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[email protected] Current Biotechnology, 2013, 2, 000-000
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Synthesis of Silver Nanoparticles Using Flaxseed Hydroalcoholic Extract and its Antimicrobial Activity Archana A. Sharbidre1 and Deepak M. Kasote*,2 1
Department of Zoology, University of Pune, Pune 411007, MS, India
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Interactive Research School for Health Affairs (IRSHA), Bharati Vidyapeeth University, Pune- Satara Road, Dhankawadi, Pune – 411 043, MS, India Abstract: Agriculture industrial waste or byproducts could be a valuable biological material for synthesis of metal nanoparticles. Deoiled flaxseed meal is a byproduct of flaxseed oil industry. In present study its hydroalcoholic extract (70%) was assessed for the synthesis of silver nanoparticles. Synthesis of silver nanoparticles was confirmed and characterized by using UV-Vis spectrophotometer, X-ray diffraction (XRD), scanning electron microscope (SEM) and fourier transform infrared spectrum (FT-IR). The antimicrobial activity of silver nanoparticles was evaluated against gram-negative (E. coli), gram-positive (S. aureus) bacteria, and mycotoxin producing fungi A. flavus and A. parasiticus. The results of XRD, SEM analysis confirmed the face centered cubic structure of colloidal silver nanoparticles having particle size 9.22 nm. FT-IR analysis showed phenolic components of flaxseed hydroalcoholic extract could be responsible for the synthesis of silver nanoparticles. The resultant potent antimicrobial activity of synthesized silver nanoparticles could corroborate its usefulness in food and health product industry including flaxseed oil.
Keywords: Synthesis, silver nanoparticles, flaxseed, antimicrobial, mycotoxin producing fungi. 1. INTRODUCTION Several physical, chemical and biological methods have been developed for synthesis of metal nanoparticles. However, these days, biological methods have been receiving great interest for synthesis of metal nanoparticles due to their cost effective and eco-friendly nature. Plant materials [1,2], algae [3,4], fungi [5,6], bacteria [7,8], etc. have been used for synthesis of range of nanoparticles. However, amongst them plant based methods are simple to scale up and more economic [9,10]. Synthesis of metal nanoparticles like silver [11,12], gold [13,14], platinum [15], zinc [16], copper [17], titanium [18], Palladium [19], etc. has been carried out by using various plant extracts. In the middle of these metal nanoparticles, silver nanoparticles reported to have wide range biomedical applications. So far, wide range of bioactivities have been reported to plant mediated synthesised silver nanoparticles such as antioxidant [12], cytotoxic [20], antimicrobial [21], larvicidal [22] and feeding deterrent activity [23]. Flaxseed is a nutritionally important oil seed crop due to its high content of omega-3 fatty acid -linolenic acid (ALA), lignan secoisolariciresinol diglucoside (SDG) and fibers. Flaxseed contains about 36–40% of oil, rich in omega-3 fatty acid ALA (55-60%) [24,25]. Deoiled flaxseed meal primarily consists of proteins, fibers and bioactive phenolics [26, 27]. Faxseed bioactive phenolics mainly *Address correspondence to this author at the Interactive Research School for Health Affairs (IRSHA), Bharati Vidyapeeth University, Pune Satara Road, Dhankawadi, Pune – 411 043. MS, India; Tel: +91-020-24366929; Fax: +91-020-24366929; E-mail:
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include lignans, flavonoids, phenylpropan-oids, phenolic acids and tannins [28-33]. Thus far, plants with known ancient medicinal properties have always been a choice for synthesis of silver nanoparticles. Agriculture industrial waste or byproducts have been comparatively much less explored for the synthesis of silver nanoparticles. Deoiled flaxseed meal is a byproduct of flaxseed oil industry rich in phenolic compounds. These phenolics could be responsible for reduction of silver ion i.e. synthesis of silver nanoparticles owing to their good reducing power [32,34]. However, the role of these flaxseed phenolics in synthesis of silver nanoparticles is still obscure. Under this investigation, we have reported a simple method for the synthesis of silver nanoparticles using flaxseed hydroalcoholic extract; and antimicrobial potential of their silver nanoparticles especially against mycotoxin producing fungi like Aspergillus flavus and Aspergillus parasiticus. These fungi are commonly growing on oil seeds including flaxseed, and make them unsafe for consumption. 2. MATERIALS AND METHODS 2.1. Chemicals All chemicals used were of analytical grade, procured from Merck, India. Nutrient agar and potato dextrose broth were purchased from Hi-Media, Mumbai, India. 2.2. Preparation of Flaxseed Hydroalcoholic Extract Authenticated flaxseed of variety NL-97 was used for the preparation of hydroalcoholic extract. The flaxseed was first © 2013 Bentham Science Publishers
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demucilaged according to the method described by Marambe et al. [35]. Briefly, seeds were mixed with 0.5 M NaHCO3 (1:8, w/v) for 1 hr. Then, they were recovered by filtration with use of muslin cloth and washed thoroughly with distilled water. The washing treatment was repeated for two more times. Seeds were dried in an oven at 45 oC for 24 h. The dried demucilaged seeds were grinded into fine powder using a home style mixer grinder. Then, the powder was deoiled by stirring with n-hexane (1:4, w/v) at room temperature for 6 h. The n-hexane layer was separated out, and the resulting de-oiled flaxseed meal was air dried. The flaxseed hydroalcoholic extract was prepared by adding 70% aqueous ethanol to deoiled flaxseed meal (1:5, w/v). The resulting extract was filtered, solidified and stored in refrigerator at 4 oC for further studies. 2.3. Biosynthesis of Silver Nanoparticles For the synthesis of silver nanoparticles, 1 g of hydroalcoholic extract was dissolved in 1 ml of dimethyl sulphoxide (DMSO). 200 l of this dissolved extract was mixed with 100 ml of 1 mM AgNO3 solution and subjected to constant mixing in a rotary shaker at 30 oC. The formation of nanoparticles was indicated by a visual color change from light yellow to brown. Synthesis of silver nanoparticles was monitored by measuring UV –Visible absorption of small aliquot of reaction mixture at different time intervals. The reactions mixture was centrifuged at 10000 rpm for 10 min and the pellet was re-suspended in a small amount of sterilized double distilled water, re-centrifuged, dried and used for further chemical characterization. 2.4. Characterization of Silver Nanoparticles In order to determine the particle size, X-ray diffraction (XRD) analysis of the prepared silver nanoparticles was done using a PXRD, Bruker D8 Advanced X-ray diffractometer, Cu-K X-rays of wavelength =1.5406 Å and data was taken for the 2 range of 10° to 80°. The surface morphology of flaxseed silver nanoparticles was analyzed by using scanning electron microscope (SEM) of model JEOL JSM 6360A. Fourier transform infrared spectrum (FT-IR) analysis was carried out for functional groups characterization (JASCO FT/IR-6100).
Sharbidre and Kasote
37 oC for 24 h in an incubator. Finally the zones of inhibition were observed. 3. RESULTS AND DISCUSSION 3.1. Formation of Silver Nanoparticles Reduction of silver nanoparticles from aqueous AgNO3 solution was visually observed by color change. Addition of 0.2% hydroalcoholic extract of flaxseed to aqueous AgNO3 solution resulted in color change from colorless to brown. This color change was associated with the excitation of surface plasmon vibrations with the silver nanoparticles [36]. Formation of silver nanoparticles was monitored by measuring UV–Vis absorption of reaction mixture at different time intervals (Fig. 1). Resultant maximum absorption within the range 385-400 nm confirmed the synthesis of silver nanoparticles. The observed broadening UV peak could be due to the aggregation of silver nanoparticles, and confirmed that flaxseed hydroalcholic extract could not act as a capping agent. The absorption intensity of silver nanoparticles formation was found to be increased with time, maximum at 96 h. This increase in the absorbance values with time indicated that synthesis of silver nanoparticles is a time dependent process; and could be dependent on the availability or efficacy of reducing biomolecules. It has been well known that biomolecules such as polyphenols have the ability to take part in redox reactions, which is associated to hydroxyl (-OH) group present in their structure. Flaxseed hydroalcoholic extract is rich in polyphenols, and could be responsible for reduction of silver ions of AgNO3 salt. Numerous factors such as pH, silver ions concentration, temperature, ionic strength, presence of oxygen or organic matter, etc. have been found to be affected by the synthesis of nanoparticles not only at the beginning, but also during and at the end of the experiments. Hence, further experiments in the context of optimization of silver nanoparticle synthetic process are warranted.
2.5. Screening of Antimicrobial Property in Synthesized Nanoparticles Antibacterial activity of synthesized silver nanoparticles was carried out by agar well diffusion method against gramnegative (Escherichia coli), gram-positive (Staphylococcus. aureus) bacteria, and mycotoxin-producing fungi Aspergillus flavus and A. parasiticus. Pure cultures of bacterial and fungal microorganisms were obtained from NCIM, NCL, Pune, India. They were sub-cultured on nutrient agar and potato dextrose agar (PDA). The bacteria were grown on nutrient broth at 37 oC, whereas fugal pure cultures were grown on PDA plates at 30 oC for 24 hours. Approximately 7 mm diameter of well was made on nutrient agar and PDA plates. Aliquot of 100 μl of each microorganism was spread on respective culture plate using a sterile glass spreader. The volume 15 to 60 μl (1 mg/ml) of synthesized particles was inoculated to the well, and then the plates were incubated at
Fig. (1). UV–Vis absorption spectra of silver nanoparticles synthesized by treating 1 mM aqueous AgNO3 solution with 0.2% flaxseed hydroalcoholic extract at different time intervals (24, 48,72 and 96 h).
3.2. Characterization of Silver Nanoparticles XRD is commonly used for the determination of crystal structure and size of the nanoparticles. Fig. (2) reveals XRD pattern of biosynthesized silver nanoparticles by flaxseed hydroalcoholic extract. A number of strong peaks were
Synthesis of Silver Nanoparticles Using Flaxseed Hydroalcoholic Extract
observed at 38.17, 44.00, 64.43 and 77.37, which corresponded to the (111), (200), (220) and (311) Bragg reflections respectively. These reflections confirmed the face centered cubic (fcc) crystal structure of silver. Full width at half maximum (FWHM) data was used with Scherrer’s formula to determine the average particle size [37]. Scherrer’s equation is provided by, d = 0.9/COS
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at 1543 cm-1, confirmed the absence of protein. The peak at 1148 cm-1 (coupled C–C and C–O vibrations), indicated the presence of carbohydrate [38]. The broad absorption peaks from 3629 to 3280 cm-1 were corresponding to hydroxyl group. The observed presence of hydroxyl group could corroborate the presence of phenolics and phenolic glycosides. These phenolic compounds might be responsible for reduction of silver ions.
where, d = mean diameter of the nanoparticles, = wavelength of X-ray radiation source, = angular FWHM of the XRD peak at the diffraction angle . The estimated average particle size was approximately 9.22 nm.
Fig. (4). FT-IR spectroscopic analysis of flaxseed hydroalcoholic extract.
3.3. Antimicrobial Activity
Fig. (2). XRD pattern of biosynthesized silver nanoparticles by flaxseed hydroalcoholic extract.
Results of surface morphological and nanostructural studies of synthesized silver nanoparticles by flaxseed hydroalcoholic extract using SEM are summarized in Fig. (3). The SEM results showed the formation of aggregated crystalline silver nanoparticles. FT-IR spectroscopic studies were carried out in order to explore the possible bioreducing agents present in the flaxseed hydroalcoholic extract. Fig. (4) shows FT-IR spectra of flaxseed hydroalcoholic extract. The medium strong peak at 1504 cm-1 (ester O–CH3 stretch) and 1721 cm-1 (ester carbonyl C=O stretch), could represent the presence of lipids or amino acids. However, absence of peak
Oil seeds including flaxseed are known to be susceptible for microbial contamination especially of molds. Microbial contamination could have harmful effects on quality, safety of oil seeds and their products. Hence, these days the use of antimicrobial agents is increasing for preservation of food and health products. The antimicrobial potential of synthesized silver nanoparticles (1 mg/ml) was tested against E. coli, S. aureus, A. flavus and A. parasiticus at different volumes (15, 30 and 60 l). Crude flaxseed hydroalcoholic extract demonstrated weak antimicrobial activity against E. coli, S. aureus and A. flavus. However, it did not show any activity against A. parasiticus. Flaxseed silver nanoparticles showed augmented antimicrobial activity against E. coli, S. aureus, A. flavus and A. parasiticus than flaxseed hydroalcoholic extract (Table 1). The observed antimicrobial activity could be due to the binding of silver nanoparticles to cell wall and cell membrane components of microorganism, which may cause upsetting membrane permeability, respiration functions of the cell [39,40]. Interestingly and notably, flaxseed silver nanoparticles showed potent activity against A. flavus and A. parasiticus, mytotoxin producing fungi were reported to grow on oilseeds [41]. The potential
Fig. (3). Scanning electron microscopic (SEM) images of biosynthesized silver nanoparticles by flaxseed hydroalcoholic extract.
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Table 1.
Sharbidre and Kasote
Antimicrobial Activity of Flaxseed Hydroalcoholic Extract and Biosynthesized Silver Nanoparticles Zone of Inhibition (mm)
Microorganisms
Volume of Flaxseed Hydroalcoholic Extract (1 mg/ml)
Volume of Silver Nanoparticles (1 mg/ml)
30 l
15 l
30 l
60 l
Staphylococcus aureus
9 ± 1.4
9.5 ± 0.7
11.5 ± 0.7
16 ± 1.4
Escherichia coli
9 ± 0.0
10 ± 0.0
11 ± 1.4
19.5 ± 0.7
Aspergillus parasiticus
-
10.5 ± 0.7
12 ± 0.0
12.5 ± 2.1
Aspergillus flavus
8 ± 0.0
7.5 ± 0.7
12 ± 0.0
15.5 ± 0.7
All values are mean ± S.D.
antimicrobial activity of flaxseed silver nanoparticles could have commercial application in contamination free storage of oilseeds including flaxseed. However, further in detail broad antimicrobial studies are essential for effective use of flaxseed silver nanoparticles in various biomedical applications. 4. CONCLUSION Deoiled flaxseed meal is a byproduct of flaxseed oil industry which is generally rich in phenolic compounds. The hydroalcoholic extract could be an effective reducing agent for the rapid synthesis of silver nanoparticles. The finding of XRD, FT-IR and SEM characterization confirmed that flaxseed phenolics could be responsible for the synthesis of colloidal silver nanoparticles. The observed potent antimicrobial activity of silver nanoparticles against E. coli, S. aureus, A. flavus and A. parasiticus, established its usefulness in food and health product industry including flaxseed oil. CONFLICT OF INTEREST
[4]
[5]
[6] [7]
[8] [9]
[10]
[11] [12]
No conflict of interest to disclose. [13]
ACKNOWLEDGEMENTS AAS is thankful to Departmental Research and Developmental Grant, Department of Zoology, University of Pune for providing financial support to carry out this work. Authors are grateful to IISER, Pune and Department of Physics, University of Pune, India for providing analytical facilities. DMK is thankful to Prof. M. V. Hegde, IRSHA, Pune and Hrishikesh Joshi, IISER, Pune for their assistance during experiment.
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REFERENCES
[17]
[1]
[2]
[3]
MataY, Bar H, Bhui DK, Sahoo GP, Sarkar P, De SP, Misra A. Green synthesis of silver nanoparticles using latex of Jatropha curcas. Colloids Surf A: Physicochem Eng Aspects 2009; 348: 212-16. Cruz D, Falé PL, Mourato A, Vaz PD, Serralheiro ML, Lino ARL. Preparation and physicochemical characterization of Ag nanoparticles biosynthesized by Lippia citriodora (Lemon Verbena). Colloids Surf B: Biointerfaces 2010; 81: 67-73. Torres E, Blázquez ML, Ballester A, González F, Mu˜noz, JA. Gold (III) biosorption and bioreduction with the brown alga Fucus vesiculosus. J Hazard Mater 2009; 166: 612-18.
[15] [16]
[18]
[19]
Ogi T, Saitoh N, Nomura T, Konishi Y. Room-temperature synthesis of gold nanoparticles and nanoplates using Shewanella algae cell extract. J Nanopart Res 2010; 12: 2531-39. Ahmad A, Mukherjee P, Senapati S, Mandal D, Khan M, Kumar R, Sastry M. Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium oxysporum. Colloids Surf B: Biointerfaces 2003; 28: 313-18. Bhainsa K, D'Souza, S. Extracellular biosynthesis of silver nanoparticles using the fungus Aspergillus fumigates. Colloids Surf B: Biointerfaces 2006; 47: 160-64. Kalimuthu K, Babu R, Venkataraman D, Bilal M, Gurunathan S. Biosynthesis of silver nanocrystals by Bacillus licheniformis. Colloids Surf B: Biointerfaces 2008; 65: 150-53. Prakash A, Sharma S, Ahmad N, Ghosh A, Sinha P. Bacteria mediated extracellular synthesis of metallic nanoparticles. Int Res J Biotechnol 2010; 5: 71-9. Shankar S, Rai A, Ahmad A, Sastry M. Rapid synthesis of Au, Ag and bimetallic Au core–Ag shell nanoparticles using neem (Azadirachta indica) leaf broth. J Colloid Interf Sci 2004; 275: 496-502. Rajasekharreddy P, Rani P, Sreedhar B. Qualitative assessment of silver and gold nanoparticle synthesis in various plants: a photobiological approach. J Nanopart Res 2010; 12: 1711-21. Nagajyothi P, Lee K. Synthesis of plant-mediated silver nanoparticles using Dioscorea batatas rhizome extract and evaluation of their antimicrobial activities. J Nanomater 2011; 1-7. Dipankar C, Murugan S. The green synthesis, characterization and evaluation of the biological activities of silver nanoparticles synthesized from Iresine herbstii leaf aqueous extracts. Colloids Surf B: Biointerfaces 2012; 98: 112- 19. Kumar K, Paul W, Sharma C. Green synthesis of gold nanoparticles with Zingiber officinale extract: Characterization and blood compatibility. Process Biochem 2011; 46: 2007-13. Montes M, Mayoral A, Deepak F, Parsons J, Jose-Yacama´n M, Peralta-Videa J, Gardea-Torresdey J. Anisotropic gold nanoparticles and gold plates biosynthesis using alfalfa extracts. J Nanopart Res 2011; 13: 3113-21. Song J, Kwon E, Kim B. Biological synthesis of platinum nanoparticles using Diopyros kaki leaf extract. Bioprocess Biosyst Eng 2010; 33: 159-64. Dutta R, Nenavathu B, Mahesh K, Gangishetty M, Reddy A. Studies on antibacterial activity of ZnO nanoparticles by ROS induced lipid peroxidation. Colloids Surf B: Biointerfaces 2012; 94: 143- 50. Harne S, Sharma A, Dhaygude M, Joglekar S, Kodam K, Hudlikar M. Novel route for rapid biosynthesis of copper nanoparticles using aqueous extract of Calotropis procera L. latex and their cytotoxicity on tumor cells. Colloids Surf B: Biointerfaces 2012; 95, 284-88. Velayutham K, Rahuman A, Rajakumar G, Santhoshkumar T, Marimuthu S, Jayaseelan C, Bagavan A, Kirthi A, Kamaraj C, Zahir A, Elango G. Evaluation of Catharanthus roseus leaf extractmediated biosynthesis of titanium dioxide nanoparticles against Hippobosca maculata and Bovicola ovis. Parasitol Res 2011; DOI 10.1007/s00436-011-2676-x. Petla R, Vivekanandhan S, Misra M, Mohanty A, Nallani S. Soybean (Glycine max) Leaf Extract Based Green Synthesis of Palladium Nanoparticles. J Biomater Nanobiotech 2012; 3: 14-19.
Synthesis of Silver Nanoparticles Using Flaxseed Hydroalcoholic Extract [20]
[21]
[22]
[23]
[24] [25] [26]
[27]
[28]
[29]
Justin S, Finub J, Narayanan A. Synthesis of silver nanoparticles using Piper longum leaf extracts and its cytotoxic activity against Hep-2 cell line. Colloids Surf B: Biointerfaces 2012; 91: 212- 14. Sathishkumar M, Sneha K, Yun Y. Immobilization of silver nanoparticles synthesized using Curcuma longa tuber powder and extract on cotton cloth for bactericidal activity. Bioresource Technol 2009; 101: 7958-65. Santhoshkumar T, Rahuman A, Rajakumar G, Marimuthu S, Bagavan A, Jayaseelan C, Zahir A, Elango G, Kamaraj C. Synthesis of silver nanoparticles using Nelumbo nucifera leaf extract and its larvicidal activity against malaria and filariasis vectors. Parasitol Res 2011; 108: 693-702. Kamaraj C, Rajakumar G, Rahuman A, Velayutham K, Bagavan A, Zahir A, Elango G. Feeding deterrent activity of synthesized silver nanoparticles using Manilkara zapota leaf extract against the house fly, Musca domestica (Diptera: Muscidae). Parasitol Res 2011; DOI 10.1007/s00436-011-2689-5 Bozan B, Temelli F. Supercritical CO2 extraction of flaxseed. J Am Oil Chem Soc 2002; 79: 231-35. Kasote D, Badhe Y, Hegde M. Effect of mechanical press oil extraction processing on quality of linseed oil. Indus Crop Prod 2013; 42: 10-13. Herchi W, Arráez-Román D, Boukhchina S, Kallel H, SeguraCarretero A, Fernández-Gutierrez A. A review of the methods used in the determination of flaxseed components. Afr J Biotechnol 2012; 11: 724-31. Kasote D, Badhe Y, Zanwar A, Hegde M, Deshmukh K. Hepatoprotective potential of ether insoluble phenolic components from n-butanol fraction of defatted flaxseed meal against CCl4 intoxication in rats. J Pharm Bioallied Sci 2012; 4: 231-35. Luyengi L, Pezzuto J, Waller D, Beecher C, Fong H. Linusitamarin, new phenylpropanoid glucoside from Linum usitatissimum. J Nat Prod 1993; 56: 2012-15. Meagher L, Beecher G, Flanagan V, Li B. Isolation and characterization of the lignans, isolariciresinol and pinoresinol, in flaxseed meal. J Agric Food Chem 1999; 47: 3173-80.
Received: March 17, 2013
Current Biotechnology, 2013, Volume 2, No. 2 [30]
[31] [32]
[33]
[34] [35]
[36] [37] [38]
[39] [40]
[41]
Revised: June 22, 2013
5
Qiu S, Lu Z, Luyengi L, Lee S, Pezzuto J, Farnsworth N, Thompson L, Fong H. Isolation and characterization of flaxseed (Linum usitatissimum) constituents. Pharm Biol 1999; 37: 1-7. Stasevich O, Mikhalenok S, Kurchenko V. Isolation of secoisolariciresinol diglucoside from lignan-containing extract of Linum usitatissimum seeds. Chem Nat Compd 2009; 45: 21-23. Kasote D, Hegde M, Deshmukh K. Antioxidant activity of phenolic components from n-butanol fraction (PC-BF) of defatted flaxseed meal. Am J Food Tech 2011; 6: 604-12. Kasote D, Hegde M, Deshmukh K. HPTLC and LC-ESI-MS characterization of ether insoluble phenolic components from nbutanol fraction (EPC-BF) of defatted flaxseed meal. Int J Drug Dev Res 2011; 3: 126-30. Zanwar A, Hegde M, Bodhankar S. In vitro antioxidant activity of ethanolic extract of Linum usitatissimum. Pharmacologyonline 2010; 1: 683-96. Marambe P, Shand P, Wanasundara J. An in-vitro investigation of selected biological activities of hydrolysed flaxseed (Linum usitatissimum L.) proteins. J Am Oil Chem Soc 2008; 85: 1155-64. Mulvaney P. Surface plasmon spectroscopy of nanosized metal particles. Langmuir 1996; 12: 788-800. Dubey M, Bhadauria S, kushwah BS. Green synthesis of nanosilver particles from extract of Eucalyptus hybrida (safeda) leaf. Dig J Nanomater Bios 2009; 4: 537-43. Thygesen L, Løkkey M, Micklander E, Engelsen S. Vibrational microspectroscopy of food. Raman vs FT-IR. Trends in Food Sci Technol 2003; 14: 50-57. Morones J, Elechiguerra J, Camacho A, Katherine H, Juan B, Jose Tapia R, Miguel J. The bactericidal effect of silver nanoparticles. Nanotechnol 2005; 16: 2346-53. Kim J, Sung S, Moon S, Choi J, Kim J, Lee D. Antifungal effect of silver nanoparticles on dermatophytes. J Micro Biotechnol 2008; 18: 1482-84. Begum F, Samajpati N, Mycotoxin production on rice, pulses and oilseeds. Naturwissenschaften 2000; 87: 275-77.
Accepted: June 24, 2013
