Nanotechnologies are involved in finding new inexpensive, rapid and safe solutions for synthesis of nanoparticles, especially silver and gold nanoparticles (1, 2, 3), efficient against spoiling or pathogenic microorganisms producing important economic loss in agriculture and food industry or seriously affecting plants, animals and human health (4, 5, 6, 7, 8).
A lot of research is devoted to biosynthesis of silver nanoparticles (AgNPs) with antimicrobial properties, mediated by various natural sources such as extracts of plant parts (root, stem, leaves, seeds) (9, 10, 11), algae (12,13), bacteria (14, 15) and fungi (16, 17) as an eco-friendly, low-cost and simple alternative to hazardous toxic and expensive chemicals (18, 19). Thus, while physical and chemical synthesis methods use scarce and expensive sources, the biosynthesis of metal nanoparticles can contribute to sustainable development goals (20). Biogenic metal nanoparticles are produced by various microorganisms by either intracellular or extracellular mechanism (21). In the first case, ions are transported into the cell and reduced to their elemental form through electrostatic and enzymatic interaction, with formation of nanoparticles within the microbial cell. Extracellular mechanism of biosynthesis is mediated either by enzymatic reduction at the cell surface or by secreted molecules that reduce metal ions into their elemental form (22).
Literature reports data concerning with biomolecules, polysaccharides acting as chelating/reducing agents and capping agents for the synthesis of nanoparticles, utilized for preventing aggregation, increasing stability and longevity of the biosynthesized nanoparticles. Considerable efforts have been done to obtain biosynthesized nanoparticles with controlled morphology, small dimensions, uniformity and stability, characteristics favorable to a high antibacterial and antifungal activity (23, 24, 25). Research has been carried out to bring new information for clarifying the mechanisms of antimicrobial action of biosynthesized AgNPs (26), very useful for medicine against clinical pathogens (27, 28).
The goal of the present paper was to present the results of the research carried out on extracellular biosynthesis of silver nanoparticles mediated by culture filtrate of a lactic acid bacteria strain and to assess the antimicrobial activity.
Materials and Methods
Materials and microorganisms
Lactic acid bacteria strain LCM5 (with origin in brined cucumbers) was cultivated on liquid media MRS broth (purchased from Liofilchem Italy) in test tubes at 360C for 24 or 48 hours (29).
The four test microorganisms, represented by mycotoxigenic fungi from species Aspergillus ochraceus, Aspergillus flavus and Penicillium expansum were isolated from contaminated bread, pickles and apples and pathogenic Gram-negative bacteria Chromobacterium violaceum was isolated from soil.
Biogenic synthesis of silver nanoparticles
Extracellular synthesis of silver nanoparticles was accomplished by mixing 50ml cell free supernatant from 48 hours liquid culture of lactic acid bacteria strain LCM5 (filtered through membrane filter with 0.2μm pore dimension) with 50ml aqueous solution of 1mM silver nitrate (AgNO3). The mixture was incubated in Erlenmeyer flasks on orbital shaker (200 rpm) at 28±20C in the dark for 5 days. A flask with cell free supernatant without AgNO3 was run along with experimental flask and utilized as control (30).
Visual analysis and spectrophotometric characterization of silver nanoparticles
Synthesized nanoparticles color in liquid was visually monitored for changing towards yellowish to brown after adding AgNO3, consequently to completion of reaction, the phenomenon being produced by excitation of Surface Plasmon vibration in silver nanoparticle (31). Aliquots of AgNPs solution (incubated in dark for 24 hours) were taken and optical density (O.D.) was read to a Carl-Zeiss Jena Spectrophotometer at wavelength between 400 and 500 nm against deionized water as blank.
Transmission electron microscopy (TEM) analysis
The size, shape and dispersion of silver nanoparticles synthesized were analyzed by Transmission electron microscopy (TEM) using a JEM – 1400 (Jeol) microscope with an accelerating voltage of 80kV. Three samples were prepared for imaging by drop-coating silver nanoparticles solution on carbon-coated copper TEM grid 40 mm x 40 mm mesh size, air-drying and loading on specimen holder (32). TEM images for each sample were taken at various magnifications and particle size distribution was performed by computer assisted analysis of representative images and grouping the particles counted in categories, according to diameter. The diameter was calculated as average value of two perpendicular diameters for particles assumed as circular or as average of minor and major axes (33).
Antimicrobial activity of silver nanoparticles
Antifungal activity of silver nanoparticles biosynthesized was assessed according to agar well diffusion method (34) against isolates of Aspergillus ochraceus, Aspergillus flavus and Penicillium expansum. Antibacterial activity was assessed against gram negative bacteria Chromobacterium violaceum.
30 mL of biosynthesized AgNPs were added in wells of 6 mm diameter in Petri plates with potato-dextrose-agar (PDA) or nutrient agar (NA) media (Merk KGaA, Germany) previously inoculated with test-microorganisms (fungi and respectively bacteria strain). Distilled water and AgNO3 were added in equal quantities in wells as control and plates were incubated at 25°C for 5 to7 days. The diameter of zone of inhibition around the well was measured.
The experiment was performed in triplicate.
Visual analysis and spectrophotometric characterization of silver nanoparticles
After 24 hours incubation of cell free culture with AgNO3 the color gradually changed to dark brown, indicating the production of silver nanoparticles (due to reduction of Ag+ to Ag0 mediated by enzyme nitrate reductase) and no color change appeared in control without silver ion Ag+(Fig.1).
The color of biosynthesized nanoparticles was dark brown due to the excitation of Surface Plasmon vibration in silver nanoparticle. The maximum absorption peak (optical density) occurred at the wavelength of 420nm, as resulted from spectrophotometric analysis.
Transmission electron microscopy (TEM) analysis of AgNPs Silver nanoparticles synthesized using culture filtrates of lactic acid bacteria Lactobacillus sp. strain LCM5 were polydisperse, presented approximately spherical shape, with clear parallel lattice fringes, confirming their crystalline nature. They were embedded in a matrix (acting as capping agent to prevent agglomeration of nanoparticles) as evidenced by transmission electron microscopy (TEM) (Fig.2).
Analysis of TEM micrographs evidenced that the size of AgNPs synthesized using culture filtrates of lactic acid bacteria Lactobacillus sp. strain LCM5 ranged between 3 and 35 nm, with an average particle size of 13.84±4.56 nm.
The particles size distribution of silver nanoparticles showed high values and similar (30%) for 5-10 nm and 10-15 nm categories (Fig.3).
Frequency distribution revealed that preponderant dimensions of biosynthesized AgNPs were below 20 nm (94%).
Antimicrobial activity of silver nanoparticles
Silver nanoparticles synthesized with culture filtrate of lactic acid bacteria Lactobacillus sp. strain LCM5 exerted an inhibitory effect against all microbial strains tested. The effectiveness (inhibition zone) as provided by agar well diffusion method and presented in Table 1, was variable depending on both species and the group of microorganisms (bacteria or fungi) involved.
Antimicrobial activity of silver nanoparticles
|Test microorganism||Inhibition zone (mm) Ag NO3||Inhibition zone (mm) Ag NPs|
|Aspergillus flavus||9.00 ± 1.06c*||12.39 ± 0.61c|
|Aspergillus ochraceus||7.00 ± 0.37d||12.86 ± 0.78c|
|Penicillium expansum||11.00 ± 0.46b||15.87 ± 1.01b|
|Chromobacterium violaceum||16.00 ± 0.67a||18.00 ± 0.69a|
Diameter of growth inhibition zone of Aspergillus flavus (Fig.4) and Aspergillus ochraceus (Fig. 5) caused by silver nanoparticles synthesized by culture filtrate of lactic acid bacteria Lactobacillus sp. strain LCM5 were similar (12.39 ± 0.61 mm and 12.86 ± 0.78 mm) but significantly stronger inhibition was registered against Penicillium expansum, with inhibition zone diameter of 15.87 ± 1.01 mm (Fig.6).
Antibacterial effect of silver nanoparticles against Chromobacterium violaceum was evidenced with larger growth inhibition zone diameter (18 ± 0.69 mm) when compared to those from fungi (Fig. 7).
Even though both chemical AgNO3 and biogenic AgNPs presented antimicrobial activity against test-microorganisms in the assay, the nanoparticles biosynthesized using culture filtrates of lactic acid bacteria Lactobacillus sp. strain LCM5 were more efficient and induced higher values of inhibition zone diameter in the case of bacteria than in the case of fungi.
The results of the research carried out demonstrated the extracellular synthesis of silver nanoparticles mediated by culture filtrate of Lactobacillus sp. strain LCM5. The color change to dark brown as a consequence of reduction of silver ions with formation of AgNPs and the maximum absorption peak occured at 420 nm weavelength were similar to those occured for AgNPs synthesized by other culture filtrates of lactic acid bacteria from genus Lactobacillus (35, 36) or using aqueous leaf extracts (37), with maximum absorption peaks at 430 nm and 437 nm weavelengths.
Biosynthesized AgNPs presented a good dispersion, approximately spherical shape, with parallel stripes certifying crystal structure. The preponderant dimensions of the AgNPs were below 20 nm, with an average particle size of 13.84±4.56 nm. Unlike chemical or physical synthesis, biosynthesis mediated by bacteria culture filtrate presents the advantage to be rapid, eco-friendly (minimal waste generating) and energy efficient as confirmed by similar research (38). Recent highly efficient (92.4% yield) chemical synthesis of AgNPs by one-pot low cost process using monoethanolamine as a strong reducing agent and poly (acrilic acid) as stabilizing agent was reported, with well dispersed, spherical particles of 14.83±5.96 nm and catalytic properties (39). A synthesis of chemical, physical and biological methods for obtaining AgNPs with various shapes and dimensions (from 2 to 300 nm) was presented in an article comparing advantages and disadvantages of these methods and the applications of the nanoparticles in different domains (40).
It is well known that the high frequency of small AgNPs confer the advantage of larger surface of contact with test-microorganisms, increasing their reactivity comparatively with larger AgNPs (41).
Higher intracellular bioavailability of AgNPs associated with increased in vitro toxicity for various microbial cells (bacteria, yeasts) was hypothesized to be due to more efficient cell-particle contact of small nanoparticles (10nm) as compared to larger ones (42, 43).
Our results are in concordance with data from research carried out on AgNPs biosynthesized using culture filtrates of lactic acid bacteria strains showing also prominently spherical shape and diameters ranging from 2-20 nm to 20-40 nm, as a function of strain (36, 44). Similar research on lactobacilli found that the ability to reduce the silver ions was strain specific, in most cases the diameter of spherical nanoparticles measured by TEM analysis was between 10 and 40 nm (89.6%) whereas 0.7% were smaller than 10 nm and 9.7% were greater than 40 nm. The monosaccharide composition of capsular heteropolysaccharides from different Lactobacillus strains could explain the ability to reduce the silver ions and produce AgNPs with different sizes and size distribution. The differences in antibacterial activity of AgNPs assayed, as in our experiment, by agar well diffusion method were explained by authors as influenced by the capsular biopolymer composition (45). Also, a recent study (46) presented the biosynthesis of AgNPs for biomedical applications, mediated by extracellular pigment produced by Thalaromyces purpurogenus, with particles size from 4 to 41 nm diameter, polydispersed and stable, exhibiting a surface plasmon resonance at around 410 nm but a more variable shape of nanoparticles (spherical, hexagonal, rod-shaped or triangular) than in the present research. As in our research, the authors reported a higher number of particles within 10 nm range and a lesser number of larger particles. Green synthesized AgNPs presented higher antibacterial activity against Gram-positive and Gram-negative species assessed by comparing with the effect of Streptomycine.
Similar results with those from our bioassay showing a significant higher antimicrobial activity of biosynthesized AgNPs as compared with AgNO3, were reported by other authors (47) for antifungal activity of biosynthesized AgNPs (assessed by agar well diffusion method, against Candida spp.) as compared with AgNO3.
In the present research, the antimicrobial activity of biosynthesized AgNPs maintained over 14 days as illustrated by the aspect of Petri dishes with Chromobacterium violaceum, Aspergillus flavus, Aspergillus ochraceus and Penicillium expansum. The presence of zone of inhibition visible over 7 days was considered as a bactericidal and respectively fungicidal effect, as stated in assays of antimicrobial activity of bacteriocin-producing lactic acid bacteria strains (48).
Recent research evidenced the antibacterial and anti-quorum sensing activity of phytosynthesized silver nanoparticles against violacein producing bacteria Chromobacterium violaceum (49) or other human pathogenic species of genera Escherichia, Staphylococcus, Pseudomonas and Bacillus, with zones of inhibition of 4-18 mm as a function of species and the concentration of AgNPs (50). In the last case, the AgNPs ranged in size from 37 to 43 nm (higher values in diameter as compared with our findings for AgNPs synthesized from bacteria strain) and exhibited varying levels of inhibition of violacein production. Other research (51) with similar results were obtained using agar well diffusion method to assess the antibacterial effect of phytosynthesized AgNPs against various bacteria species causing human urinary tract infections (UTI). The authors reported significant inhibition of all UTI pathogens, with the maximum antibacterial activity of AgNPs synthesized from ginger (zone of inhibition of 18 mm against Staphylococcus spp.).
Results from literature reported zones of inhibition from 15 to 20 mm diameter of six fungal species of Aspergillus and Fusarium caused by antifungal action of AgNPs synthesized using culture filtrates of Aspergillus foetidus strain MTCC8876 (52) and powerful antifungal activity of mycosynthesized AgNPs against Penicillium digitatum, Aspergillus flavus and Fusarium oxysporum in vitro (53).
Other research reported AgNPs synthesis mediated by species of genus Lactobacillus and their antibacterial action against Gram-positive and Gram-negative human pathogenic species was evidenced by variable growth inhibition zones of 16 to 24 mm in diameter, as a function of species (54). Antifungal action of biogenic AgNPs synthesized using culture filtrates of Escherichia coli and Bacillus subtilis was suggested to be produced by penetration of AgNPs into the fungal cell and inducing cellular death of clinical isolates of Candida albicans, Trichophyton rubrum and Aspergillus fumigatus (55). Zones of inhibition varying from 8 mm to 14 mm caused by antifungal action of silver nanoparticles biosynthesized using Allium ampeloprasum leaf extract were reported for five mycotoxigenic isolates of Aspergillus (56).
Recent reviews of antimicrobial activity of AgNPs obtained by green synthesis mediated by various plants and microbes, assessed mainly by agar well diffusion method (57, 58) are consistent with the results from our study concerning the size and shape of AgNPs, and present comparable values reflecting antibacterial and antifungal action.
Results of the present research evidenced the antimicrobial activity of silver nanoparticles biosynthesized using culture filtrate of the bacterial strain LCM5 and recommend it for utilization in biotechnological strategies against pathogenic and food spoilage microorganisms.
Silver nanoparticles biosynthesis was accomplished using culture filtrates of lactic acid bacteria Lactobacillus sp. strain LCM5 and aqueous solution of 1 mM AgNO3.
Transmission electron microscopy (TEM) analysis showed a good dispersion of biosynthesized silver nanoparticles, approximately spherical shape, with parallel stripes certifying crystal structure and preponderant dimensions below 20 nm.
Silver nanoparticles biosynthesized using culture filtrate of Lactobacillus sp. strain LCM5 presented antifungal activity against Penicillium expansum, Aspergillus flavus, Aspergillus ochraceus, the diameter of zone of inhibition differing depending on fungal species.
Antibacterial activity of biosynthesized silver nanoparticles has been observed against Chromobacterium violaceum with more extended zone of inhibition.
Antimicrobial activity of biosynthesized silver nanoparticles was maintained more than 14 days, confirming their bactericidal and fungicidal effect.
Results recommend the silver nanoparticles biosynthesized using culture filtrate of Lactobacillus sp. strain LCM5 for biotechnological purposes, as antimicrobial agents.
The results were obtained under the frame of European Social Fund, Human Resources Development Operational Program 20072013, project no. POSDRU/159/1.5/S/132765 and Research Program NUCLEU, the project PN 19 34 04 01/2019.
The authors thank to engineer Alexandru Brînzan from Institute of Biology, Bucharest for technical support and expertness in obtaining TEM images of AgNPs.
Rai M, Ingle AP, Birla S, Yadav A, Santos CAD. Strategic Role of Selected Noble Metal Nanoparticles in Medicine. Crit Rev Microbiol 2016; 42: 696-719.
Awah JI, Ukwuru MU, Alum EA, Kingsley TL. Bio-preservative potential of lactic acid bacteria metabolites against fungal pathogens. Afr J Microbiol Res 2018; 12 (39): 913-922. on- line, DOI: 10.5897/AJMR2018.895
Jalal M, Ansari MA, Alzohairy MA, Ali SG, Khan HM, Almatroudi A, Siddiqui MI. Anticandidal activity of biosynthesized silver nanoparticles: effect on growth, cell morphology, and key virulence attributes of Candida species. Int J Nanomedicine 2019; 14: 4667-4679.
Krishnaraj C, Jagan EG, Rajasekar S, Selvakumar P, Kalaichelvan PT, Mohan N, 2010. Synthesis of silver nanoparticles using Acalypha indica leaf extracts and its antibacterial activity against water borne pathogens. Coll Surf B Biointerfaces 2010; 76: 50-56.
Singh R, Shedbalkar UU, Wadhwani SA, Chopade BA. Bacteriagenic silver nanoparticles: synthesis, mechanism, and applications. Appl Microbiol Biotechnol 2015; 99: 4579-4931. Doi: 10.1007/ s00253-015-6622-1
Khalil N. Biogenic silver nanoparticles by Aspergillus terreus as a powerful nanoweapon against Aspergillus fumigatus Afr J Microbiol Res 2013; 7(50): 5645-5651.
Ishida K, Cipriano T, Rocha GM, Weissmüler G, Gomez F, Miranda K, Rozental S. Silver nanoparticle production by the fungus Fusarium oxysporum nanoparticle characterization and analysis of antifungal activity against pathogenic yeasts. Mem Inst Oswaldo Cruz 2014;109(2): 220-228.
Ingale AG, Chaudhari AN. Biogenic synthesis of nanoparticles and potential applications: An eco-friendly approach. J Nanomed Nanotechnol 2013; available online at: http://www.omicsonline.org/2157-7439/2157-7439-4-165.digital/2157-7439-4-165.html
Raileanu-Plugaru V, Pomastowski P, Meller K, Zloch M, Rafinska K, Buszewski B. Lactococcus lactis as a safe and inexpensive source of bioactive silver composites. Appl Microbiol Biotechnol 2017; 101(19): 7141-7153. Doi:10.1007/s00253-017-8443-x
Cueva M, Horsfall L. The contribution of microbially produced nanoparticles to sustainable development goals. Microb Biotechnol 2017; 10: 1212-1215.
Marooufpour N, Alizadeh M, Hatami M, Lajayer BA. Chapter 3. Biosynthesis of nanoparticles by different groups of bacteria. In: R Prasad (ed.). Microbial Nanobionics Nanotechnology in the Life Sciences, Springer Nature Switzerland AG 2019: 63-85. available online at: https://doi.org/10.1007/978-3-030-16383-9_3
Hulkoti N, Taranath TC. Biosynthesis of nanoparticles using microbes. Coll Surf B Biointerfaces 2014; 121: 474-483.
Prabhu S, Poulose EK. Silver nanoparticles: mechanism of antimicrobial action, synthesis, medical applications and toxicity effects. Int Nano Lett 2012; available online at: http://link.springer.com/article/10.1186/2228-5326-2-32/fulltext.html
De Man JC, Rogosa M, Sharpe ME. A medium for the cultivation of lactobacilli. J. Appl. Bacteriol 1960; 23: 130.
Saifudin N, Wong W.C, Nur Yasumira AA. Rapid biosynthesis of silver nanoparticles using culture supernatant of bacteria with microwave irradiation. CODEN ECJHAO E-J Chem 2009; 6(1): 6170.
Singh P, Raja RB. Biological synthesis and characterization of silver nanoparticles using the fungus Trichoderma harzianum Asian J Exp Biol Sci 2011; 2(4): 600-605.
Dwivedi AD, Gopal K. Biosynthesis of silver and gold nanoparticles using Chenopodium album leaf extract. Colloids Surf A Physicochem Eng Asp 2010; 369(1-3): 27-33.
Woehrle G, Hutchinson J, Ozkar S, Finke R. Analysis of nanoparticle transmission electron microscopy data using a public-domain image-processing program, Image Turk J Chem 2006; 30: 1-13.
Roy K, Sarkar CK, Gosh CK. Apium graveolens leaf extract-mediated synthesis of silver nanoparticles and its activity on pathogenic fungi. Dig J Nanomat Biostruct 2015; 10(2): 393-400.
Thiruneelakandan G, Vidya S, Vinola J, Jayasudha S, Babu V, Sevasundhari L, Sivakami R, Anthoni SA. Antimicrobial activity of silver nanoparticles synthesized by marine Lactobacillus sp against multiple drug resistance pathogens. Sci Technol Arts Res J 2013; 2(4):5-9.
Ranganath E, Rathod V, Banu A. Screening of Lactobacillus spp., for mediating the biosynthesis of silver nanoparticles from silver nitrate. IOSR J Pharm 2012; 2(2): 237-241.
Sani NJ, Aminu BM, Mukhtar MD. Eco-friendly synthesis of silver nanoparticles using Lactobacillus delbruckii subsp. bulgaricus isolated from kindrimoo (locally fermented milk in Kano state, Nigeria. Bayero J Pure Appl Sci 2017; 10(1): 481-488.
Hussain A, Alajmi M, Khan M, Pervez S, Ahmed F, Amir S, Husain F, Khan M, Shaik G, Hassan I, Khan R, Rehman MT. Biosynthesized silver nanoparticle (AgNPs) from Pandanus odorifer leaf extract exhibits anti-metastasis and anti-biofilm potentials. Front Microbiol 2019; available online at: https://doi.org/10.3389/fmicb.2019.00008
Nakamura S, Sato M, Sato Y, Ando N, Takayama T, Fujita M, Ishihara M. Synthesis and application of silver nanoparticles (AgNPs) for prevention of infection in healthcare workers. Int J Mol Sci 2019; 20, 3620, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6695748/
Auffan M, Rose J, Bottero JY, Lowry GV, Jolivet JP, Wiesner MR. Towards a definition of inorganic nanoparticles from an environmental, health and safety perspective. Nat Nanotech 2009; 4(10): 634-641.
Mavaani K, Shah M. Synthesis of silver nanoparticles by using sodium borohydride as a reducing agent. Int J Eng Res Technol 2013; 2(3): 1-5.
Prabhu SS, Mohan RK, Sanhita P, Ravindran R. Production of bacteriocin and biosynthesis of silver nanoparticles by lactic acid bacteria isolated from yoghurt and its antibacterial activity. Scrut Int Res J Microbiol Bio Technol 2014; 1(3): 7-14.
Garmasheva I, Kovalenko N, Voychuk S, Ostapchuk A, Livins’ka O, Oleschenko L. Lactobacillus species mediated synthesis of silver nanoparticles and their antibacterial activity against opportunistic pathogens in vitro BioImpacts 2016; 6(4): 219-223.
Paul S, Mohanram K, Kannan I. Antifungal activity of curcumin-silver nanoparticles against fluconazole-resistant clinical isolates of Candida species AYU 2018; 39(3): 182-186. available online at:https://wwww.ayujournal.org/text.asp?2018/39/3/182/255253
Adebayo CO, Aderiye BI. Antifungal activity of bacteriocins of lactic acid bacteria from some Nigerian fermented foods. Res J Microbiol 2010; 5: 1070-1082.
Hussain F, Shaban S, Kim J, Kim DH. One-pot synthesis of highly stable and concentrated silver nanoparticles with enhanced catalytic activity. Korean J Chem Engineering 2019; 36(6): 988-995.
Arunkumar M, Suhashini K, Mahesh N, Ravikumar R. Quorum quenching and antibacterial activity of silver nanoparticles synthesized from Sargassum polyphylum Bangladesh J Pharmacol 2014; 9: 54-59.
Sheikh S, Tale V. Green synthesis of silver nanoparticles: its effect on quorum sensing inhibition of urinary tract infection pathogens. Asian J Pharm Clin Res 2017; 10(5): 302-305.
Roy S, Mukherjee T, Chakraborty S, Kumar Das T. Biosynthesis, characterization & antifungal activity of silver nanoparticles synthesized by the fungus Aspergillus foetidus MTCC8876. Dig J Nanomat Biostruct 2013; 8(1): 197-205.
Al-Zubaidi S, Al-Ayafi A, Abdelkader H. Biosynthesis, characterization and antifungal activity of silver nanoparticles by Aspergillus niger isolate. J Nanotechnol Res 2019; 1(1): 023-036.
Salman JAS. Synthesis of silver nanoparticles by some locally Lactobacillus spp. and detection of their antibacterial activity. Al-Mustansiriyah J Pharm Sci 2013; 13(2):164-173.
Arjun TV, Bholay AD. Biosynthesis of silver nanoparticles and its antifungal activities. J Environ Res Develop 2012; 7(1A): 338-345.
Al-Zahrani SS, Al-Garni SM. Biosynthesis of Silver Nanoparticles from Allium ampeloprasum Leaves Extract and Its Antifungal Activity. J Biomat Nanobiotechnol 2019; 10(01): 11-25. https://file.scirp.org/pdf/JBNB_2019012317102817.pdf
Roy A, Balut O, Some S, Mandal Kumar A, Ylmaz D. Green synthesis of silver nanoparticles: biomolecule-nanoparticle organizations targeting antimicrobial activity. RSC Advances 2019; 9: 2673-2702.
Siddiqi KS, Husen A, Rao R. A review on biosynthesis of silver nanoparticles and their biocidal properties. J Nanobiotechnol 2018; 16: 14.
Data represent the mean of three replicates ± standard deviation; values in each column followed by the same letter are not significantly different for p<0.05 (Student test)