Physicochemical and Antimicrobial Properties of Lactic Acid-Based Natural Deep Eutectic Solvents as a Function of Water Content

Abstract

The interest in natural deep eutectic solvents (NADESs) in green technology as an alternative to organic solvents has grown over the past decades. In this work, for the first time, the effect of dilution with water on the physicochemical and antimicrobial properties of lactic acid-based NADES with choline chloride (NADES1), sorbitol (NADES2), and glucose (NADES3) was systematically studied. According to FTIR data, after the dilution of NADESs with water, the strong hydrogen bonds weakened, however, were not destroyed after dilution of up to 40% water. The dilution of NADES with water resulted in a linear decrease in density and refractive index and in a linear increase in pH. The equations for the prediction of NADES density, pH, and refractive index as a function of water content were calculated. The viscosity decreased by half after adding approximately 10% water. The initial viscosity of NADES2 and NADES3 was significantly different. However, after adding 20% of the water, the viscosity was almost the same. The most pronounced decrease in surface tension (by 46.7%) was found for NADES1. The water activity was decreased in the following order: NADES3 > NADES1 > NADES2. The dilution of NADES with water caused a gradual increase in water activity. NADES1 showed the lowest minimal inhibitory concentrations (MIC) (7.8, 3.9, and 0.98 mg/mL) and minimum bactericidal concentrations (MBC) (15.6, 7.8, and 1.95 mg/mL) for Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, respectively. The antimicrobial activity was decreased by 2–8 times after the addition of 40% water. The water activity for all tested NADES together with low pH could explain the antimicrobial effect. The revealed regularity can be useful for the prediction of NADES properties and for the selection of green solvents on a laboratory and industrial scale.

1. Introduction

Solvents play a crucial role in practically all phytochemical strategies and are frequently utilized in huge amounts in each extraction, separation, and purification stage. Larger volumes of solvents are required to achieve high yields of target components and sufficient purity of active metabolites. Solvents have a substantial influence on the environment as pollutants and on energy consumption in concentration processes, which, therefore, hurt climate change. Although common organic solvents have several advantages, their use requires special care due to their high volatility, explosiveness, fire hazard, and toxicity to humans, flora, and fauna. Traditionally used organic solvents are not biodegradable and cause toxicity and carcinogenicity for live organisms and environmental toxicity [1]. The reduction of the use and production of hazardous substances in industry, decrease in energy consumption, and switching to renewable sources are the main challenges of green chemistry. To protect human health and the environment from the risks related to toxic reagents, many efforts are focused on the quest for alternative green solvents [2].

Recently, a new class of deep eutectic solvents (DESs) has attracted the vigorous attention of the scientific community as a promising alternative to organic solvents [3,4]. DESs were first proposed at the beginning of the XXI century by Abbott et al. (2004) as a biodegradable green alternative to organic liquids [5]. DESs are systems obtained by the interaction of a hydrogen bond donor (HBD) with a hydrogen bond acceptor (HBA) through hydrogen bonds to form a eutectic mixture [6]. The special term, natural deep eutectic solvent (NADESs), was proposed for DESs prepared from components that represent normal metabolites of live cells [7,8,9].

Although NADESs are a relatively young group of solvents, different classes of plant metabolites, such as alkaloids [10], anthocyanins [11], carotenoids [12], coumarins [13], isoflavones [14], polyphenols [15], steroidal [16] and triterpene saponins [17], and polysaccharides [18], were successfully extracted from plants. High viscosity is one of the limitations of NADES. The high viscosity of NADES slows solvent penetration into the plant matrix, mass transfer when dissolving metabolites within plant cells, and transfer of extract within the solvent [19]. This problem could be overcome by increase of temperature, stirring [12], in-process grinding of plant material in vibrocavitation equipment [17], application of ultrasound waves [11], or microwaves [10]. Another efficient way to decrease their viscosity is dilution by water [11,13,14,19,20]. Even though many commonly used NADESs are prepared from components that could be consumed by microorganisms, several publications report some antimicrobial activities of NADES. However, this aspect has not been sufficiently studied. Water is used for the tuning of NADES properties. The systematic study of the effect of water content on the properties of NADES and their antimicrobial activity is required.

In this paper, we characterized the physicochemical and antimicrobial properties of NADES, composed of lactic acid in combination with sorbitol, glucose, and choline chloride, depending on the water content in NADES.

2. Materials and Methods

2.1. Chemicals

Choline chloride (>98.0%), D-sorbitol (>98.0%), and D-glucose anhydrous (>98.0%) were purchased from Sigma-Aldrich, L-lactic acid (88.0–92.0%) was purchased from Panreac Química SLU (Barcelona, Spain). Water was purified by Milli-Q system (Millipore, Bedford, MA, USA).

2.2. NADES Preparation

Natural deep eutectic solvents, NADES1: lactic acid–choline chloride (LA:ChCl, molar ratio 3:1), NADES2; lactic acid–sorbitol (LA:SL, molar ratio 1:3), and NADES3: lactic acid–glucose–water (LA:GL:W; molar ratio 3:5:1) were prepared by heating with stirring until a transparent liquid was formed according to the previously reported method [19]. The composition of NADES was selected according to our previous experience and literature data [21,22,23]. For future physicochemical properties’ tests, NADESs were diluted with ultrapure water. The water content below indicates the percentage of added water.

2.3. Physicochemical Properties Tests

2.3.1. Fourier-Transform Infrared Spectrometry (FTIR)

Fourier-transform infrared spectrometer Spectrum 3 (FTIR, L1280127, PerkinElmer Inc., Shelton, CT, USA) was used for the analysis of molecular interactions between components of NADESs. The spectra of NADES samples (0.05 mL aliquots) were recorded at room temperature at a wavelength of 4000–400 cm⁻¹.

2.3.2. Density

The measurements of the concentration dependence of density of the NADESs and their water solutions were performed by the pycnometer method [24].

2.3.3. pH Analysis

The digital pH meter AMT28F (Amtast USA Inc., Lakeland, FL, USA) was operated at 25 °C to measure pH in NADESs before and after dilution with water.

2.3.4. Refractive Index (RI)

RI of the NADESs were determined using the Abbe refractometer IRF-454B2M (OJSC Kazan Optical and Mechanical Factory, Kazan, Russia) with measurement limits in transmission light $n_D^{25}$ = 1.3–1.7, $∆n_D^{20}$ = ±0.0001, the accuracy of temperature control ΔT = ±0.2 K.

The molar refraction (Rm), the specific refraction (R_D), electronic polarization (E), and polarizability constant (δ) for NADES with different water content at constant temperatures were calculated by the following equations [25,26]:

$$R_D={\displaystyle \frac{n_D^2-1}{n_D^2+2}}·{\displaystyle \frac{1}{\rho }}$$

(1)

$$R_m={\displaystyle \frac{n_D^2-1}{n_D^2+2}}·V$$

(2)

$$E=n_D^2$$

(3)

$$\delta ={\displaystyle \frac{3}{4}}·{\displaystyle \frac{R_m}{\pi N_A}}$$

(4)

where ρ, V, and $n_D$ are density, molar volume, and refractive index of NADES, respectively. δ is the polarizability constant of liquid NADES. N_A is the Avogadro constant.

2.3.5. Viscosity

Brookfield viscometer (DV-III) equipped with CP-40 cone and spindle (Brookfield Engineering Laboratories, Inc., Stoughton, MA, USA) was used for the viscosity analyses of the NADES. The viscosimeter was calibrated with ultrapure water at 25 °C in triplicates. The external thermostatic water bath (Brookfield, TC-502P) was used for the temperature control in the system. Rheocalc software (V3.3, Brookfield Engineering Laboratories, Middleboro, MA, USA) was used for the calculation of viscosity.

2.3.6. Surface Tension

The surface tension of all studied NADESs was measured at 25 °C using a K6 Force tensiometer by the Du Noüy ring method (Krüss GmbH, Hamburg, Germany).

2.3.7. Water Activity

The water activity (a_w) for NADES as a complex solution was described by Equation (5), identical to that of Norrish (1966) with a correlating constant K, defined by Equation (6) [27]:

$${\left(a_w\right)}_M=X_1·exp\left[-K_{NADES}·X_2^2\right]$$

(5)

$$K_{NADES}={\sum }_s{K}_s{C}_s({\displaystyle \frac{\overline{M}}{Ms}})$$

(6)

where $C_s$ is the weight ratio of solute s to total solid; $\overline{M}$ is an average molecular weight of NADES; M_s is the molecular weight of s component; X₁ and X₂ are molar fractions of water and solute.

2.4. Antimicrobial Activity Test

Gram-positive bacteria (Staphylococcus aureus 6538P ATCC) and Gram-negative bacteria (Escherichia coli 25922 ATCC and Pseudomonas aeruginosa ATCC 27835) were from the Collection of Microorganisms of the Department of Microbiology and Biochemistry (Murmansk Arctic University, Russia), frozen at −70 °C in nutrition broth (State Research Center for Applied Microbiology and Biotechnology, Obolensk, Russia). S. aureus and E. coli were grown in Mueller Hinton broth (MHB, Obolensk, Russia) and agar (MHA, Obolensk, Russia); P. aeruginosa was grown in brain heart infusion (BHI, Obolensk, Russia) and agar (BHIA, Obolensk, Russia).

The broth dilution method was employed to determine the minimal inhibitory concentrations (MIC) for NADESs [28,29]. Briefly, twofold serial dilutions of the substances ranging from 0.25 up to 125 mg/mL were prepared in threefold repetitions. An equivalent volume of the bacterial suspension with the density of 1 × 10⁶ CFU/mL (prepared using the McFarland standard) was added to each well. The tubes were incubated at 37 °C for 24 h. The lowest drug concentration, which inhibited the visible bacterial growth, was accepted as MIC. Gentamicin (0.03125–1024 µg/mL) was used as the reference antibiotics (positive control). Tubes containing inoculated and non-inoculated media were prepared as growth and purity controls simultaneously. The MIC values were determined as the lowest concentrations that inhibited bacterial growth compared with the compound-free control and are expressed in mg/mL. Minimum bactericidal concentrations (MBC) were determined after seeding of the samples treated from 1/2 × MIC up to the highest concentration on Petri dishes with agar [30]. These samples were incubated at 37 °C for 24 h. MBC was defined as the lowest drug concentration reducing colony growth of the initial bacterial inoculum by ≥99.9%. All tests were performed as three independent experiments, each carried out in triplicate, and the results were presented as median/modal values. According to the widely accepted norm in MIC testing, the mode and median were used for the final value calculation when triplicate endpoints were within the two- and three-dilution ranges, respectively.

2.5. Statistical Analysis

The statistical analysis of all experimental results was performed with STATGRAPHICS Centurion XV (StatPoint Technologies Inc., Warrenton, VA, USA). Data are expressed as mean ± standard deviation (±SD), and error bars in the figures indicate standard deviation.

3. Results and Discussion

In this study, the physicochemical and antimicrobial properties of lactic acid-based NADESs were evaluated. The components for NADESs were selected following our previous experience in application for extraction of different classes of plant metabolites, and due to the ease of production, low cost, and good compatibility of the components. According to Dai et al. (2013), carboxyl groups of organic acids are allowed to form NADESs as a stable liquid [8]. Being a liquid, lactic acid reacts quickly with other components of NADESs to form clear fluids at low temperatures. Our results and literature data suggest that tailoring NADESs with water affects the physicochemical properties of solvents. However, the addition of more than 50% water led to the disappearance of intermolecular interactions responsible for the eutectic network, and a simple aqueous solution of NADES components was formed [19,31,32]. Therefore, we have investigated native NADESs and solvents diluted with 10, 20, 30, and 40% water.

3.1. Physicochemical Properties

3.1.1. FTIR Analysis

In the case of NADES, it is critical to investigate the strength of hydrogen bond interactions between HBD and HBA. FTIR spectrometry is one of the widely used methods for analyzing the interaction of different groups and identifying the structure of a material [33,34]. Due to its sensitivity to the change of microstructure [35], FTIR is suitable for characterizing the hydrogen bond interactions.

The FTIR spectra were recorded for HBD, HBA, and NADES1 (LA:ChCl), NADES2 (LA:SL), and NADES3 (LA:GL:W), and NADES diluted with 10–40% water (Figures S1–S3). The FTIR spectra of lactic acid, choline chloride, sorbitol, and glucose (Figures S1–S3) agree with those reported in the literature [36,37,38,39].

According to the FTIR spectra (Figures S1–S3), it is evident that the NADES spectra overlap the spectra of respective HBA and HBD with slight differences. In the spectra of all NADESs, peak broadening and shifting are observed. The most pronounced differences are observed in the region corresponding to the stretching vibrations of the O–H bond (3200–3600 cm⁻¹). A peak around 1730 cm⁻¹ is ascribed as C=O stretching vibrations, while the signals in the 1000–1300 cm⁻¹ region can be related to C–O, C–C–O, and C–O–C stretching vibrations, and C–O–H bending vibrations. The shift and the broadening of the peaks indicate the formation of hydrogen bonds between molecules, which confirms intermolecular bonding between components.

Regarding NADES1 (Figure S1), the shift of O–H stretching in FTIR spectra was observed after the formation of the NADES to higher wavenumber (3323 cm⁻¹). The hydroxyl groups of lactic acid were attracted to chlorine anion in choline chloride, consequently producing an O–H–Cl bond. It was also observed that the C=O bond and C–O stretching shifted to the lower wavenumber with increasing the ratio of water in the NADES.

The FTIR spectral analysis of NADES2 revealed several deviations from the spectra of the individual components (Figure S2). The broadening of all signals in the NADES spectra suggests a more complex hydrogen-bonding network within the NADES, as multiple functional groups from both components interact more extensively. Originally, a characteristic peak for lactic acid at 1727 cm⁻¹ was associated with C=O stretching vibrations. The decrease in intensity suggests a possible interaction or reaction involving the carboxylic group, potentially through hydrogen bonding with hydroxyl groups of sorbitol. Additionally, a deviation in the signal at 1217 cm⁻¹ is typically noted in lactic acid spectra for C–O stretching. It indicates altered molecular environments, likely due to interactions with sorbitol. This shift not only supports the presence of altered bonding but also may influence the reaction dynamics and solvation capabilities of the NADES. The peak at 1122 cm⁻¹, typically a sharp feature due to lactic acid’s C–O stretching, shows a notable decrease in intensity. This alteration suggests a modification potentially influenced by the interaction with sorbitol.

In the FTIR spectra of NADES3 (Figure S3), several deviations from the spectra of individual components were also observed. A new peak at 1650 cm⁻¹ likely represents the formation of complex intermolecular hydrogen bonds between the hydroxyl groups of lactic acid and the anomeric center of glucose. The broadening signal at 1028 cm⁻¹ could reflect a change in the environment around the C-O stretching vibrations in glucose and the C-OH groups in lactic acid. It will be possible due to the formation of a more complex hydrogen-bonding network. Peak disappearance at 838 cm⁻¹ and 612 cm⁻¹ on glucose FTIR spectra typically associated with specific vibrational modes of glucose suggests significant alterations in its structural conformation or interactions that prevent these vibrations within the NADES matrix.

The impact of water addition on the stability of hydrogen bonds in NADESs was examined. The change in the IR absorption wavenumber of the O–H functional group was conducted according to [34]

Δσ_OH = σ_OH,HBD − σ_OH,NADES

(7)

where Δσ_OH is the change of IR absorption wavenumber of O–H functional group; σ_OH,HBD and σ_OH,NADES are IR absorption wavenumber of O–H functional group of HBD and NADES, respectively. After the dilution of NADESs with water, the strong hydrogen bonds between HBD and HBA weakened (Figure 1), and the frequencies of the OH bands shifted to increasingly higher values as water content increased (Figures S1–S3). Analysis of variance (ANOVA) with repeated measurements revealed a statistically significant influence of the “NADES” factor (F6,35 = 58.7, p < 0.0000) and the “amount of water” factor (F4,140 = 19.1, p = 0.0004).

3.1.2. Density

Density is one of the key indicators of NADESs, which can provide information about intermolecular interactions in eutectic solvents. As a rule, eutectic solvents have a higher density than water [20,40]. Being a part of the structure of NADES, HBD, or HBA and having a lower density compared to NADES, water reduces the density and viscosity of solvents. The densities of the tested NADES as a function of added water content after dilution are presented in Figure 2.

The density values ranged from 1.226 ± 0.010 g/cm³ (NADES1 at 0% water) to 1.510 ± 0.050 g/cm³ (NADES2 at 0% water) and decreased linearly with the increase in water content. The density of NADES2 following a function y₂ = −0.0051x₂ + 1.5062, R² = 0.996 (y_i: density; x_i: water content percentage for relevant NADES). A similar linear relationship was also observed in NADES3 (y₃ = −0.0041x₃ + 1.3904, R² = 0.99) and NADES1 (y₁ = −0.0022x₁ + 1.2213, R² = 0.989). Using these equations, the density of NADES can be calculated for a known amount of dilution with water.

Notably, the coefficient for water content (equation for NADES1) was lowest among other tested NADES. This indicates a weak impact of water on the density of NADES1 (ChCl:LA 1:3) (Figure 2). Similar trends for NADES containing ChCl:LA were observed previously. The density of NADES containing ChCl:LA (at the molar rations 1:1–1:2.5) diluted with water (up to 22 wt%) [41] and ChCl:LA 1:1 with water contents up to 50 wt% [42] has a linear dependence on the amount of added water and varies slightly [20]. We suppose that the increase in LA content in NADES resulted in a decrease in NADES density.

The literature data evidence the influence of composition and molar ratio of HBA and HBD on the density of the eutectic solvents. Shafie et al. (2019) compared the density of different molar ratios of ChCl and DES based on citric acid (CA) [37]. It was reported that the density of the eutectic mixture decreased with increasing ChCl relative to CA. The study [43] showed that increasing density either by replacing HBD or increasing the molar amount of HBD promotes an association between HBD molecules. The addition of water up to 40% decreases the density of NADES. According to the results [44], the polar components of eutectic mixtures interact with water, and an increase in the percentage of water leads to a gradual weakening of the strength of hydrogen bonds. For solvents containing ChCl, the NADES nanostructure remains stable up to 42 wt% water due to solvophobic water sequestration into nanostructured domains around choline cations. However, increasing water to 51% leads to the destruction of the DES nanostructure, and the solvent is converted into an aqueous solution of the DES components [45].

3.1.3. pH Analysis

The dilution of all tested NADES with water led to a linear statistically significant increase in pH. The pH of LA-based NADES ranges from 0.11 (NADES1) to 1.74 (NADES2 with 40% water at room temperature (25 °C), which refers to acidic NADES (Figure 3). The pH value as a function of water content could be described by the following functions: y₁ = 0.019x + 0.170 (R² = 0.977), y₂ = 0.011x + 1.269 (R² = 0.959), y₃ = 0.018x + 0.509 (R² = 0.950) for NADES1, NADES2, and NADES3, respectively (where y_i: pH; x_i: added water content percentage for relevant NADES). The pH values of NADES can be predicted for a known quantity of diluted water by following the formulas above:

The pH is a vital parameter for the design and implementation of laboratory and industrial processes and their optimization [46,47]. pH has an important impact on the extraction of plant metabolites. The acid-based properties of some DES have been addressed [48]; however, the impact of pH on extraction is underinvestigated. Depending on the components, the dilution of NADES with water resulted in a dual effect. The increase in pH was observed for NADES of betaine with CA (1:1) and ChCl with proline and malic acid (1:1:1) after water addition. In contrast, the acidy of NADES comprising ChCl with CA or NADES (proline–malic acid) was decreased after dilution with water [49]. The nature of the HBD significantly affects the acidity of the DES [44]. The ChCl which contained NADES with organic acid HBDs (such as CA, glycolic acid, LA, malic acid, malonic acid, and oxalic acid) had the highest acidity. The pH was increased with polyols HBDs (ethylene glycol, glycerol) > sugars (fructose and glucose) > amines (ethanolamine and diethanolamine).

The Pearson correlation analysis of our results showed a strong positive correlation between the strength of the hydrogen bond and pH (r = 0.674 p = 0.0082 < p = 0.05). Thus, the observed change in pH with the addition of water can be interpreted by the fact that the pH property depends significantly on the interaction at the molecular level between HBA and HBD.

3.1.4. Refractive Index

The refractive index is one of the significant optical properties of materials used to check the purity of objects, identification and quantification of soluble compounds, etc. [50,51]. The experimental refractive indexes of NADESs are presented in Figure 4 as a function of water content. The refractive index decreased with increasing water content. These results were expected since the mixture as a whole became less dense as the water content of the solution increased (Figure 2). The refractive index of NADES1 was similar to the data given in [52]. As far as we know from the available literature, the refractive indices for NADES2 and NADES3 and their water solutions were measured by us for the first time.

The corresponding equations for the calculation of refractive indexes (y) as a function of water content (x) were calculated (Equations (8)–(10) for NADES1–NADES3, respectively).

Y₁ = −0.0012 × X + 1.4523 (R² = 0.998)

(8)

Y₂ = −0.0016 × X + 1.4945 (R² = 0.982)

(9)

Y₃ = −0.0012 × X + 1.4633 (R² = 0.996)

(10)

The equations could be used for the control of water content in NADES for the industrial-scale application of relevant NADES.

The other parameters closely associated with refractive indexes such as molar refraction, specific refraction, electronic polarization, and polarizability constant for studied NADESs were calculated according to equations presented in Section 2.3.3. The aforementioned properties are useful in chemical, medical, biotechnical, and engineering fields.

The data on electronic polarization are important for the understanding of forces between molecules and their behavior in solutions [37]. Since the electronic polarization (E) is a power state of the refractive index, it is expected that the dilution of NADES with water will lead to a decrease in electronic polarization. In our experiments, the values of E upon the dilution of NADES change insignificantly by no more than 10% (

Table 1. Calculated molar refraction, specific refraction, electronic polarization, and polarizability constant of the tested NADES at room temperature (mean ± sd).

NADES Sample	Molar Refraction, Rm (cm3/mol)	Specific Refraction, RD (cm3/g)	Electronic Polarization, E	Polarizability Constant, δ·10−23 (cm3/mol)
NADES1	19.4 ± 0.1	0.220 ± 0.005	2.11 ± 0.05	0.770 ± 0.004
NADES1 + 10% H2O	17.5 ± 0.1	0.220 ± 0.005	2.07 ± 0.03	0.693 ± 0.003
NADES1 + 20% H2O	15.5 ± 0.2	0.219 ± 0.004	2.04 ± 0.04	0.614 ± 0.004
NADES1 + 30% H2O	13.4 ± 0.2	0.218 ± 0.002	2.01 ± 0.03	0.533 ± 0.003
NADES1 + 40% H2O	11.3 ± 0.1	0.214 ± 0.004	1.97 ± 0.04	0.450 ± 0.002
NADES2	28.8 ± 0.4	0.187 ± 0.011	2.22 ± 0.09	1.144 ± 0.013
NADES2 + 10% H2O	27.3 ± 0.2	0.196 ± 0.012	2.19 ± 0.04	1.081 ± 0.012
NADES2 + 20% H2O	24.6 ± 0.1	0.199 ± 0.008	2.16 ± 0.03	0.977 ± 0.008
NADES2 + 30% H2O	21.3 ± 0.3	0.197 ± 0.005	2.10 ± 0.05	0.846 ± 0.004
NADES2 + 40% H2O	18.3 ± 0.3	0.198 ± 0.006	2.04 ± 0.04	0.728 ± 0.007
NADES3	24.2 ± 0.2	0.198 ± 0.006	2.14 ± 0.05	0.962 ± 0.004
NADES3 + 10% H2O	22.0 ± 0.1	0.199 ± 0.007	2.10 ± 0.03	0.874 ± 0.005
NADES3 + 20% H2O	19.8 ± 0.2	0.202 ± 0.003	2.06 ± 0.03	0.785 ± 0.004
NADES3 + 30% H2O	17.4 ± 0.3	0.203 ± 0.004	2.04 ± 0.04	0.692 ± 0.004
NADES3 + 40% H2O	14.9 ± 0.2	0.202 ± 0.003	2.00 ± 0.02	0.590 ± 0.005

).

We noted that the calculated specific refraction for all tested NADESs and their water solutions was not dependent on water content and was in narrow diapason (mean 0.205 ± 0.011 cm³/g) for all tested samples (Table 1). Similar trends for specific refraction were observed for binary mixtures of the C60-tris-malonic derivative with water [25].

The information on the RI and δ for NADES used as a solvent or extragent helps to explain the behavior of NADES, such as dispersion forces. The high δ constant is evidence of strong dispersion forces [53]. The δ values for tested NADESs are shown in Table 1. It was established that the polarizability constants for NADESs decreased linearly with the increase in water content in NADES. Similar results were reported by Ghaedi et al. (2018) for DES analogues [26]. The solvents with a high δ constant and large polarizability should be capable of enjoying powerful dispersion forces [54], being suitable solvents for compounds with elevated polarizabilities.

3.1.5. Viscosity

The viscosity of solvents largely determines the efficiency of extraction; it is important in the development of industrial technologies and in the calculations of fluid flow systems. The choice of the optimal proportion of HBD and HBA and the amount of water in NADES is also carried out taking into account viscosity [55]. The high viscosity of NADESs is beneficial for the design of lubricants [56], supramolecular eutectogel in medicines [57], etc. However, the high viscosity of NADES causes several problems in extraction procedures due to delayed mass transfer during extraction. To overcome this problem, NADES is usually diluted with a certain amount of water. If the amount of ± water is low, its molecules are adsorbed into the molecular matrix of NADES, and H-bonds are formed between the ions and HBD. With an increase in water concentrations, the components of NADES actively interact with water, which leads to a decrease in inter- and intramolecular interactions in NADES and destroys its structure [19]. The viscosity of tested NADES diluted with a certain amount of water was measured at room temperature and is presented in Figure 5.

The composition and water content significantly affect the viscosity of NADES [58]. The viscosity of the tested NADESs was high and decreased in the following order: NADES2 > NADES3 > NADES1. The dilution with water weakened the hydrogen bonds between the NADES components and led to a significant reduction in viscosity. The viscosity decreased by half after adding approximately 10% water. The initial viscosity of NADES2 and NADES3 containing different saccharides was significantly different. However, after adding 20% of the water, the viscosity was almost the same (Figure 5). Similar results were reported for NADES containing ChCl–diethylene glycol, ChCl–triethylene glycol, ChCl:PEG 200, and 1,2-propanediol–ChCl–water [59]. Pires et al. (2022) have observed a viscosity reduction by 83% in LA–glycine mixture [60].

3.1.6. Surface Tension

The number of hydrogen bonds between HBD and HBA, molecular weight, viscosity, and temperature are among the major factors affecting the surface tension of NADES [26,61]. Surface tension functions for NADES and its water solutions measured by the Du Noüy ring method are presented in Figure 6. The surface tensions of investigated NADESs were 81.4 ± 3.2, 91.5 ± 6.8, and 95.9 ± 5.8 N/m for the NADES1, NADES2, and NADES 3, respectively. The dilution of all solvents leads to a statistically significant decrease in surface tension. The most pronounced reduction of surface tension (by 46.7%) was found for NADES1.

The high surface tension, as well as the high viscosity of the solvents, slows down the solvent penetration in the plant cell capillaries during the extraction. The penetration of the solvent into the raw material is associated with capillary forces. The plant matrix has amphiphilic properties, and the hydrophilic properties of fiber are more pronounced than the hydrophobic ones. Plant tissue has a large number of capillary-type pores. The solvent penetrates through capillaries, filling the cells and space inside the plant material. The penetration of the solvent is accompanied by humidification inside the cells, which depends on the chemical affinity of the raw material and the solvent [62].

The liquid phase spreads over the surface of plant cells, and the driving force of the humidification process is determined by the value of the spreading coefficient. The reduction of solvent surface tension improves the humidification of plant material cells, increasing the contact surface and the depth of its penetration into the cells. Based on the above, NADES1 will be preferable for extraction compared with NADES3. The tuning of solvents with water can also improve the efficiency of extraction. Similar recommendations were provided by Savi et al. (2019) for some organic acid-based NADESs [20].

3.2. Water Activity

Water activity was estimated as the ratio of the partial pressure of water vapor in a sample to the partial pressure in pure water in standard conditions. The calculated water activity of NADES3 was the highest (a_w = 0.097), followed by NADES1 and NADES2 with a_w = 0.034 and a_w = 0.031, respectively. This is probably due to the fact that NADES3 had water already added at the preparation stage. The dilution of NADES with water caused a gradual increase in water activity. The water activity in all samples diluted with 40% water increased up to 0.75 (Figure 7). A similar increase in water activity according to water content was observed for several NADESs [19,63].

3.3. Antimicrobial 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. Antimicrobial activity of NADES without and after dilution with water (mg/mL).

Sample	Water Content, %	E. coli		P. aeruginosa		S. aureus
		MIC	MBC	MIC	MBC	MIC	MBC
NADES1	0	7.8	15.6	3.9	7.8	0.98	1.95
	10	15.6	31.3	7.8	15.6	1.95	3.9
	20	15.6	31.3	15.6	31.3	1.95	3.9
	30	31.3	62.5	15.6	31.3	3.9	7.8
	40	31.3	62.5	31.3	62.5	3.9	7.8
NADES2	0	15.6	31.3	7.8	15.6	7.8	15.6
	10	15.6	31.3	7.8	15.6	7.8	15.6
	20	31.3	62.5	7.8	15.6	15.6	31.3
	30	31.3	62.5	15.6	31.3	15.6	31.3
	40	31.3	62.5	15.6	31.3	31.3	62.6
NADES3	0	15.6	31.3	1.95	3.9	1.95	3.9
	10	15.6	31.3	3.9	7.8	1.95	3.9
	20	31.3	62.5	7.8	15.6	3.9	7.8
	30	31.3	62.5	15.6	31.3	3.9	7.8
	40	31.3	62.5	31.3	62.5	7.8	15.6
Gentamicin	-	1 × 10−3	2 × 10−3	2 × 10−3	4 × 10−3	0.25 × 10−3	0.5 × 10−3

.

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). Radošević 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 significant effect on antimicrobial activity, and S. aureus was the most sensitive to the pH of NADES (Pearson’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). The bacteriostatic effect of sirups, which provides self-preservation and helps to limit the addition of preservatives, is also associated with low water activity [76]. Some authors believe that NADES and honey have comparable supramolecular structures [77]. High osmotic pressure associated with low water activity and acidity were considered responsible for the antimicrobial activity of honey [78]. We suggest that water activity value can be a reasonable parameter for the choice of the best extraction solvent for a particular purpose.

4. Conclusions

In this work, for the first time, the effect of dilution with water on the physicochemical and antimicrobial properties of lactic acid-based NADESs with choline chloride (NADES1), sorbitol (NADES2), and glucose, (NADES3) was systematically studied. Several physicochemical parameters, including intermolecular interactions, density, pH, refractive index, viscosity, surface tension, and water activity, were evaluated. The formation of hydrogen bonds in NADES was confirmed using the FTIR method. After the dilution of NADESs with water, the strong hydrogen bonds weakened, however, were not destroyed after dilution with 40% water. The linear decrease in density and refractive index was observed for all NADES with the increase in water content. The dilution of NADES with water led to a statistically significant linear increase in pH. The equations for the prediction of NADES density, pH, and refractive index as a function of water content were calculated. A significant reduction in the viscosity of NADES was observed after the increase in water. The viscosity decreased by half after adding approximately 10% water. The initial viscosity of NADES2 and NADES3 containing different saccharides was significantly different. However, after adding 20% of the water, the viscosity was almost the same. The surface tension gradually and statistically significantly decreases after the dilution of all solvents. The most pronounced decrease in surface tension (by 46.7%) was found for NADES1. The calculated water activity of NADES3 was higher (a_w 0.097), followed by NADES1 and NADES2 with a_w 0.034 and a_w 0.031, respectively. The dilution of NADES with water caused a gradual increase in water activity.

All the tested NADESs inhibited the growth of both Gram-positive and Gram-negative microorganisms. NADES1 showed the lowest MIC (7.8, 3.9, and 0.98 mg/mL) and MBC (15.6, 7.8, and 1.95 mg/mL) for E.coli, P. aeruginosa, and S. aureus, respectively. The Gram-positive opportunistic pathogen was more sensitive to all NADESs in the current study. 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. We found that pH has a significant impact on antimicrobial activity. S. aureus was the most sensitive to the pH of NADES. The water activity concept could be applied in predicting the growth of bacteria. The a_w for all tested NADESs together with low pH could explain the antimicrobial effect.

The revealed regularity for physicochemical and antimicrobial properties of lactic acid-based NADES after their dilution with water can be useful for the prediction of NADES properties and for the selection of green solvents for extraction both in the laboratory and on an industrial scale.