world journal of pharmaceutical sciences a study on …wjpsonline.org/admin/uploads/spgzfa.pdf ·...

*Corresponding Author Address: Dr. I. Arulpandi, Research Department of Microbiology, Asan Memorial College of Arts and Science,

Jaladampet, Chennai – 600100, Tamilnadu, India; E-Mail: [email protected]

World Journal of Pharmaceutical Sciences ISSN (Print): 2321-3310; ISSN (Online): 2321-3086

Published by Atom and Cell Publishers © All Rights Reserved

Available online at: http://www.wjpsonline.org/

Original Article

A study on evaluation of antimicrobial property of biologically synthesized silver and

zinc oxide nanoparticles against human pathogens

Vidya Pradeep and I. Arulpandi*

Research Department of Microbiology, Asan Memorial College of Arts and Science, Jaladampet, Chennai –

600100, Tamilnadu, India

Received: 11-09-2016 / Revised: 24-10-2016 / Accepted: 26-10-2016 / Published: 31-10-2016

ABSTRACT

In the present study, antimicrobial activity of silver (Ag NPs) and Zinc oxide (ZnO NPs) nanoparticles

synthesized from Pichia fermentans were tested against common human pathogens. The nanoparticles were

biosynthesized and characterized by XRD, TEM, EDX and FTIR analysis. The average size of Ag NPs was

60nm and ZnO NPs was 28nm. The study on antimicrobial activity of individual and combined nanoparticles

showed that the ZnO NPs had greater antimicrobial activity than Ag NPs. The combined Ag NPs and ZnO NPs

showed greater antimicrobial properties than their individual performance.

Key Words: Nanoparticles, Silver nanoparticles, Zinc oxide nanoparticles, Pichia fermentans

INTRODUCTION

One of the major threats in health care industry is

the emergence of microbial resistance to various

antibiotics, and other treatment methods [1].

Researchers have tried to develop new, effective

antimicrobial agents that are free of resistance and

cost. Such problems needs to the resurgence in the

use of Nanoparticle-based treatments that may be

linked to broadspectrum activity and far lower

propensity to induce microbial resistance than

antibiotics. Inorganic nanocrystalline metal oxides

such as Zinc oxide (ZnO) are particularly

interesting because they can be prepared with

extremely high surface areas, and are more suitable

for biological applications. The inorganic

antibacterial materials have advantages over

organic antibacterial materials that the former

shows superior durability, less toxicity and greater

selectivity and heat resistance [2]..

Recently, need for designing new materials with

improved properties have forced fast development

of nanostructured materials, especially

nanocomposites. Thus, researches have been

focused on investigation of materials at the atomic,

molecular and macromolecular level, with the aim

to understand and manipulate the features that are

substantially different from the processing of

materials on micro-scale [3]. Polymer-based

nanocomposites, with inorganic nanoparticles

dispersed in polymer matrix, are interesting

because of their improved properties, simple

processing steps and relatively low costs [4].

However, newly developed nanocomposites with

bactericidal properties occupy considerable

attention in recent years, not only due to their

impact on human health and safety but also due to

the possibility of extended lifetime of materials

used in everyday life. Possible applications of these

materials are very broad: i) different types of sterile

materials are important in hospital, where often

wounds are contaminated with microorganisms, in

particular fungi and bacteria [5], ii) purification of

water, i.e. the removal or inactivation of pathogenic

microorganisms, is necessary for the treatment of

wastewater, etc [6]. During the past few decades,

several investigations have been carried out

concerning the use of polymer films, synthetic and

natural zeolites and particles with different metal

ions (Ag, Cu, Zn, Hg, Ti, Ni, Co) as materials with

bactericidal properties [6,7].

Among inorganic antibacterial agents, silver

nanoparticles have been employed most

extensively. Since it liberates silver ions in liquids

that shows a broad spectrum of antimicrobial

activities [8]. The mechanism of antimicrobial

effect of silver is still not fully understood. It is

believed that DNA loses its replication ability and

cellular proteins become inactivated upon treatment

Vidya and Arulpandi, World J Pharm Sci 2016; 4(11): 183-194

184

with silver ions. In addition, it has also been shown

that silver ions bind to the functional groups of

proteins, resulting in protein denaturation [9].

It is also believed that ZnO nanoparticles have

bactericidal properties primarily due to its

photocatalytic activity. The main advantage of

using ZnO nanoparticles are its excellent stability

and long shelf life with organic antimicrobial

agents [10]. In particular, the antimicrobial

properties of nanoscale ZnO particles have been the

focus of industrial applications in biocides coating

in water treatment, paints and cosmetic products

[11]. ZnO in its nanoscale form has a strong

toxicity towards a wide range of micro-organisms

including bacteria, fungi, fish, algae and plants

[12]. Of the inorganic antibacterial materials, metal

oxides such as zinc oxide (ZnO) have received

increasing attention in recent years, not only

because their stablity under harsh processing

conditions, also they are generally regarded as safe

materials to human beings and animals [13, 14].

Recent studies have shown ZnO have selective

toxicity to bacteria but exhibit minimal effect on

human cells [15, 16].

In the present study, the silver and Zinc oxide

nanoparticles were biologically synthesized using

yeast Pichia fermentans. The nanopariticles were

characterized and subjected to evaluation of in vitro

antimicrobial activity against common human

pathogens. The antimicrobial activity was

investigated for individual activity of ZnO NPs and

Ag NPs, and also in the combined state of both

nanoparticles.

MATERIALS AND METHODS

The yeast strain Pichia fermentans was procured

from culture bank, Research Department of

Microbiology, Asan Memorial College, Chennai.

The yeast strain was cultivated using sterile

Sabouraud’s Dextrose broth. The strain was

characterized by microscopic and biochemical

tests.

The active Pichia fermentans culture was freshly

inoculated in sterile Sabouraud’s Dextrose broth

and incubated in room temperature at 200 rpm for

3 days. After the incubation period, the broth

culture was centrifuged 12000 rpm for 10 min for

cell separation. The clear supernatant was collected

without cell debris and the cells free supernatant

was used to synthesize the nanoparticles.

Biosynthesis of Silver Nanoparticles: The

collected supernatant (1 %) was added to conical

flask containing 1 mM Silver nitrate (AgNO3) and

was incubated in a rotator shaker for 3 hrs and

centrifuged at 10000 rpm for 10 minutes. The

supernatant was discarded and the pellet was

washed thrice using deionised water by

centrifugation. Finally, the pellet was dried at 60°C

for 2 hours and collected in a vial and stored for

further use.

Biosynthesis of Zinc oxide Nanoparticles: 50 ml

of the culture solution was added with equal

volume (50ml) of sterile distilled water in a sterile

conical flask and gently heated in a steam bath for

15 min and incubated overnight in an orbital

shaker. After incubation, Sodium bicarbonate was

added till the pH reached 8 and then 20ml of Zinc

chloride was added. The flasks were heated on a

steam bath upto 80°C for 5-10min. Cloudy

haziness in culture solution and white deposition

was appeared at the bottom of the flask. The flask

was further incubated for 9hrs till white clusters

deposited at the bottom of the flask. The solution

was centrifuged at 2000 rpm for 20 min. The

supernatant was discarded and the pellet was

washed thrice with sterile distilled water, air dried

and stored for further use.

Characterization of Nanoparticles: Synthesized

AgNPs and ZnONPs were characterized by

Scanning Electron Microscope (SEM), Fourier

Transform Infrared Spectroscopy (FTIR) and X-ray

Diffraction (XRD). The characterization was

carried out at Sophisticated Instrumention Facility,

IIT, Chennai.

X-ray diffraction (XRD) analysis: The X-

ray diffraction was carried out using a ISO

Debyeflex (2002) INELCPS with a resolution of

120 and the intensity was 2θ. The nanoparticles

redispersed sterile deionized water, freeze dried,

and the topology was analyzed by X-ray

diffraction.

Fourier Transmission Infrared (FTIR)

Spectroscopy analysis: FTIR analysis was carried

out using a BRUKER RFS system with a scan

range of MIR 50-4000 cm-1 and with the resolution

of 2.0cm-1. The sample was mixed with potassium

bromide and ground well with a pestle and mortar.

Then the mixture was made to a pellet and

analysed.

Scanning Electron Microscopy (SEM) analysis: Scanning Electron Microscope analysis was carried

out using a FEI Quanta FEG200, Netherland with a

magnification maximum of 10,000x. The filtrate

embedded with nanoparticles was dried under

vacuum and subjected to SEM studies.

Energy- dispersive x-ray (EDX) spectroscopy

analysis: Energy-dispersive X-Ray (EDX)


185

spectroscopy analysis was carried out using a

EDAX for the confirmation of elements in the

sample.

Evaluation of Antimicrobial Activity of

nanoparticles: The common human pathogenic

microbes such as Escherichia coli, Klebsiella sp.,

Proteus sp., Pseudomonas sp., Shigella sp.,

Salmonella sp., Steptococcus sp, Staphylococcus

sp., Yeast Candida sp. and fungi Aspergillus sp.,

Penicillium sp., Mucor sp., and Rhizopus sp. were

selected for the study The bacterial cultures were

enriched in sterile nutrient broth and incubated

overnight at 37°C and the other fungal and yeast

strains were enriched in sterile Sabouraud’s

dextrose broth at room temperature for 36 hours.

Sterile Mueller Hinton agar plates were swabbed

with overnight broth culture. Three wells of 5mm

size was made using well sterile puncture device.

Thirty microliter of nanoparticles (100µg/ml

Dimethyl sulfoxide) solution was added in the test

well, Dimethyl sulfoxide was added in the second

well as negative control and phenol (1%) solution

was added in the third well as positive control. The

bacterial plates were incubated in an upright

position at 37°C for 24hrs and the fungal plates

were incubated at room temperature for 36 hours.

After incubation, zone of inhibition was observed.

The diameter of the inhibition zones were

measured in mm and the results were recorded. The

experiments were performed separately for

individual activity of Silver nanoparticles, Zinc

nanoparticles and mixed nanoparticles.

RESULTS AND DISCUSSION

Yeast strain: Pichia fermentans, the yeast

commonly found on fruit surfaces like grapes was

selected for the study since it has the ability to

synthesize both silver and zinc nanoparticles

extracellularly [17]. The colonies of Pichia

fermentans in agar medium was cream colored

and ovoidal in shape (Figure 1&2) and fermented

Glucose and assimilated D-Xylose, Succinate,

Lactose, Citrate.

Nanoparticles: The silver nanoparticles

synthesized from Pichia fermentans appeared as

pale brown colour during synthesis and it appeared

blackish brown crystalline powder with metallic

shining after drying (Figure.3A) [5]. During

biosynthesis of ZnO nanoparticles, white cloudy

haziness was observed in the solution which

deposited at the bottom of the flasks as

nanoparticles. The biosynthesized zinc oxide

nanoparticles were white powdery crystal and

insoluble in water (Figure. 3B). The colour and

texture of the particles may vary based on the

method of synthesis [15].

Characterization of Nanoparticles

X-ray diffraction (XRD) analysis: The XRD

pattern of silver nanoparticles indicated that the

presence of three diffraction peaks, which agreed

well with 111, 200 and 220 diffractions confirmed

topology of silver nanoparticle (Figure.4A) [5].

The XRD pattern of the zinc oxide nanoparticles

showed peaks in the whole spectrum of 2θ values

ranging from 10-70. The XRD peaks indicated that

the material consist of particles in nanoscale range.

The diffraction peaks located at 100, 002, 101, 102,

110, 103, 200, 112 and 004 indicated the hexagonal

wurtzite phase of ZnO (Figure. 4B). The intensity

of the peaks increased with the calcination

temperature, indicating increased crystallinity.

Further, it also confirmed that the synthesized

nanoparticles were free of impurities as it does not

contain any characteristic XRD peaks other than

ZnO peaks [18,19]. Thus, the results of XRD

diffraction peaks of Silver and Zinc oxide

nanoparticles showed in a good agreement with

results reported in JCPDS file.

Fourier Transmission Infrared (FTIR)

Spectroscopy analysis of zinc oxide

nanoparticles: In the FTIR spectra of silver

nanoparticles, the absorption bands centered at

1076, 1384, 1631, 3433cm-1 is associated with

vibration and assigned to ester linkages (Figure.

5A). The peak at 1631cm-1 corresponding to amide

I, arising due to carbonyl stretch vibrations in the

linkage of the protein the peak at 3433 cm-1 refers

to the stretching vibration of primary amines

[20,21]. FTIR spectra of ZnO nanopartilces

exhibited prominent peaks at 543, 885, 1633 and

3399 cm-1. The absorption peak at 543 cm-1

indicated the presence of Zinc oxide nanoparticles

(Figure.5B) [18].

Scanning Eelectron Microscopy (SEM) analysis

of Zinc oxide nanoparticles: The SEM analysis

was performed to measure the size of silver

nanoparticles of about 42µm (Figure. 6A).

Generally the size of the silver nanoparticle will be

1-100µm in size. In several reports the size of

synthesized nanoparticles was found to be 20-

60nm and they are spherical and rectangular with

curved edges and well distributed in solution

[20,22]. The SEM study of ZnO nanoparicles

revealed that the shape was irregular, rod shaped

and with the average size of 28 nm (Figure. 6B)

[23,24].

Energy Dispersive x-ray (EDX) spectroscopy

analysis: EDX spectroscopy analysis was

performed to confirm the presence of elementals in

nanoparticles. In silver nanoparticles, the optical

absorption band peak showed 3Kev which is

typical for the absorption of metallic silver


186

nanocrystallites (Figure.7A) [25]. The EDAX result

of ZnO nanoparticles indicated the presence of

elements Zn and O with the indication of sharp

signals (Figure.7B). The weight percentage was

found to be 88.73% and 11.27% for Zn and O

respectively [23,26].

Antimicrobial studies: The antimicrobial

properties of individual silver nanoparticles showed

maximum activity against the bacteria E.coli sp.

followed by Klebsiella sp. and fungi Penicillium

sp. and showed no activity against Candida sp

(Figure.8). In the studies of Kim et al. [5], AgNPs

showed antimicrobial activity against E. coli and S.

aureus where the E. coli inhibited even at low

concentrations, while the inhibitory effect on the

growth of S. aureus was less. AgNPs have been

shown to have definitely an effective antimicrobial

property against E. coli, S. typhi, Staphylococcus

epidermidis and S. aureus (Figure.9).

The ZnO nanoparticles showed greater inhibitory

activity against the bacterial strains E.coli,

Klebsiella sp. and Proteus, and the fungal strains

Rhizopus sp. followed by Penicillium sp. and

Aspergillus sp. (Figure.10) Comparatively

(Figure.8), the ZnO nanoparticles have shown

higher antimicrobial activity than Silver

nanoparticles. ZnO NPs were relatively well

dispersed with slight agglomeration in water.

Concentration and size are two important factors

affecting antimicrobial properties of ZnO NPs.

During the synthesis processing of ZnO NPs, they

may exist in the form of agglomerates [27].

Therefore, ultrasonication and dispersants, such as

polyethylene glycol (PEG), polyvinylpyrolidone

(PVP) and bovine serum albumin (BSA) are often

used to disintegrate nanoparticle agglomerates [15].

In the study of Yamamoto et al., [28] , the presence

of reactive oxygen generated by ZnO nanoparticles

is responsible for their bactericidal activity. Zhang

et al., [27] reported that the antibacterial behaviour

of ZnO nanoparticles could be due to chemical

interactions between hydrogen peroxide and

membrane proteins, or between other chemical

species produced in the presence of ZnO

nanoparticles and the outer lipid bilayer of bacteria.

The hydrogen peroxide produced enters the cell

membrane of bacteria and kills them.

The cumulative activity of Silver and Zinc

nanoparticles have showed enhanced activity in

the bacterial strains Shigella sp. and Salmonella

sp., and fugal strain Mucor sp. and Rhizopus sp.

than their individual activity (Figure.11).

CONCLUSION

The present study proved the ability of Pichia

fermentans to biosynthesize both Ag NPs and Zno

NPs. The Ag NPs showed greater antimicrobial

activity against common human pathogens E.coli

sp., Klebsiella sp. and Penicillium sp. and ZnO NPs

showed against E.coli sp., Klebsiella sp., Proteus

sp. and Rhizopus sp. The combined activity of Ag

NPs and ZnO NPs showed higher inhibitory

activity than individually. Hence it is concluded

that these nanoparticles in combination may be

used as antimicrobial agents.

ACKNOWLEDGEMENT

The authors express their gratitude to Sophisticated

Analytical Instrumentation Facility, Indian Institute

of Technology, Chennai India for the nanoparticles

characterization study.

Figure.1 Pichia fermentans in PDA plate


187

Figure.2 Morphology of Pichia fermentans in negative staining

Figure.3 Biosynthesized nanoparticles

(A) Silver Nanoparticles

(B) Zinc Oxide nanoparticles


188

Figure.4 XRD spectrum of nanoparticles



Figure.5 FTIR spectrum of Nanoparticles



189


Figure. 6 SEM analysis of Nanoparticles



190



191

Figure 7. EDAX profile of nanoparticles



Figure.8 Antimicrobial properties of nanoparticles


192

Figure.9 Antimicrobial properties of Ag NPs

E.coli Klebsiella sp. Penicillium sp

NC – Negative Control PC-Positive control

Figure.10 Antimicrobial properties of ZnO NPs

E.coli Klebsiella sp. Candida sp.



193

Figure.11 Antimicrobial properties of mixed Ag NPs and ZnO NPs

Shigella sp. Salmonella sp.

Mucor sp. Rhizopus sp.


REFERENCES

1. Edmond MB. Vancomycin-resistant Staphylococcus aureus Perspectives on Measures needed for control. Annual Int Medica

1996; 124: 329-334

2. Nagarajan P, Rajagopalan V. Enhanced bioactivity of ZnO nanoparticles- anantimicrobial study. Scientific Technol Adv

Materiol 2008; 7(4): 354-358.

3. Bhattacharyya et al. Nanotechnology, Big things from a Tiny World: a Review. Int J Sci and Technol 2009; 2: 3-8

4. Gurjas K et al. Nanotechnology: A Review. IJEAR 2012; 2(1):50-54. 5. Kim JS et al. Antimicrobial effects of silver nanoparticles. Nanomedi 2007; 3: 95–101.

6. Jain P, Pradeep T. Potential of silver nanoparticle coated polyurethane foam as an antibacterial water filter. Biotechnol Bioeng

2005; 90:59–63. 7. Shearer AEH et al. Potential of an antibacterial ultraviolet irradiated nylon film. Biotechnol Bioeng 2000; 67: 141–146.

8. Kawashita M et al. Antibacterial silver containing silica glass prepared by sol–gel method. Biomater 2000; 21:393–398.

9. Feng QL et al. A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J Biomed Mater Res 2000; 52: 662–668.

10. Kolodziejczak RA, Jesionowski T. Zinc Oxide-From Synthesis to Application. Materials 2014; 7: 2833-2881.

11. Allahverdiyev AM et al. Coping with antibiotic resistance: combining nanoparticles with antibiotics and other antimicrobial agents. Expert Rev Anti Infect Ther 2011; 9: 1035-1052.

12. Huang Z et al. Toxicological effect of ZnO nanoparticles based on bacteria. Langmuir 2008; 24: 4140-4144.

13. Stoimenov PK et al. Metal oxide nanoparticles as bactericidal agents. Langmuir 2002; 18: 6679–6686. 14. Fu G et al. Anatase TiO2 nanocomposites for antimicrobial coatings. J Phys Chem B 2005; 109: 8889–8898.

15. Brayner R et al. Toxicological impact studies based on Escherichia coli bacteria in ultrafine ZnO nanoparticles colloidal

medium. Nano Lett 2006; 6: 866–870. 16. Thill A. Cytotoxicity of CeO2 nanoparticles for Escherichia coli. Physico-chemical insight of the cytotoxicity mechanism.

Environ Sci Technol 2006; 40: 6151– 6156.

17. Ritika C et al. Biosynthesis of silver and zinc oxide nanoparticles using Pichia fermentans JA2 and their antimicrobial property. Appl Nanosci 2015; 5(1): 63–71.


194

18. Selvarajan E, Mohanasrinivasan V. Biosynthesis and characterization of ZnO nanoparticles using Lactobacillus planatarum VITES07. Mater Lett 2013; 112: 180-182.

19. Talam S et al. Synthesis, characterization and spectroscopic properties of zinc oxide nanoparticles. Int Schol Res Net 2012 ;

10(12): 1-6 20. Monali G et al. Fungus-mediated synthesis of silver nanoparticles and their activity against pathogenic fungi in combination with

fluconazole. Nanomed 2009; 5(4) : 382–386.

21. Narasimha GB et al. Mushrooms (Agaricus bisporus) mediated biosynthesis of sliver nanoparticles,characterization and their antimicrobial activity. Int J Nano Dimens 2011; 2(1): 29-36.

22. Nithya R, Ragunathan R. Synthesis of silver nanoparticle using Pleurotus sajor caju and its antimicrobial study. Dig J

Nanomater Biostruc 2009;4 (4):623 – 629. 23. Kumar S et al. Synthesis, characterization and optical properties of zinc oxide nanoparticles. Int Nano Lett 2013; 3(30): 1-6.

24. Wilhelmi V et al. Zinc Oxide Nanoparticles induce Necrosis and Apoptosis in Macrophages in a p47 phox and Nrf2 independent

Manner. Plos One 2013; 8(6): 1-15 25. Manish D et al. Green synthesis of nanosilver particles from extract of Eucalyptus hybrida leaf. Dig J Nanomater Biostru 2009;

4(3):537 – 543.

26. Renugadevi K et al. In vitro evaluation of antibacterial activity and cytotoxicity effect of chemically synthesized ZnO nanoparticle. Int J Pharma Sci Res 2012; 3(8): 2639-2643

27. Zhang LL et al. Investigation into the antibacterial behaviour of suspensions of ZnO nanoparticles (ZnO nanofluids). J Nanopart

Res 2007; 9: 479–489 28. Yamamoto O. Influence of particle size on the antibacterial activity of zinc oxide. Int J Inorg Mater 2001; 3:643–68.

world journal of pharmaceutical sciences a study on …wjpsonline.org/admin/uploads/spgzfa.pdf ·...

Documents