## 2 Materials and Methods
### Microorganisms and Inoculum Preparation  
All microorganisms used in this study were isolated from food: bacteria--Escherichia coli BTLB0001 and Pseudomonas aeruginosa BTLB0015, yeasts--_Candidia parasilosis BTLYCP1_ and _Yarrowia lipolytica BTLY0054_--and fungi--_Aspergillus terreus BTLF0001, A. fumigatus BTLF0002_ and _Penicillium expansum BTLF0003_. Bacteria for antimicrobial tests were grown on nutrient agar (Oxoid, Hampshire, UK), and yeasts and fungi were grown on Sabouraud dextrose agar (Oxoid, Hampshire, UK). Inoculum were obtained from overnight bacterial strains on nutrient agar slants at 28 \({}^{\circ}\)C. Yeasts were cultured for 3 days, and fungi were cultured for 7 days. The turbidity of the microorganism cell suspensions was measured at 610 nm by spectrophotometer (Thermo Scientific, Waltham, MA, USA). The resulting suspensions of microorganisms were mixed for 15 s with a vortex.  
Evaluation of the Antimicrobial Activity of the Beetroot-Gelatin Films Containing Silver Nanoparticles  
The activity of gelatin-beetroot films containing silver nanoparticles was screened for antimicrobial activity using the agar diffusion method. For the agar diffusion assay, 100 \(\upmu\)L of each suspension of microorganisms was uniformly spread on Mueller-Hinton agar (bacteria) or on Sabouraud dextrose agar (yeasts) in a Petri dish (90 mm). The suspension micromycete spores (one by one mL) were poured into Petri dishes with Sabouraud dextrose agar at 45 \({}^{\circ}\)C. The gelatin-beetroot films containing silver nanoparticles (5 \(\times\) 5 mm) were placed on the surface of each Petri dish after absorption of inoculum by agar. The plates with yeasts and fungi were incubated at 26 \(\pm\) 1 \({}^{\circ}\)C and were placed for 3 days (for fungi) or for 5 days for yeasts, while the plates with bacteria were incubated at 37 \({}^{\circ}\)C for 2 days. All tests were triplicated for all strains. Beetroot juice for the synthesis of  
Figure 3: An example of beetroot–gelatin free-standing film containing silver nanoparticles.  
silver nanoparticles was used as negative control. The diameters of the inhibition zones after the incubation period were measured in millimeters.  
Evaluation of the pH Effect on the Antimicrobial Properties of the Gelatin Films with Silver Nanoparticles  
Activities of gelatin-beetroot films containing silver nanoparticles were tested in four pHs, i.e., 3, 4, 7 and 8. The 35% wt. of HCl and 5 M NaOH were used in order to obtain necessary pH values; the amount of base or acid needed in each case was previously determined. For the agar diffusion assay, 100 \(\mu\)L of each suspension of microorganisms was uniformly spread on Mueller-Hinton agar (bacteria) or on Sabouraud dextrose agar (yeast) in a Petri dish (90 mm). The suspensions of spores of micromycetes (one by one mL) were poured into Petri dishes with Sabouraud dextrose agar at 45 \({}^{\circ}\)C. The gelatin films with silver nanoparticles (5 \(\times\) 5 mm) were placed on the surface of each Petri dish after absorption of inoculum by agar. The plates with yeast and fungi were incubated at 26 \(\pm\) 1 \({}^{\circ}\)C for 3 days or for 5 days (for fungi), while plates with bacteria were incubated at 37 \({}^{\circ}\)C for 2 days. All tests were triplicated for all strains. Beetroot juice used for the synthesis of silver nanoparticles was used as negative control.