THE STUDY OF ANTIMICROBIAL ACTIVITY OF AgNPs MADE BY USING GREEN SYNTHESIS AGAINST SPECIFIC MICROORGANISMS

This work is focused on studying the antimicrobial effect of silver nanoparticles made by green synthesis. For the green synthesis, extracts from Salvia officinalis were used. Four different temperatures (20, 40, 60, 80 °C) were used to prepare the extracts from the plant material. The antimicrobial capability of the AgNPs was assessed using different yeasts (Saccharomyces cerevisiae, Zygosaccharomyces bailii), gram-positive (Lactobacillus plantarum, Listeria innocua, Staphylococcus Aureus) and gram-negative (Escherichia coli, Acetobacter aceti) bacterial strains. The range of AgNPs concentrations 0-2,500 μg/mL were tested. The antimicrobial effect was studied using two methods: 1) the viable cell count method and 2) the inhibition zone method. The method of direct counting showed a small inhibitory effect (inhibition at concentrations 1,250 and 2,500 μg/ml) only for E. coli, Z. bailii and S. cerevisiae. The inhibition zone results displayed better antibacterial activity of the silver nanoparticles (inhibition at all concentrations 50, 100, 150 and 200 μg/mL) prepared by using the extract obtained at 20 oC against E. coli and S. aureus.


INTRODUCTION
The process of green synthesis in nanoparticle production has become a more and more popular alternative to chemical synthesis in recent years. It is a simple, inexpensive and environmentally friendly method for producing nanoparticles. Biosynthesised nanoparticles show high biocompatibility and antimicrobial, antioxidant, antidiabetic, anticancer and other effects, and thus have the potential to be widely used in many fields [1,2].
Silver nanoparticles produced by green synthesis are abundantly studied for their antimicrobial properties [3,4]. This is primarily based on the biological matrix of the sample. Parameters in the production of nanoparticles such as temperature, pH, extraction time and individual production processes play an important role [5,6].
Salvia officinalis, used in our work as a reducing agent, contains a significant amount of antioxidant components. It contains essential oils, tannins, phenolic substances and flavonoids [7,8]. The production of essential oils, which are contained in the leaves, is significant [9].
This work aimed to determine the inhibitory effect of AgNPs on selected bacteria and yeasts. In the second part, the aim was to test the effect of AgNPs prepared at various temperatures against S. aureus and E. coli. https://doi.org/10.37904/nanocon.2019.8766

Preparation of AgNPs by green synthesis
Preparation of AgNPs by green synthesis is shown in Figure 1 A. Plant materials (Salvia officinalis) were dried at 60 °C for 48 h and homogenised by grinding to dust. For the preparation of the plant extract, the mixture was stirred in water (20, 40, 60, 80 °C) for 60 minutes in a ratio of 1:10 followed by centrifugation (15 min; 4,000 g). The extract was mixed with 0.1 M AgNO3 (1:1, 24 h), and the prepared particles were purified with methanol (1:1; 1 h). After precipitation, the methanol was removed, and the AgNPs were dried at 60 °C for 24 h in VWR (Radnor, Pensylvania, USA) dryer (model VDL23). The purified AgNPs were dispersed in a medium (purified water 18 MΩ or a mixture of water/acetone) by using ultrasound (40 minutes until a homogenous mixture was obtained) to obtain a stock solution of nanoparticles of 10 mg/mL.

Culture conditions and strains
The reference strains for the first experiment (S.

Antimicrobial susceptibility assays
For the viable cell count method: The antimicrobial activity of the AgNPs prepared by green synthesis was determined by the microdilution method [10]  For the inhibition zone method [11]: Petri dishes with agar medium were covered by 50 µL of bacteria suspension (OD620 = 0.01). AgNPs (c = 10 mg/mL) dispersion was pipetted into a Petri dish in amounts of 5, 10, 15 and 20 µL. This resulted in variants with concentrations of 50, 100, 150 and nd of 200 µg/mL. The bacteria were cultivated for 12 h at 37 °C. After 12 h of incubation, photographs were taken, and the total area of the inhibition zone in cm 2 was calculated in the LIS programme (Mediapro, s.r.o., Brno, Czech Republic). When the inhibition zone began to overgrow with bacterial colonies, a constant value of 0.01 cm 2 was selected.

Viable cell count method results
This method allows for identifying the number of viable cells in a given culture. Specifically, it involves counting colonies that are visible to the naked eye. Table 1 shows the microbial counts of the studied bacteria and yeast in the presence of different concentrations of the AgNPs.
The results of the study show us that the silver nanoparticles have a relatively small antimicrobial effect. This occurred only in the bacterial culture of E. coli and yeast cultures of Z. bailii and S. cerevisiae. The effect was observed only at concentrations of 1,250 and 2,500 µg/mL, and in E. coli only at concentrations of 2,500 µg/mL.

Zone inhibition results
In the next part of the experimental work, the effect of the AgNPs on E. coli and S. aureus was studied using the zone inhibition method. In this method, the size of the area around each disc indicates the antimicrobial effect of the individual samples of AgNPs. For this, nanoparticles produced at 20, 40, 60 and 80 °C were used and pipetted into Petri dishes at concentrations of 50, 100, 150 and 200 µg of AgNPs/mL (Figures 2A, 2B). Box plots comparing the area of the inhibition zones of AgNPs to S. aureus and E. coli. The purified water was used as a control. Inhibitor zone sizes ranged from 0.01 cm 2 to 1.5 cm 2 . The best antibacterial effect on S. aureus was found with the AgNPs prepared by using the extract that was prepared at 20 °C (value 0.9 cm 2 at concentration 200 µg/mL). The best effect on E. coli also had AgNPs prepared by using the extract at 20 °C (value 1.5 cm 2 at concentration 200 µg/mL). A better antibacterial effect of AgNPs was obtained against E. coli.

CONCLUSION
In this experimental work, silver nanoparticles were prepared using green synthesis. In the first part of the experimental work, when the 100 µL nanoparticle solution was spread on Petri dishes and subsequently calculated by CFU, we were able to show a small inhibitory effect only in the bacterial culture of E. coli and yeast cultures of Z. bailii and S. cerevisiae.
In the second part of the experiment, four different amounts (50, 100, 150 and 200 µg of AgNPs/mL) produced at four different temperatures (20, 40, 60 and 80 °C) were pipetted onto the Petri dishes. The detection of differences in the size of the inhibition zones in the preparation at different temperatures was critical. The best antibacterial effect to E. coli and also S. aureus was found with the AgNPs prepared by using the extract that was prepared at 20 °C. In the future we anticipate the study of antimicrobial activity using different extraction times of the biological matrix and other solvents.