2.1 Chemicals and reagents used
In this study, sodium caprylate, a salt of food-grade weak organic acid was electrochemically activated and procured from Sigma Aldrich, India. Caprylic acid, sodium chloride, Muller Hinton agar, and Muller Hinton broth were purchased from HI Media (Mumbai, India). At end molar concentration ranging from 0.5 to 0.03125 mol/L, the NaCl and sodium caprylate crystals were dissolved in 1 L of distilled water. All the compounds used were of purely analytical and HPLC grade. All samples of investigated salt were made on the day when the experiment was conducted. ZoBell’s standard solution was obtained from Sigma Aldrich, India.
2.2 Test microorganisms
Four nontyphoidal Salmonella strains that were multidrug resistant were obtained from the (IMTECH) Institute of Microbial Technology, Chandigarh. The bacterial strains used were Salmonella enterica subsp. arizonae (MTCC 660), Salmonella Newport (MTCC 3225), Salmonella enterica ser. Typhi (MTCC 733), and Salmonella enterica ser. Paratyphi (MTCC 735). The standard conditions for the growth of these bacteria were incubation at 37 ºC for 24 hours on MHA (Mueller Hinton Agar) Petri plates.
2.3 Electrodes used for the tests
Ruthenium-iridium-coated titanium (MMO) electrodes from (Exotic Metal Tech, Kovilambakkam, Chennai, India) were employed in tests for the electrochemical activation of aqueous solutions and to prevent metal electrode erosion. Undoubtedly, in brine solution electrolysis operations, the RuO2-IrO2-TiO2 (MMO) anode plate is an excellent fouling prevention electrode and firm in terms of its dimension toward erosion. The electrode dimensions were 50 mm × 100 mm, and the thickness of the Titanium ASTM Grade 2B 265 plate type with MMO coating was 1.5 mm.
2.4 Design of the electroactivation reactor
The reactor was a three-section, segmented electrochemical activation cell. The reactor comprised three Plexiglas compartments with dimensions of 50 mm (Length) × 50 mm (Width) × 120 mm (height). To prevent any disturbance between the current flow and the reactor, Plexiglas was used. The anodic and cathodic portions were partitioned with the middle compartment region by an anionic exchange membrane (AMI 7001S) and a Cationic exchange membrane (CMI 700S), alternately (Membrane International Inc. Ringwood, New Jersey, USA). For a 3 cm × 7 cm exposed transfer area, the membranes used to preferentially flow ions had a standard thickness of approximately 0.45 ± 0.025 mm. The anion exchange membrane (AEM) placed near the anode side prohibited cations, e.g., H+ and Na2+, from exiting and enabled anions to transfer, e.g., caprylate ions (C4H7O1-), between the anodic and central portions. The cation exchange membrane (CEM) prevents unwanted anions from transferring from the cathode to the other compartments, allowing analytes with unique properties such as the ORP, pH, DO, and pKa, to be created (Liato et al., 2015). The perforations in the three compartments are 24 mm wide and 10 mm deep. RuO2-IrO2-TiO2 electrodes with a 40 cm2 active surface area were inserted into the reactors to provide a functional active area of 40 cm2. They were placed on both sides of the reactor’s extreme compartments. The positive terminal of the direct electric current (DC electric generator GeNei TM) source was linked to the anodic compartment, while the negative side was attached to the cathodic compartment. Mechanical stirring was used to agitate all three compartments.
2.5 The electrochemical activation reactor configuration
The electrochemical activation of the investigated solutions was performed following the setup of the electrochemical activation reactor, as shown in:
Configuration No.1. Throughout every test, solutions of NaCl like (0.5, 0.25, 0.125, 0.0625, and 0.03125 mol/L) were employed in all compartments.
Configuration No.2. The alignment of membranes remained unchanged as that of configuration 1, however, in the central and anodic cells, identical molar concentrations (0.5, 0.25, 0.125, 0.0625, and 0.03125 mol/L) of sodium caprylate solutions were being used. All of the experiments were performed in the cathode chamber using a 0.1-molar NaCl solution.
Configuration No.3. Both cation and anion exchange membrane cells were arranged in the same manner as in prior experiments, but 0.1-molar NaCl solution was placed in the cathodic and central chambers. Simultaneously, sodium caprylate solutions with molar values of 0.5, 0.25, 0.125, 0.0625, and 0.03125 mol/L were placed in the anodic chamber.
Configuration No.4. In this instance, the AEM was installed on the cathode corner of the electrochemical activation reactor, whereas the CEM was installed on the anodic corner. All chambers were present in sodium caprylate with molar values of 0.5, 0.25, 0.125, 0.0625, and 0.03125 mol/L.
2.6. Electrochemical activation procedure
An electrochemical activation reactor/cell was constructed as an electrooxidation device for the production of aqueous solutions that were electrochemically activated, specifically anolytes and catholytes. Thus, every microvolume of the tested solution had to be adequately accessible to the surface of the electrodes during the procedure. Therefore, due to this interaction, unbalanced structural alternations of water occur, and the solution is enriched with the products of electrochemical processes. The effects of aqueous solutions of NaCl and sodium caprylate at varying concentrations and mixtures were systematically examined. The variations in the output voltage were also monitored to determine the power usage of the electrochemical activation reactor. A 300 ml aliquot of the investigated solution was placed in each compartment of the reactor at time zero. After being loaded with the specified solutions, the electroactivation reactor was attached to a DC electric generator (GeNei TM), which was then observed to provide currents such as 100 mA, 150 mA, 200 mA, and 250 mA to the reactor. Electroactivation was carried out for a maximum of 45 minutes under each current value. All observations were obtained just after the solutions were prepared. Commercially accessible organic acid (caprylic acid) and NaCl were used as positive and negative controls, respectively.
2.7. Assessment of antibacterial action
For the assessment of antibacterial activity, agar diffusion.was performed the well-variant method was used. The examination was carried out on the same day, and various concentrations (1%, 3%, and 5%) of electroactivated sodium caprylate solutions were formulated. All bacterial isolates were cultured in Mueller Hinton broth for 18 hours at 37ºC using McFarland.Turbidity standards, the bacterial cell density was adjusted to 105 CFU/ml [13]. A 20 µl culture of the selected strains was distributed over all the MHA plates to create homogenous bacterial colonies after the solidification wells were punched into it, into which 100 µl of the tested electroactivated sodium caprylate solutions were incorporated. The media plates were kept for 18 hours at 37ºC under an aerobic environment. The zone. of inhibition (ZOI) of the examined solutions was measured in millimeters and was defined as the clear regions surrounding the wells. This procedure was carried out in triplicate.
2.8 Evaluation of the minimum inhibitory concentration (MIC) and bactericidal concentration (MBC) of the electrochemically activated solutions
A microtitration plate experiment was used to evaluate the minimum inhibitory concentration (MIC) of electroactivated sodium caprylate solutions [9,14]. In a flat-bottom microtiter plate, the evaluated solutions were twofold serially diluted in 125 µl of growth medium. In this process, 5% of the examined solutions were diluted serially to achieve different concentrations, such as 2.5 %, 1.25 %, 0.625 %, 0.312 %, and 0.156 %) respectively, before adding 50 µl of bacterial suspension (i.e., 5 log CFU/ml) to each well, excluding that which contained 175 µl of broth, i.e., the negative control. The controls that were considered positive were those media that were inoculated with no sample solutions. Following inoculation, the tested specimens (175 µl) were incubated for 24 hours at 37ºC, and bacterial growth was recorded. The MIC value was estimated as the lowest quantity of the examined solution (electrochemically activated solutions or commercially available organic acids) at which there was no growth of bacteria after incubation [14]. Following the MIC assessment, 100 µl of each specimen was distributed on agar plates and maintained at 37ºC for 24 hours. The minimum bactericidal concentration was defined as the lowest solution concentration, i.e., organic acid and electrochemically activated solutions, that lowered the survivability of the original bacterial suspension by ≥ 99.9% [14]. This procedure was carried out in triplicate.