextrose broth and

was incubated on an orbital shaker at 27 °C with agitation of 150 rpm. The biomass was

harvested after 72 h of growth by sieving through a plastic sieve. Twenty gram of wet biomass

was brought into contact with 100 mL of sterile double-distilled water for 48 h at 27 °C. After

the incubation, the biomass was filtered by Whatman filter paper no. 1 and cell filtrate was used

for biosynthesis of AgNPs.

BIOSYNTHESIS OF SILVER NANOPARTICLES

For biological synthesis of AgNPs, 50 ml of cell filtrate was mixed with 10 ml AgNO3 solution

(10 mM) and reaction mixture without AgNO3 was used as control. Then solution was incubated

at 28°C for 24 hrs in dark to avoid any photochemical reaction. The change in colour from white

to dark brown colour indicated the synthesis of silver nanoparticles. The AgNPs were purified by

centrifugation at 10,000 rpm for 10 min twice, and collected for further characterization.

CHARACTERIZATION OF SILVER NANOPARTICLES

A. Ultraviolet-Visible spectral analysis

Ultraviolet-Visible spectral analysis of reaction mixture was done by using UV-Visible double

beam spectrophotometer of Systronics Ltd. within the range of 200-800 nm.

B. X-ray diffraction analysis

Chemically and biologically synthesized silver nanoparticles was dried and used for X- ray

diffraction analysis. The XRD spectra was recorded using X-ray diffractometer (Bruker GXS D-

8) operated at voltage of 40 kV and a current of 30 mA with CuKα radiation by using 2θ from

10-80°. The crystallite domain size was calculated from the width of the XRD peaks, assuming

that they are free from non-uniform strains, using the Scherrer formula.




D= 0.94 λ / β Cos θ

Where, D is the average crystallite domain size perpendicular to the reflecting planes, λ is the X-

ray wavelength, β is the full width at half maximum (FWHM), and θ is the diffraction angle.

C. Topographical analysis by SEM

Scanning Electron Microscopic (SEM) analysis was done using Hitachi S-4500 SEM machine.

Film of the sample were prepared on a carbon coated copper grid by dropping a very small

amount of sample on the grid, extra solution was removed using a blotting paper and then the

film on the SEM grid were allowed to dry by putting it under a mercury lamp for 5 min.

ANTIMICROBIAL ACTIVITY BY WELL DIFFUSION METHOD

Muller Hinton (MH) Agar plates were prepared, sterilized and allowed to solidify. Plates were

then spreaded with bacterial cultures (S. aureus, P. aeruginosa, S. typhi, E. coli). After

spreading, sterilized 6 mm cork borer was used to make agar wells. The 100 µl of biologically

synthesized AgNPs were placed into the wells. Assay was performed in triplicate. The plates

were incubated at 370C for 24 hrs and zone of inhibition was measured. The results were

compared with standard antibiotic Gentamycin (10 µg/ml).

PREPARATION OF SODIUM ALGINATE FILM CONTAINING SILVER

NANOARTICLES

Sodium alginate was weighed to 2.5 g and dissolved in 25 mL of double distilled water by

mixing slowly with a magnetic stirrer for 30 min; 1 mL of glycerol was added to the mixture,

and boiled for 5 min. After the mixture had cooled to room temperature, silver nanoparticles

were added to it. This mixture was further stirred for 20 min and then added evenly on sterilized

Petri plates. The plates were placed at room temperature for 24 hrs for drying purpose and films

were stored for further use.

ANTIBACTERIAL ACTIVITY OF SILVER NANOPARTICLE INCORPORATED

SODIUM ALGINATE FILM BY DISC DIFFUSION METHOD

A diffusion method was used to assay the antibacterial activity of silver nanoparticle

incorporated sodium alginate thin film against test strains such as S. aureus, P. aeruginosa, S.

typhi, E. coli on MH agar plates. Overnight grown bacterial cultures were spreaded on separate

agar plates and part of silver nanoparticle incorporated sodium alginate film was kept at center of

each plate. Then plates were incubated at 37 °C for 24 h, the zones of inhibition were observed

and recorded. Assay was performed in triplicate.




FILM COATING ON FRUITS

Grapes and chikku were purchased from a local market and surface sterilized using 6 ppm

chlorine dioxide (ClO2) for 20 mins [18]. Above fresh fruits were covered completely into silver

nanoparticles incorporated sodium alginate film and without silver nanoparticles incorporated

sodium alginate film. Uncoated fruits were served as control. Coated and control grapes and

chikku were stored at 27 °C then weight loss and soluble protein content was analyzed for next

five days.

DETERMINATION OF WEIGHT LOSS AND SOLUBLE PROTEIN CONTENT

The water loss (weight loss) and soluble protein content were monitored regularly in grapes and

chikku stored at room temperature (27 °C). They were weighed every day for next five days.

Soluble protein content was estimated by using Bradford’s method [19]. The grapes and chikku

were weighed to about 1 g, mixed with 2 mL of distilled water, and homogenized for 5 min at

room temperature. After centrifugation at 5000 rpm for 20 min, supernatant was collected in

Eppendorf tubes and stored at 4 °C for 2 h before analysis. The protein extract was diluted 50

times, and from that 1.0 mL of protein extract was mixed with 5 mL of Coomassie brilliant blue

G-250 (100 mg) and incubated for 15min at room temperature; the absorbance was read at 595

nm using a spectrophotometer. Total protein concentration was calculated on basis of standard

graph.

DECOLORIZATION STUDIES

For decolorization study, 250 mL Erlenmeyer flasks containing 125 mL solutions of 50µM

congo red was prepared in the decolorization media [20]. Silver nanoparticles were added to the

above media which is indicated as test. Similarly, A. niger culture was also added to this media

separately which serves as control for the above. The flasks were incubated at 30°C under

shaking conditions. After 48 hr interval samples were withdrawn, filtered and centrifuged at

4400 rpm for 5 mins and the supernatants was analyzed using UV-Visible spectrophotometer at

498 nm.

RESULTS AND DISCUSSION

In the present study, biosynthesis of AgNPs was carried out using fungal species isolated from

spoiled bread. The isolated fungus was identified and confirmed as A. niger by morphological

characteristics and Lactophenol cotton blue staining (Figure 1).




Fig. 1: Lactophenol Cotton Blue Staining.

The aqueous extract of A. niger mixed with AgNO3 (1 mM) for 24 hrs showed a rapid change in

the colour of the solution from pale white to dark-brown. The appearance of brown color

indicates the reduction of silver ions by fungus which forms silver nanoparticles (Figure 2).

Mukherjee et al. (2001) [21] suggested that cell wall and cell wall sugars play important role in

reduction of metal ions. A number of studies have suggested role of protein in nanoparticles

formation. Bansal et al. (2004) [22] observed that fungal species secretes an enzyme, which

brings about reduction of silver ion thereby forming the silver nanoparticles. UV-

Fig. 2: Fungal cell filtrate (A) before and (B) after treatment with solutions of AgNO3

Vis spectroscopy of this solution confirmed the synthesis of AgNPs, as revealed by a

characteristically distinct and fairly broad absorption peak at 420 nm [23]. The presence of broad

resonance indicated an aggregated structure of the AgNPs and another absorbance peak at 270

nm is also clearly visible and is attributed to aromatic amino acids of proteins (Figure 3).




Fig. 3: UV-Vis spectrophotometer analysis

Scanning Electron Microscope (SEM) is used to decide size and shape of NPs. Scanning

Electron Microscope analysis revealed development of silver nanostructures and it also

confirmed its spherical shape (Figure 4). The SEM result showed that the size of AgNPs ranges

between 1 – 100 nm.

Fig. 4: Scanning electron micrograph of silver nanoparticles synthesized by A. niger

Figure 5 showed the XRD patterns obtained for the AgNPs synthesized by A. niger. The

crystalline size for AgNP was calculated using Scherrer’s formula [24]. A broad and blunt peak

in the XRD patterns suggested that the particles were extremely smaller (∼7 nm) in size and

exhibits amorphous nature.




Fig. 5: X-ray diffraction pattern in 2-theta (2θ) scale with counts to depict number of silver

nanoparticles synthesized by Aspergillus niger

The antibacterial activity of silver nanoparticles was evaluated against Gram-positive (S. aureus)

and Gram negative (E. coli, P. aeruginosa, S. typhi) bacteria. The results are shown in the

Table1. The maximum zone of inhibition was observed with S. aureus which was about 28 mm

in diameter, whereas the cultures of S. typhi, E. coli, and P. aeruginosa had also shown zones of

inhibition which was about 21 mm, 23 mm and 16 mm in diameter, respectively. The growth

inhibitions against bacteria were compared with Gentamycin. Synthesized AgNPs independently

showed efficient antimicrobial activity against Gram positive and Gram negative bacteria

compared to Gentamycin. Thus, AgNPs could be considered as excellent broad-spectrum

antibacterial agents. Antibacterial activity of biosynthesized AgNPs in the present study are

found to be higher than that reported by A. K. Gade et al (2008) [25] and Kalaivani.M. et al

(2009) [26]. Antibacterial activity of silver nanoparticle incorporated sodium alginate films were

also tested against above four test strains. A clear zone was observed around the silver

nanoparticle incorporated sodium alginate film; S. typhi (7 mm), S. aureus (8 mm), E. coli (7

mm), P. aeruginosa (6 mm). These results indicated that AgNPs retains its antimicrobial

potential with sodium alginate which can be used efficiently for fruit preservation application.

Table No. 1 - Antimicrobial Activity of AgNPs (n =3)

ORGANISM PARAMETER ZONE OF INHIBITION (mm)

P. aeruginosa Antibiotic 25

Biosynthesized AgNPs 16

S. typhi Antibiotic 26


E. coli Antibiotic 26


S. aureus Antibiotic 25





Figures 6 showed that in chikku and grapes, for five days, weight losses was significantly higher

in control and significantly lower in wrapped fruit with film of sodium alginate containing

AgNPs. It indicated that wrapping of fruits with sodium alginate film containing AgNPs

effectively controls water loss than control and film without AgNPs. In chikku and grapes,

interactive effect of time and different condition affected % weight loss. However, this

interactive effect was higher in grapes than that of chikku (Table 2). In addition to above,

wrapping of fruits with sodium alginate film containing AgNPs may control growth of fruit

spoilage causing microorganisms, as it is having potent antimicrobial activity, helps to increase

shelf life of fruits (Table 1).

Table No. 2: The results of Bonferoni multiple comparisons for Grapes (upper right)

and chikku (lower left) for % weight loss in the one-way ANOVA of 15 levels.

In the one-way ANOVA of 15 levels (groups) of days (Dk), condition (Ci) where, k = 1:

day1, 2: day2, 3: day 3, 4: day 4; 5: day 5; i = 1: Control (Uncoated fruits), 2: Sodium

alginate film coated fruit, 3: Silver nanoparticles incorporated sodium alginate film coated

fruit. significant difference at P < 0.05. Blank blocks represent not significantly different

cases (P > 0.05).

D1

C1

D2

C1

D3

C1

D4

C1

D5

C1

D1

C2

D2

C2

D3

C2

D4

C2

D5

C2

D1

C3

D2

C3

D3

C3

D4

C3

D5

C3

Gra

pe

D1 C1 * * * * * * * * * * *

D2 C1 * * * * * * * * *

D3 C1 * * * * * * * * *

D4 C1 * * * * * * * * * *

D5 C1 * * * * * * * * * * * *

D1 C2 * * * * * * * * * * *

D2 C2 * * * * * * * * * *

D3 C2 * * * * * * *

D4 C2 * * * * * * *

D5 C2 * * * * * * * * *

D1 C3 * * * * * * * * * * * *

D2 C3 * * * * * * * * * *

D3 C3 * * * * * * * *

D4 C3 * * * * * * *

D5 C3 * * * * * *

Chikku

The soluble protein content was evaluated from fruits wrapped by sodium alginate film with and

without AgNPs on day 5. A significant decrease in soluble protein content of unwrapped i.e.

control fruits were due to utilization of proteins for metabolic activities such as




Fig. 6: % Weight losses of fruits wrapped with AgNPs Incorporated Sodium Alginate

Films, film without Silver nanoparticles and unwrapped fruits as control.

substrate for respiration, due to inadequate carbohydrate source [27]. The soluble protein content

of grapes and chikku wrapped in film without AgNPs was 0.28 ± 0.04 and 1.05 ± 0.02 mg/ml,

respectively, which was significantly less than soluble protein content of grapes and chikku

wrapped in film with AgNPs, 0.34 ± 0.01 and 1.18 ± 0.08 mg/ml respectively, indicated that

AgNPs might decrease rate of metabolism during storage period and which may increase shelf

life of fruits (Figure 7).

Fig. 7: Soluble Protein Concentration

In addition to reduction in weight loss of fruits and antimicrobial activity, AgNPs also showed

potent dye degradation capability. Congo red degradation was observed higher in AgNPs

synthesized from A. niger (51%) than only A. niger filtrate (45%) (Fig. 8). Similar results were

reported by Nithya [20] where the AgNPs from Pleurotus sajor caju was used and by Swetha S.

[28] where AgNPs from Phaseolus vulgaris was used to bring about extensive degradation of

Congo red. It suggested that synthesized AgNPs can used to degrade hazardous dyes from textile

industries. Congo red is a benzidine-based dye and benzidine has been classified by IARC as

Group 1 carcinogen. It is a recalcitrant and a known carcinogen.




Fig. 8: Dye decolorization after 48 hrs incubation

CONCLUSION

Aspergillus niger isolated from spoiled bread sample has shown potential for extracellular

synthesis of AgNPs having size of 7 nm. The results observed in present study emphasize on

novel application of biologically synthesized AgNPs in preservation of fruits and enhancement in

shelf life of fruits; as well as provide cheap and eco-friendly way of hazardous dye degradation.

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