2. Materials and Methods
2.1. Materials
2.1.1. Preparation of Culture Media, Solutions, and Laboratory Materials
All culture media and solutions intended for microbiological procedures, together with materials such as microtubes, test tubes, Falcon tubes, tweezers, micropipette tips, among others, were sterilized in a Prismatec® CS-A line vertical autoclave (Prismatec Indústria e Comércio, Itu, SP, Brazil) for 30 min at 121 °C. Tap water was purified in a Master System All (model MS2000, Gehaka, São Paulo, SP, Brazil) to a final resistivity of ca. 18.18 MΩ·cm and conductivity of 0.05 µS·cm−1, and was used in the preparation of all media and solutions. Media and reagents were weighed on an analytical scale from Marte Científica® (model OHAUS AS200S, Santa Rita do Sapucai, MG, Brazil). A biological safety cabinet (model Filterflux® Class II B2, SPLab, Piracicaba, SP, Brazil) was used for manipulations with bacteria, bacteriophages, culture media, and microbiological activity measurements.
2.1.2. Biological Materials
Cefar Diagnóstica (São Paulo, SP, Brazil) provided the Salmonella enterica CCCD-S004 strain collection bacterium used as a bacterial host.
Phage SentS01L was isolated from lake water, whereas phage SentS01T was isolated from a soil sample, both collected in the surroundings of the Veterinary Hospital of UNISO, Sorocaba, SP, Brazil (geographic coordinates: 23°29′58.7″ S; 131 47°23′45.2″ W).
2.1.3. Chemicals
The reagents were purchased from (i) Dinâmica Química Contemporânea Ltd. (Diadema, SP, Brazil) (anhydrous dibasic sodium phosphate, monobasic sodium phosphate, calcium chloride, and sodium chloride); (ii) Sigma-Aldrich (St. Louis, MO, USA) (culture media Tryptic Soy Agar (TSA) and Tryptic Soy Broth (TSB), polyethylene glycol (PEG) 8000, d-gluconolactone (GDL), magnesium sulfate, Trizma hydrochloride (Tris-HCl), sodium alginate, and uranyl acetate); (iii) Gibco Diagnostics (Madison, WI, USA) (microbiological solid agar); (iv) Merck-Millipore (Darmstadt, Germany) (sterilizing filtration systems/Stericup™-GP); (v) Labsynth (Diadema, SP, Brazil) (magnesium sulfate); (vi) Anidrol (Diadema, SP, Brazil) (calcium carbonate (CaCO3)); and (vii) BioRad (Santo Amaro, SP, Brazil) (disruption buffer, molecular weight markers, and Coomassie Brilliant Blue G-250).
2.2. Experimental Procedures
2.2.1. Preparation of a Suitable Bacterial Suspension of Salmonella enterica CCCD-S004
The host bacteria were incubated at 37 °C for 12 h in solid TSA and were hydrated after in TSB liquid medium.
2.2.2. Bacterial Lawns (Salmonella enterica CCCD-S004) Using the Pour Plate Technique
For the preparation of host bacterial lawns, 100 µL of bacterial suspension were added in a tube with 5 mL of molten top agar-TSB (MTA-TSB). After homogenization, these samples were poured into plates with solid TSA, allowed to dry out, and then incubated at 37 °C during 12 h.
2.2.3. Spot Test Verification of Lytic Activity in the Two Phage Suspensions
For the verification of the lytic activity of the isolated phages, 10 µL of each phage suspension were poured onto host bacterial lawns and incubated overnight at 37 °C. After this time, clear lysis zones, which are a sign of the existence of lytic bacteriophages, could be seen.
2.2.4. Phage Virion PEG-Precipitation
In a sterile mixture of polyethylene glycol (PEG) 8000 (10%, w/w) and NaCl (1 mol/L) (2:1), phage suspension samples were added. These suspensions were incubated at 4 °C during 12 h and, after this time, the samples were centrifuged (11,000 rpm, 4 °C, 45 min). The pellet was resuspended in 5 mM MgSO4, and the supernatant discarded.
2.2.5. Bacteriophage Enumeration
In accordance with Adams [18], the bacteriophage titer was determined. Serial dilutions were prepared employing stock bacteriophage suspension (50 µL) and SM phage buffer (450 µL) (200 mM NaCl; 10 mM MgSO4; and 50 mM Tris-HCl in pH 7.5). In sequence, 50 µL of each dilution was added to 100 µL of bacterial culture grown and 4 mL of MTA-TSB. The suspension was added to a TSA Petri plate and incubated at 37 °C for 12 h. Following this time frame, lytic plaques were seen in each dilution and counted taking into account dilutions containing 20–200 bacteriophage plaques. Next, the bacteriophage titer (PFU/mL) was computed as number of phage plaques formed×1dilution×1Vbacteriophage inoculum(mL)
. The resulting phage titers were 1.40 × 1012 PFU/mL (phage SentS01L) and 1.72 × 1012 PFU/mL (phage SentS01T).
2.2.6. Transmission Electron Microscopy (TEM) Analyses
The phage particles were centrifuged in a micro-ultracentrifuge from Beckman-Coulter (model Optima TLX, Indianapolis, IN, USA) for 150 min at 45,000 rpm and 4 °C. The samples were negatively stained employing uranyl acetate at 2% (w/v) and pH 7.0, [21,22] and photomicrographed in a Transmission Electron Microscope from JEOL (model JEM 2100, Tokyo, Japan) using a high-resolution CCD camera from GATAN Inc. (model ORIUS™ 832.J4850 SC1000B, Pleasanton, CA, USA). The software Gatan Microscopy Suite (DigitalMicrograph from GATAN Inc., version 2.11.1404.0) was used to obtain digital photos of the phage virions.
2.2.7. Sodium DodecylSulphate PolyAcrylamide Gel Electrophoresis (SDS-PAGE) Analysis of Phage Virion Structural Proteins
SDS-PAGE was used for determining the molecular weights of the bacteriophage SentS01L and SentS01T structural proteins, employing a Mini-PROTEAN® Tetra Cell from Bio-RAD (Hercules, CA, USA). In an Eppendorf, 500 µL of disruption buffer was added to each sample of bacteriophage suspension, and the mixture was boiled for ten minutes. After, 20 µL of sample supernatant and 5 µL of molecular weight markers were applied to the 5% acrylamide–bisacrylamide concentration gel/12% acrylamide–bisacrylamide separation gel, and electrophoresis was run for 60 min. Coomassie Brilliant Blue G-250 was used to dye the gel, after which it was photographed in high resolution for further analysis.
2.2.8. Formulation and Characterization of the Edible Antibacterial Coating (Ediphage) Integrating the Lytic Phage Cocktail
Formulation of the EBP Film. The EdiPhage formulation (Table 1) was prepared via internal gelification as described by Balcão et al. [23,24] and Łętocha et al. [25]. The internal gelification method was used to polymerize alginate at room temperature for 72 h. To initiate alginate polymerization, a fresh aqueous δ-gluconolactone (GDL) solution was used to release of the calcium ions dispersed in the formulation. Calcium carbonate (CaCO3) was employed as a source of calcium ions. The previously made polymeric dispersion was homogenized with this hydrolysis solution, and the cheese samples were dipped in it. Following this period, the cheese matrices were kept at 4 °C until further analyses were in order.
Table 1. Formulations of the edible antibacterial coating (EdiPhage) with (or without) phage particles.
The appropriate phage virion/bacterial cell ratio (i.e., Multiplicity Of Infection—MOI), aiming at the inactivation of host bacteria (viz. MOI 100 and MOI 1000), was used based on a previous work by Pereira et al. [21].
EdiPhage Thickness. The average thickness of the polymerized coatings was determined using a caliper with a resolution of 0.001 mm, through five random measurements on the area of each EdiPhage.
Evaluation of the Maintenance of the Lytic Viability of the Bacteriophage Particles Integrated in the Edible Antibacterial Coating. A sample of EdiPhage was removed from a cheese matrix sample and positioned in the center of a bacterial lawn of Salmonella enterica CCCD-S004. After incubation for 24 h at 37 °C, a macroscopical analysis was carried out to observe the presence of clear zones of lysis in the bacterial lawn surrounding the EdiPhage samples.
Evaluation of the Potential for Cytotoxicity of the Ediphage, Via Disc Diffusion Assay. The disc diffusion assay employing cell lines of immortalized human keratinocytes (HaCaT) and mouse fibroblasts (3T3) [26,27,28] was used for the evaluation of the cytotoxicity potential of the EdiPhage integrating the cocktail of lytic phages. A Petri plate containing the cells was filled with a small disc of sample (EdiPhage) and was incubated for 24 h at 37 °C in a 5% CO2 atmosphere. The same protocol was used for a negative control (a disc of innocuous paper) and positive control (latex). The presence of cytotoxicity was detected by the formation of a transparent halo due to cell lysis surrounding the sample tested [29].
Determination of the Ediphage Elemental Composition Via Energy Dispersive X-ray Fluorescence (EDXRF) Analyses. An X-ray fluorescence spectrometer with energy dispersion (EDXRF) from Amptek (Bedford, MA, USA) was used for the determination of the elemental composition of the EdiPhage formulations. Every measurement was performed with ambient air, and each sample’s measuring time was fixed at 300 s (live time).
Fourier Transform Infrared Spectrophotometry (FTIR) Analyses. A Fourier Transform Infrared Spectrophotometer from Agilent (model Cary 630, Santa Clara, CA, USA) was used for the FTIR spectra of EdiPhage samples. The measurements were obtained in the range from 4000 cm−1 to 400 cm−1 and a resolution of 2 cm−1.
Thermal Analyses Via Differential Scanning Calorimetry (DSC). Shimadzu’s DSC-60 microcalorimeter (model DSC-60, Kyoto, Japan) was used to conduct DSC analyses combined with a Thermal Analyzer TA 60W (Shimadzu, Kyoto, Japan). The parameters used were a temperature increase from ca. 25 °C up to 300 °C, at a heating rate of 10 °C min−1, under an inert atmosphere (argon of 50 mL min−1 [26]. The samples weighed 1.080 mg (plain EdiPhage) and 1.820 mg (bioactive EdiPhage).
Tomographic Analyses Via X-ray Transmission (XRT). An X-ray transmission tomograph from Bruker microCT (model SkyScan 1174, Kontich, Belgium) was employed for tomographic images. The software NRecon™ from Bruker (version 1.6.9.4, Kontich, Belgium) used the algorithm of Feldkamp et al. [30] in the process of reconstructing the tomographic images. The software CTVox™ (version 2.6.0 r908-64 bit, from Bruker microCT), CTan™ (version 1.13.5.1-64 bit, from Bruker microCT) and CTvol (version 2.2.3.0-64 bit, from Bruker microCT) were used for the processing of the tomographic images.
Scanning Electron Microscopy Analyses (SEM). The images of the EdiPhage sample were obtained using a scanning electron microscope (JEOL, model JSM-IT200, Tokyo, Japan), with an Energy Dispersive X-ray Spectrometer (EDS) detector (JEOL, model DRY SD™25 Detector Unit, Tokyo, Japan). The sample coatings were prepared through the cathodic pulverization of Au (92 Å thickness) in a metalizing device.
Mechanical Resistance Properties. A texturometer from Stabile Micro Systems (model TA-TX Plus, Godalming, UK) was used to assess the mechanical qualities of the EdiPhage, employing a maximum force of 5 kg and a distance of 5 mm. All experiments were performed in triplicate, using sample dimensions of 3 cm × 2 cm.
2.2.9. Statistical Analyses
The statistical analysis of the data was carried out utilizing GraphPad Prism 7.04 software (GraphPad Software, San Diego, CA, USA). The data’s normal distribution was examined using a Kolmogorov–Smirnov test. The homogeneity of variance was assessed using Levene’s test. The significance of bacterial concentrations was tested using two-way ANOVA and the Bonferroni post-hoc test. A value of p < 0.05 was considered to be statistically significant.