Biosynthesis of silver nanoparticles mediated by culture filtrate of lactic acid bacteria, characterization and antifungal activity

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.

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.