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Green methods have become vital for sustainable development of the scientific and commercial sphere; however, they can bring new challenges, including the need for detailed characterization and elucidation of efficacy of their products. In this study, green method of silver nanoparticles (AgNPs) production was employed using an extract from grapevine canes. The aim of the study was to contribute to the knowledge about biosynthesized AgNPs by focusing on elucidation of their antifungal efficiency based on their size and/or hypothesized synergy with bioactive substances from Vitis vinifera cane extract. The antifungal activity of AgNPs capped and stabilized with bioactive compounds was tested against the opportunistic pathogenic yeast Candida albicans. Two dispersions of nanoparticles with different morphology (characterized by SEM-in-STEM, DLS, UV-Vis, XRD, and AAS) were prepared by modification of reaction conditions suitable for economical production and their long-term stability monitored for six months was confirmed. The aims of the study included the comparison of the antifungal effect against suspension cells and biofilm of small monodisperse AgNPs with narrow size distribution and large polydisperse AgNPs. The hypothesis of synergistic interaction of biologically active molecules from V. vinifera extracts and AgNPs against both cell forms were tested. The interactions of all AgNPs dispersions with the cell surface and changes in cell morphology were imaged using SEM. All variants of AgNPs dispersions were found to be active against suspension and biofilm cells of C. albicans; nevertheless, surprisingly, larger polydisperse AgNPs were found to be more effective. Synergistic action of nanoparticles with biologically active extract compounds was proven for biofilm cells (MBIC80 20 mg/L of polydisperse AgNPs in extract), while isolated nanoparticles suspended in water were more active against suspension cells (MIC 20 mg/L of polydisperse AgNPs dispersed in water). Our results bring new insight into the economical production of AgNPs with defined characteristics, which were proven to target a specific mode of growth of significant pathogen C. albicans.

Rhamnolipids are extensively studied biosurfactants due to their potential in many industrial applications, eco-friendly production and properties. However, their availability for broader application is severely limited by their production costs, therefore the optimization of efficacy of their cultivation gains significance as well as the information regarding the physio-chemical properties of rhamnolipids resulting from various cultivation strategies. In this work, the bioprocess design focused on optimization of the rhamnolipid yield of Pseudomonas aeruginosa DBM 3774 utilizing the response surface methodology (RSM). Six carbon sources were investigated for their effect on the rhamnolipid production. The RSM prediction improved the total rhamnolipid yield from 2.2 to 13.5 g/L and the rhamnolipid productivity from 11.6 to 45.3 mg/L/h. A significant effect of the carbon source type, concentration and the C/N ratio on the composition of the rhamnolipid congeners has been demonstrated for cultivation of P. aeruginosa DBM 3774 in batch cultivation. Especially, changes in presence of saturated fatty acid in the rhamnolipid congeners, ranging from 18.8% of unsaturated fatty acids (carbon source glycerol; 40 g/L) to 0% (sodium citrate 20 g/L) were observed. This demonstrates possibilities of model based systems as basis in cultivation of industrially important compounds like biosurfactants rhamnolipids and the importance of detailed study of interconnection between cultivation conditions and rhamnolipid mixture composition and properties.

The ever-growing range of possible applications of nanoparticles requires their mass production. However, there are problems resulting from the prevalent methods of nanoparticle production; physico-chemical routes of nanoparticle synthesis are not very environmentally friendly nor cost-effective. Due to this, the scientific community started exploring new methods of nanoparticle assembly with the aid of biological agents. In this study, ethanolic Vitis vinifera cane extract combined with silver nitrate was used to produce silver nanoparticles. These were subsequently characterized using UV-visible (UV-Vis) spectrometry, transmission electron microscopy, and dynamic light-scattering analysis. The antimicrobial activity of produced nanoparticles was tested against the planktonic cells of five strains of Gram-negative bacterium Pseudomonas aeruginosa (PAO1, ATCC 10145, ATCC 15442, DBM 3081, and DBM 3777). After that, bactericidal activity was assessed using solid medium cultivation. In the end, nanoparticles’ inhibitory effect on adhering cells was analyzed by measuring changes in metabolic activity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay - MTT). Our results confirmed that ethanolic Vitis vinifera cane extract is capable of mediating silver nanoparticle production; synthesis was conducted using 10 % of extract and 1 mM of silver nitrate. The silver nanoparticles’ Z-average was 68.2 d.nm, and their zeta potential was –30.4 mV. These silver nanoparticles effectively inhibited planktonic cells of all P. aeruginosa strains in concentrations less than 5 % v/v and inhibited biofilm formation in concentrations less than 6 % v/v. Moreover, minimum bactericidal concentration was observed to be in the range of 10–16 % v/v. According to the results in this study, the use of wine agriculture waste is an ecological and economical method for the production of silver nanoparticles exhibiting significant antimicrobial properties.

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