INTRODUCTION:
Nanoparticles (Nps) are small, 1 micrometer in at least one dimension. These particles can be spherical,tubular or irregularly shaped. A nanoparticle is considered as nanomaterial when at least two dimensions are between 1and100 nanometers. Different types of nanomaterials like copper, zinc, titanium, magnesium, gold, alginate and silver have come up but silver nanoparticles have proved to be most effective as it has good antimicrobial efficacy against bacteria, viruses and other eukaryotic microorganisms. One of the most studied aspects of nanotechnology nowadays is their ability to offer the opportunity to fight microbial infections via synthesis of nanoparticles. The mechanism of prevention of bacterial growth by antibiotics is quite different from the mechanisms by which nanoparticles inhibit microbial growth. The antimicrobial activity of silver nanoparticles appears significantly high.
Size of nanoparticle is directly proportional to the atoms. Nanoparticles exhibit quantum mechanical behavior. They are also called artificial atoms because free electrons in them behaves in a way similar to electrons bound by atoms in that they can occupy only certain permitted energy states (Logeswari et al., 2012).
Metal nanoparticles are synthesized by several physical and chemical methods such as physical vapour deposition, ion implantation method, electrochemical techniques, chemical reduction, photochemical reaction, etc. But all these process may not be ecofriendly. So green chemistry method uses of microorganisms are considered novel as it is ecofriendly and also cost effective. In these methods no toxic chemicals are employed and also there is no need of using high temperature, pressure and energy. Microbes such as bacteria, fungi and actinomycetes are used for the synthesis of silver nanoparticles (Gavhane et al., 2012).
Human beings are often infected by microorganisms such as bacteria, molds, yeasts, and viruses present in their living environments. Because of the emergence and increase in the number of multiple antibiotic-resistant microorganisms and the continuing emphasis on health-care costs, many scientists have researched methods to develop new effective antimicrobial agents that overcome the resistances of these microorganisms and are also cost-effective. Such problems and needs have led to resurgence in the use of silver-based antiseptics that may be linked to a broad-spectrum activity and considerably lower propensity to induce microbial resistance compared with those of antibiotics. In particular, silver ions have long been known to exert strong inhibitory and bactericidal effects as well as to possess a broad spectrum of antimicrobial activities (Kushwaha and Malik, 2013).
It has a broad application in medical field also due to its broad spectrum antimicrobial activity. Scientists in the University of Hong Kong say that the antibacterial effect of silver nanoparticles is due to its penetration of microbial cell wall and alteration of DNA. A new generation of wound dressing incorporating silver nanoparticles is being formulated to reduce or prevent infections. Silver nanoparticles have been found to promote faster wound healing. The particles can be incorporated in materials and cloth rendering them sterile.
The coating of medical instruments is one recent silver nanoparticles medical application in study. A combination of the bacteriolytic action of lysozyme and biocidal activity of silver nanoparticles synthesized together as a form of stainless surgical steel blades and needles through an electrophoretic process. The treated instruments were observed to have a powerful bactericidal action against K. pneumonia, B. anthracis Sterne, and B. subtilis.
The use of silver nanoparticles as antibacterial agent is relatively new. Because of their high reactivity due to the large surface to volume ratio, nanoparticles play a crucial role in inhibiting bacterial growth in aqueous and solid media.
Actinobacteria will provide a valuable resource for novel products of industrial interest, including antibacterial agents. Thus, in the present study an attempt was carried out to see the antibacterial activity of silver nanoparticles synthesized from the isolated marine derived Streptomyces. The aim of this study is to biosynthesize silver nanoparticle from Streptomyces and confirmation using UV spectrophotometer. Characterization study was performed using SEM and FTIR. The antimicrobial activity of the produced silver nanoparticles was checked against pathogens.
MATERIALS AND METHODS:
Sample collection
Soil sampleswere collected from different locations in Pudukkottai, Tamil Nadu, S. India. The collected samples were carefully stored in polythene bags and transported to the laboratory for the experiment.
Isolation of Actinomycetes
Isolation of actinobacteria was performed by traditional serial dilution and plating technique using actinomycetes isolation agar medium Selvakumar et al., (2012). One gram of the soil sample was suspended in 25 ml sterile water in a conical flask, stirred thoroughly with the help of a glass rod and left for some time.
Distilled
water (9 ml) was taken in each of 7 test tubes and labeled from 1to7. The
supernatant liquid from the dissolved soil sample was transferred into the test
tubes so as to achieve the serial dilutions of 10-1 to
10-7. 0.1 ml of the diluted sample was inoculated in the
Actinomycetes isolation agar medium plates from each dilution of 10-4,
10-5 and 10-6. The petriplates are then rotated to spread
the sample uniformly. Plates were then incubated at room temperature (28 to 30
) for 7 days.
Preparation of culture
Actinomycetes
isolated from soil were grown in yeast malt broth at 37
C for 5 days in a shaking incubator at 200 RPM (Hemath
Naveen et.al., 2010). After 5 days of incubation, the bacterial mass
was separated with the help of a centrifuge and the pellet was washed thrice
with sterile distilled water. The pellet was resuspended in sterile distilled
water and incubated in a shaking incubator for 24 hrs.
Biosynthesis of Silver Nanoparticles
After incubation it was again centrifuged and the supernatant was collected and with that 1mM silver nitrate was added and incubated in a dark place at room temperature for 3 days. After the incubation period it was observed for the colour change. The colour change from white to brown showed the synthesis of silver nanoparticles (Gaikwad Sagar and Bhosale Ashok, 2012).
Characterization of Silver Nanoparticles
Biologically synthesized silver nanoparticles underwent for characterization by UV- visible spectrophotometer, Fourier transform infrared (FTIR), and Scanning Electron Microscopy (SEM). A scanning electron microscope was used to record the micrograph images of synthesized silver nanoparticles. The interaction between protein and silver nanoparticles was analysed by Fourier transform infrared (FTIR) analysis. The FTIR spectrum of the dried sample was recorded in the range of 450 to 4000cm-1.The bio reduction of Ag+ ions in solution was monitored by optical measurements, which were carried out by usingUV- visible spectrophotometer and scanning the spectra between 200-800 nm at the resolution of 1nm.
Determination of Antimicrobial activity
Antibacterial activity was assayed using standard well diffusion method against human pathogen bacteria collected from a diagnostic lab (Guangquan Liet. al., 2012). Muller – Hinton agar plates were prepared and the test organisms (B. subtilis, P. auginosa, E. coli ) were swabbed evenly on the medium. With a sterile borer 1 mm holes were punched in the medium for the test organisms to be loaded.
In a plate 4 wells of 1 mm were cut out. In the first well (a) 20µl of actinomycetes synthesized solution containing silver nanoparticles was filled and in the rest of the wells named b, c and d were loaded with antibiotic (chloramphenicol), sterile distilled water and pure culture of actinomycetes respectively.
In a second plate 40µl of the test solution with all the other set up being the same was also performed. Both the plates then were incubated for 24 hrs. After incubation the zone of inhibition was measured. Control plates were also maintained with all the set up being same except the test organisms being mated on the surface of the medium.
RESULT AND DISCUSSION:
In the UV- Vis absorption spectrum, a strong, broad peak located at 426nm was absorbed. Observation of this peak, assigned of this peak, assigned to surface Plasmon, is well documented for various metal nanoparticles with sizes ranging from 2-100 nm (Fig. 1).
Fig. 1. UV- Vis absorption spectrumof Nps
The result obtained from the SEM image gave the clear shape and size of the Ag-NPs produced. The diameter of the Ag-NPs in the solution was found to be in the range of 20-100nm (Fig.2).
Fig. 2.Scanning electron microscopyof Nps
Fig.3 FTIR measurement of Nps
FTIR measurements were carried out to identify possible interaction between silver salts and protein molecules, which could account for the reduction of silver ions and stabilization of silver nanoparticles formed after 72 hrs. The amide linkages between amino acid residues in proteins give rise to the well known signatures in the infrared region of the electromagnetic spectrum. Peaks are as follows 3297.26, 2982.39, 2934.34, 2356.90, 1732.82, 1653.94, 1539.43, 1454.09 1382.21, 1285.93, 1225.37, 1185.83, 1134.00, 1096.16, 1055.63, 976.15, 900.51, 829.02 and 621.59 (Fig.3).