SOCIETY

Preparation of Treatments

Experimental treatments: The films evaluated for antimicrobial activity were the following:

Reference film – without antimicrobial agent – "Control"
Film with 1% oregano extract – "OREGANO 1%"
Film with 1% mixed extract – "FLAVOMIX 1%"

All films were cut into small squares measuring 4.0 x 4.0 cm², and for each treatment three different films were used (n=3).

Preparation of initial bacterial suspension (initial inoculum) and inoculum solution

For the evaluation of the antimicrobial activity of the films, the Gram-negative bacterium Escherichia coli ATCC 1775 strain was selected.

The above strain was received in lyophilized form, reconstituted, and appropriately cultured in Brain Heart Infusion Broth to obtain an initial bacterial suspension (inoculum) of approximately 10⁹ CFU/g for E. coli. Then, three successive decimal dilutions in BHI broth were performed to obtain a final inoculum solution concentration of approximately 10⁵ cells/ml. The measurements of the initial bacterial suspension and inoculum solution are presented in the Table.

Bacterium Initial bacterial suspension Inoculation Volume Concentration of inoculum solution Target concentration (cells range) of inoculum solution acc. to ISO
E. coli 1.3 x 10⁹ cfu/ml 0.1 ml 1.3 x 10⁵ cells/ml 2.5 - 10 x 10⁵ cells/ml

R (FLAVOMIX) = U(control t=24) – A (treated flavomix) = 1.12 Log cells/cm²

Table 6. Calculation of antibacterial activity of films based on the reduction of E. coli population compared to the control.

2.3 Conclusions

The films containing oregano oil and FLAVOMIX exhibited antimicrobial activity against E. coli as the final bacterial population decreased by approximately 2.0 Log CFU/g compared to the reference film. According to the equation proposed by ISO 22196, the antimicrobial activity R of the films is as follows:

R (oregano) = 1.31 Log cells/cm²
R (Flavomix) = 1.12 Log cells/cm²

Given the strong antimicrobial activity of the bags, both were selected for application in the containment of medical waste.

Physicochemical Properties

The encapsulation of substances at micro/nano scale has attracted researchers’ attention due to the remarkable structural characteristics of the produced films and their advanced applications. To understand the structural and morphological characteristics of the polymer films, infrared spectroscopy analyses were conducted, while DSC thermal measurements were presented in Deliverable 1.4.

Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR)

The ATR technique is one of the most important reflection techniques developed and is widely used in infrared spectroscopy (FTIR), mainly for surface analyses.

The operation of ATR is based on the phenomenon of total internal reflection, which occurs when a radiation beam passes from a medium with a high refractive index into a medium with a lower refractive index. The fraction of the incident radiation that is reflected increases with the angle of incidence. The resulting infrared absorption spectrum is primarily used for qualitative analysis and to characterize the molecular structure of a compound, providing information about the nature and spatial arrangement of atoms in the molecule.

ATR accessories use a transparent crystal with a high refractive index as the Internal Reflection Element (IRE), upon which the sample is placed. The infrared beam incident on the crystal (usually at a 45° angle) undergoes multiple total internal reflections inside the crystal, passing through the sample several times and being absorbed accordingly. This internal reflection creates an evanescent wave that penetrates and decays within the sample, leading to absorption at specific IR wavelengths.

ATR-FTIR spectroscopy is among the most important non-destructive and versatile methods, providing high-quality spectra for a wide range of materials. Its main advantage lies in the fact that it requires no sample preparation (Khoshhesab, 2002).

The quantitative and chemical characteristics of the films, as well as the interaction of the antimicrobial agents, were evaluated by recording infrared spectra using ATR and FT-IR techniques. This method was also used to study the interaction between essential oils and biopolymeric pellets produced during extrusion. Specifically, ATR-FTIR confirmed the successful incorporation of antimicrobial oil into the polymer matrix and investigated their interaction.

Measurements were carried out using a Fourier Transform Infrared Spectrophotometer (FT/IR-4200, JASCO International Co., Ltd., Japan) equipped with an ATR unit (ATR PRO-410-S, JASCO International Co., Ltd., Japan). Samples were scanned over a wavelength range of 700 to 4000 cm⁻¹. Each measurement consisted of an average of 32 scans at a resolution of 4 cm⁻¹. For the measurement, the fibers were folded approximately four times to achieve sufficient size and then placed in the device for analysis.