## 3 Results and Discussion
### Anttimicrobial Activity  
NADES has attracted much attention in various fields of science, including the extraction of biologically active metabolites. However, publications on the biological activity of the solvents themselves are rare. The antimicrobial potential of NADES, especially after dilution with water, requires additional attention. The antimicrobial activity of the tested NADES against common opportunistic pathogens is presented in Table 2.  
All tested lactic acid-based NADES inhibited the growth of both Gram-positive and Gram-negative microorganisms (Table 2). NADES1 showed the lowest MIC (7.8 and 0.98 mg/mL) and MBC (15.6 and 1.95 mg/mL) for _E.coli_ and _S. aureus_, respectively, while minimal MIC and MBC for _P. aeruginosa_ were observed for NADES3. Some authors consider Gram-negative bacteria to be more sensitive to acid-based eutectic solvents [64]. In other studies, acid-based NADES has similar efficacy against Gram-positive and Gram-negative strains [65]. However, LA: ChCl (1:3) was not studied in the above-mentioned articles. The antimicrobial activity was correlated with water content in NADES and was decreased on average by 2-8 times after the addition of 40% water (Table 2). [PERSON] et al. (2018) also showed that adding water to the NADES composition leads to a loss of antimicrobial activity [66]. It was reported that citric acid-based NADES in combination with choline chloride showed more favorable antimicrobial activity than NADES with choline chloride in combinations with amine, alcohol, and sugar [64]. According to previous publications, organic acid-based NADESs possess higher antimicrobial activity due to increased acidity, which impairs the cell membrane and injuries cell protein [67; 68]. We found that pH has a sig  
Figure 7: Water activity of NADES after dilution with water. NADES1—LA:ChCl (3:1); NADES2—LA: SL (1:3); NADES3—LA:GL:W (3:5:1). nificant effect on antimicrobial activity, and _S. aureus_ was the most sensitive to the pH of NADES ([PERSON]'s correlation coefficient r = 0.804, \(p\) < 0.05). Although some components of the studied NADESs are used by bacteria as nutritional sources [69; 70], the literature data indicate that individual components such as LA show MIC from 1.25 mg/mL for _P. aeruginosa_ to 2.5 mg/mL against _E. coli_ and _S. aureus_[71]. Previously, malic acid-based NADES, containing sugars such as fructose, glucose, and water, have shown antimicrobial effects against _E. coli_ and _S. aureus_[72]. Although, it was reported that NADES can increase the permeability of the lipid membrane of eukaryotic cells [73; 74]. The mechanisms of NADES interaction with bacterial membranes is still not clearly investigated. The synergistic effect of the components of NADESs, as well as the delocalization of charges as an impact of the hydrogen bond formation, predominantly in acid-based NADESs [50; 64], could be one of the explanations for the promising antimicrobial activities of the tested NADESs. Alternatively, the solubility of some bacterial membrane components in NADESs, low pH, osmolality, or chelation of membrane-bound divalent cations [31] may also contribute to the observed activity. The water activity concept could be applied in predicting the growth of bacteria, yeasts, and molds. Each microorganism is sensitive to the indicator of water activity. For example, pathogenic bacteria lose the ability to grow when \(a_{w}<0.85\)[75]. The \(a_{w}\) value for all initial NADESs in the current study was bellow of 0.1 and could explain their high antimicrobial effect. The \(a_{w}\) for all NADESs after dilution with up to 40% was still below 0.85. This may also explain some of the antimicrobial activity of diluted NADESs. We found that \(a_{w}\) correlates with the antimicrobial activity of NADES, and this is more pronounced for Gram-negative microorganisms (Pearson correlation coefficients r = 0.858 and 0.745, \(p\) < 0.05 for _E. coli_ and _P. aeruginosa_, respectively).