Inactivation of Staphylococcus epidermidis in a Cotton Gauze with Supercritical CO₂ Modified with Essential Oils

Abstract

The bacterium Staphylococcus epidermidis is the main cause of most infections related to medical devices and prosthesis. However, current disinfection methods are not satisfactory; a new one is proposed here. S. epidermidis was spiked in a cotton gauze and then treated with supercritical CO₂ mixed with essential oils, such as lemon, cinnamon, oregano, clove, and peppermint, and isolated thymol. The operation took 30 min at 10.0 MPa and 40 °C. Concentrations of 1000, 500, and 200 ppm of the essential oils were used. These additives, which have antimicrobial power by themselves, improved the inactivation with supercritical CO₂. The peppermint essential oil was the most effective. The presence of water from 200 ppm also improved the disinfection. Thus, S. epidermidis total inactivation was achieved with the supercritical CO₂ containing 200 ppm of peppermint essential oil and 200 ppm of water. An evaluation of the gauze before and after disinfection was realized by DSC, FTIR, and SEM. At the optimal conditions, there were no significant physical or chemical changes. Furthermore, no essential oil residuals were found. This disinfection method could be established in the healthcare field as an alternative to toxic liquid chemicals.

1. Introduction

Staphylococcus epidermidis is the main cause of most infections related to medical devices and the second major cause of infection related to prosthesis insertion. This is because the bacterium colonizes human skin, increasing the chances of contamination during the insertion of any type of prosthesis/implant inside the human body. S. epidermidis is facultative anaerobic, catalase-positive, coagulase-negative, and gram-positive. Colonies can be up to 1 μm in diameter and are organized in pairs or groups of four. Its colonies measure between 2.5 and 4 mm.

Although S. epidermidis infections do not represent a risk to human life (class I), they occur very frequently and are difficult to treat. In catheters and implants, bacteria colonize the external and internal regions of the material and proliferate at 0.5 cm² per hour, being able to form a thick biofilm in 24 h. The infection route begins with the migration of the microorganisms from the skin to the insertion site, where they adhere to the catheter or implant, forming small colonies and eventually detaching themselves and migrating to the rest of the body [1]. The formation of biofilms by S. epidermidis makes it very difficult to treat, since biofilms protect the bacteria from antibiotics and the immune system. In addition, S. epidermidis has developed resistance to several antibiotics (rifampicin, gentamicin, and fluoroquinolones), further complicating its elimination [2].

Disinfection is considered a process that eliminates many or all pathogenic microorganisms, except bacterial spores. In healthcare settings, objects are usually disinfected by liquid chemicals or wet pasteurization. Many disinfectants are used alone or in combinations (e.g., hydrogen peroxide and peracetic acid). These include alcohols, chlorine and chlorine compounds, formaldehyde, glutaraldehyde, ortho-phthalaldehyde, hydrogen peroxide, iodophors, peracetic acid, phenolics, and quaternary ammonium compounds. Commercial formulations based on these chemicals are considered unique products and must be registered and cleared by the European Medicines Agency (EMA) [3]. However, occupational diseases among cleaning personnel have been associated with the use of several disinfectants (e.g., formaldehyde, glutaraldehyde, and chlorine). Asthma and reactive airway diseases can occur in sensitized persons exposed to germicides. In fact, asthma can even occur at levels below the ceiling regulated by the European Agency for Occupational Safety and Health Administration (EU-OSHA) [4].

The growing resistance to antibiotics, such as third-generation cephalosporins and aminoglycosides, highlights the urgent need to develop new alternatives for hospital disinfection. Despite the effectiveness of antiseptics like chlorhexidine, surgical infection rates remain high, especially in patients with medical devices or complicated wounds [5]. In this context, the development of new disinfection and sterilization techniques is of great interest.

Supercritical CO₂ (scCO₂) microbial inactivation has many advantages over these techniques. It is a process that works with an inert, odorless, incombustible, non-toxic, accessible, inexpensive gas that leaves no residues once the process is finished [6]. In addition, it allows working with thermosensitive and hydrosensitive materials. The operation temperature is low, and the pressure is moderate due to the mild critical point conditions of CO₂ (31 °C and 7.4 MPa). Above the critical point, a dense fluid phase is reached, with low viscosity and zero surface tension, thus granting penetrating properties to reach every nook, cranny, or pore of the structures to be disinfected [7].

The scCO₂ disinfection proceeds through serial complex mechanisms which include the modification of the cell membrane, the decrease in intracellular pH (due to the formation of carbonic acid) that causes inactivation of key enzymes and the inhibition of cellular metabolism, etc. CO₂ itself can cause other intracellular disorders such as electrolyte imbalance. Finally, the removal of vital constituents from the cell and cell membranes can also take place [7,8].

The conditions at which scCO₂ is applied affect the antimicrobial degree achieved. The rise in pressure leads to an increase in CO₂ solubilization (in aqueous environments), improving its penetrating power in the microorganism. The increase in process temperature makes the cell membrane more fluid, allowing CO₂ to penetrate more easily. Temperature also has an impact on the microorganism’s death per sé, due to the profound effect of heat on its structures and physiological properties [9]. However, high temperatures can have an adverse effect on the materials to be disinfected. So, this variable must be kept as low as possible. A prolonged operation time has a positive influence. Additionally, a high depressurization rate enhances the transfer of matter across the membrane and may even cause the cell to burst [7,10].

Essential oils (EOs) are substances obtained from plants that have antimicrobial power with less toxicity and less chemical aggressiveness than conventional antibacterial agents [11,12]. These oils contain different components, usually two or three in high concentrations, which determine the biological activity whilst the rest are found in traces. For example, in oregano oil, the major components are carvacrol (30%) and thymol (27%). In peppermint oil, the major components are menthol (59%) and menthone (19%) [11]. With a highly diversified group of phytochemicals, these components are largely classified into two groups: terpenes and terpenoids, and aliphatic and aromatic compounds, characterized by their low molecular weight. Among the terpenes and terpenoids (terpenes with an alcohol group), monoterpenes (C₁₀H₁₆) and sesquiterpenes (C₁₅H₂₄) stand out, although there are terpenes of even longer chains.

In addition, studies carried out by Bagheri et. al. have confirmed that there may be a difference in the antimicrobial activity of the same essential oils from different suppliers, and this could be due to the different origin, harvesting, and distillation conditions, and to the synergistic effect of the components that are found in lower concentration [13].

Although the mechanism of antimicrobial action of each essential oil is specific depending on the microorganism, reviews in the field point to cell membrane damage as the initiating event. Other phenomena may follow due to the considerable number of functional groups contained in essential oil components, including leakage of ions and metabolites, and of cytoplasmic structures, coagulation of the cytoplasm, etc. [14]. On the other hand, synergy with essential oils has been witnessed with other food preservatives, or antibiotics as it was recently reported for peppermint essential oil (PEO) [14,15].

So far, the incorporation of essential oils in food and in active packaging has been mostly investigated to prevent microbial proliferation and increase durability. But lately the use of essential oils in medical applications is attracting attention. Rosu et al. prepared emulsions based on PEO with various polymeric matrices to treat cotton fabrics spiked with cultures of Staphylococcus aureus and Escherichia coli [16]. Likewise, Robu et al. developed bone cements of two distinct kinds, impregnated in PEO demonstrating its biocompatibility and antimicrobial properties against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans [17,18].

This work proposes a new method of disinfection for the hospital environment based on the use of scCO₂ in combination with essential oils in small quantities between 200 ppm (0.02%) and 1000 ppm (0.1%) in volume. Both reagents are extremely low toxic and chemically unaggressive, and have not been used in conjunction in this field up to now. The mixture was evaluated to inactivate S. epidermidis on a cotton fabric widely used in dressings and hospital clothing, specifically, on a sterile gauze.

The proposal is based on our previous work performed on food products. The first consisted of the inactivation of paprika natural microflora using scCO₂ mixed with oregano essential oil [19]. The second was carried out on the inactivation of Clostridium spores in honey using scCO₂ supplemented with cinnamon oil [12]. Satisfactory results were obtained in both cases. In the first case, 99.5% microbial inactivation (mainly thermoresistant bacillus type spores) was achieved by applying a treatment of 30 min and 80 °C with scCO₂ mixed with 2.58% of the oregano oil. In the second case, a degree of inactivation of almost 4 log units was obtained in the Clostridium spores at 60 °C in a time of 60 min using scCO₂ modified with 0.4% in mass fraction of cinnamon oil. Another research group chose to immerse almonds in 50 mL of thyme oil for 10 min and treated them further with the scCO₂ to inactivate Escherichia coli. A log reduction of 5.16 colony forming units per gram (cfu/g) was achieved after treatment at 70 °C and 10 MPa for 30 min [20].

In this research, supercritical CO₂ was applied at 10.0 MPa as this moderate pressure is enough for this procedure. In addition, a very mild temperature of 40 °C was used since we were pursuing a technique friendly with thermolabile materials. In the healthcare system, many instruments and devices are made of materials (such as polymers) that cannot withstand elevated temperatures.

After the treatment, the gauze was evaluated using Differential Scanning Calorimetry (DSC) and Scanning Electron Microscopy (SEM) techniques. When using essential oils, it is also important to consider the possible residues that could impair olfactory or color changes in the disinfected material, so the presence of EOs residuals in the gauze was analyzed using Fourier Transform Infrared (FTIR) spectroscopy.

2. Materials and Methods

2.1. Chemical and Reagents

Essential oils: oregano CAS 8007-11-2, lemon CAS 8008-56-8, clove CAS 8000-34-8, cinnamon CAS 8015-91-6, and thymol CAS 89-83-8 were purchased from Sigma Aldrich-Merck^® (USA). Peppermint essential oil organic and conventional CAS 8006-90-4 was kindly donated from Ventos^® (Barcelona, Spain). The differences in the composition of the organic oil compared to the conventional one are shown in

Table 1. Comparative composition of organic and conventional peppermint essential oil.

Peppermint Essential Oil	Origin	Composition
Organic	Mentha piperita L. (Flowering tops)	Menthol racemic (≥25%; <50%), menthone (≥25%; <50%), eucalyptol (≥1%; <10%), isomenthone (≥1%; <10%), menthyl acetate (≥1%; <10%), menthofuran (≥1%; <10%), linalool (≥1%; <10%), D-limonene (≥1%; <10%), beta-caryophyllene (≥1%; <10%), pulegone (≥1%; <10%), beta-pinene (≥1%; <10%), alpha-pinene (≥1%; <10%).
Conventional	Mentha piperita L. (Leaves and flowers)	L-Menthol (≥25%; <50%), L-menthone (≥10%; <25%), menthofuran (≥1%; <10%), menthyl acetate (≥1%; <10%), isomenthone (≥1%; <10%), neo-menthol (≥1%; <10%), limonene (≥1%; <10%), pulegone (≥1%; <10%), para-cymene (≥1%; <10%).

.

Other reagents used for the preparation of nutrient broths were as follows: meat extract CAS 68990-09-0, casein trypsin peptone CAS 91079-40-2, and Baird Parker agar from Scharlau^® (Spain); yeast extract CAS 8013-01-2 and peptone CAS 73049-73-7 from Fluka^® (Spain); and sodium chloride CAS 7647-14-5 with purity ≥ 99.5% from Sigma-Aldrich^®. The sterile gauze was from Codix^® (Spain). The microorganism, Staphylococcus epidermidis was obtained from Spanish Type Culture Collection (Spain) (CECT 231, ATCC 12228). Carbon dioxide with purity ≥ 99.98% was supplied by Air Products^® (Spain).

2.2. Culture and Growth of the Microorganism

S. epidermidis was recovered from the lyophilized state. For this, it was reconstituted by adding nutrient broth culture medium, sown, and then kept frozen (−80 °C) until used. For reactivation, it was inoculated in an Eppendorf with 1 mL of nutrient broth for 2 h, then it was escalated in a Falcon tube to a volume of 10 mL with the same medium where it remained for 24 h. Subsequently, 5 mL of this culture was inoculated in a bottle with 200 mL of nutrient broth and kept in a bath at a temperature of 37 °C equipped with gentle lateral shaking for 24 h (VWR International, Grant Instruments, Leighton Buzzard, UK) to achieve a concentration of bacteria high enough to verify the inactivation. Nutrient broth to reactivate the microorganism was prepared with 5 g of meat extract, 10 g of peptone extract, 5 g of sodium chloride, and 1 L of Milli-Q water. This preparation was heated to boiling point to dissolve all components and then sterilized at 121 °C for 15 min in an autoclave Med 12 (J. P. Selecta, Barcelona, Spain).

2.3. Biocontamination Procedure

The gauze was atomized with a suspension of S. epidermidis in a laminar flow cabinet (Mini-V/PCR, Telstar, Spain). On average, the contamination achieved was 10⁵ ± 10 colony forming units per gram (cfu/g).

The contaminated material was then divided into two parts and dried at room temperature within the laminar flow cabinet. One part was subjected to the CO₂ treatment and the other part was used as control to compare the degree of growth of the microorganism.

2.4. High-Pressure Equipment and the Disinfection Method

The equipment mainly consisted of a CO₂ bottle, a refrigerated bath (Selecta, Frigiterm-30, Barcelona, Spain), a pump (Milroyal D, Dosapro Milton Roy, Tresses, France), a high-pressure vessel of 50 mL of 316 SS (Autoclave Engineers, MicroClave™, Series 401A-8067, PA, USA) equipped with agitation, and a pressure control valve. The schematic is depicted in Figure 1. All connections were fabricated from 1/8 inch of 316 SS.

The contaminated gauze and a piece of cotton impregnated with a certain amount of additive, in the presence or absence of water, were placed inside the vessel. Glass beads were used as supports to separate them, avoiding direct contact between the additive and the gauze and reducing the void volume of the vessel. The CO₂ left the bottle at its vapor pressure, 5.5–6.0 MPa. Then it was subcooled in a bath at −10 °C to avoid cavitation during pumping and was pressurized to 10.0 MPa. Then it was heated isobarically to 40 °C through a heating jacket. At the end of the established time of 30 min, the pressure was gradually decreased by opening the BPR valve. A constant slow depressurization rate was maintained, the average being 1.0 MPa/min.

2.5. Seeding and Counting

After decompression, the vessel was removed by opening its closure, immediately covered with a sterile petri dish and taken to laminar flow cabinet. Then the gauze was removed, placed in a Falcon tube with 10 mL of sterilized water and mixed for 30 s. Several dilutions were successively prepared by taking 1 mL of the suspension and adding 9 mL of sterilized water. Then 1 mL was sown on a plate with Baird Parker agar and incubated at 37 °C for 48 h (Incubator IN30, Memmert, Büchenbach, Germany) side down. Finally, the colonies were counted (Colony counter SC6 Stuart, VWR, Hayes, UK).

The total number of microorganisms (N) was calculated according to UNE-EN ISO 7218 [21] using the following formula:

N = ΣC/(V × 1.1 × d)

where ΣC is the sum of the colonies of the plates in two consecutive dilutions (where the most diluted is the first plate to have more than 10 colonies among all those seeded), V is the inoculum volume (1 mL), and d is the dilution factor of the first dilution chosen.

The effectiveness of microbial inactivation was expressed in terms of logarithmic reduction, using the count of microorganisms in the control (N₀) and treated (N) samples and calculating the logarithm of the quotient between N and N₀, obtaining a negative value.

2.6. Analysis of the Material after Treatment

The gauze was evaluated before and after inactivation using DSC, FTIR, and SEM techniques, to ensure its integrity and to verify any structural changes that could affect the viability when using or reusing it.

SEM was performed in JEOL6400 equipment (Tokyo, Japan), with resolution: 3.5 nm (35 kV, WD: 8 mm) and magnification: 10x to 300,000x (WD = 39 mm). As the gauze was not conductive, it was coated with gold (sputtering).

A Differential Scanning Calorimeter (DSC Q20 TA Instruments, New Castle, DE, USA), connected to a refrigerating cooling system, in dry nitrogen, flowing at 50.0 mL/min, was used for thermal analysis. An MT5 Mettler microbalance (Madrid, Spain) was used to weigh the samples, ranging between 3 and 10 mg (with an error of ± 0.001 mg). Temperature and enthalpy of the calorimeter were previously calibrated using standard samples of Indium (purity > 99.999%). The samples of gauze were heated from −50 °C to 275 °C at 10 °C min⁻¹.

For the FTIR analysis, a Perkin Elmer Precisely Spectrum 100 FTIR equipment (MA, USA), with the universal attenuated total reflection (ATR) sampling accessory at a resolution of 4 cm⁻¹, was used. Spectra were recorded between 650 to 4000 cm⁻¹.

2.7. Statistical Analysis

Data shown are an average of two or three replicates with double plate readings for each dilution. Tests were run six times to estimate an average standard deviation that was ±0.3 logs.

Data were statistically analyzed by a multifactor analysis of variance (multifactor ANOVA). The LSD (least significant difference) procedure was used to test the differences with a 95% confidence interval using Statgraphics Centurion 19 (Statpoint Technologies, Inc., Warrenton, VA, USA). The significance level was set at p ≤ 0.05. The result of this analysis is shown in Supplementary Material.

3. Results and Discussion

Inactivation tests of S. epidermidis were carried out on a gauze using CO₂ mixed with three concentrations (1000, 500, and 200 ppm in volume) of different essential oils (oregano, lemon, clove, cinnamon, and peppermint). In addition, experiments were carried out to compare the effect of the use of an essential oil with that of an isolated component, thymol, a principal component of oregano essential oil.

Figure 2 shows the degree of logarithmic reduction achieved according to the additive. The red line in the graphs indicates the decimal logarithm of the initial load of S. epidermidis. It is drawn to indicate what the degree of complete inactivation would be. Note that it is different according to the culture used in each set of experiments. To facilitate interpretation, the degree of inactivation achieved with scCO₂ alone is also shown in each graph with the silver bar.

In general, the effectiveness of inactivation increased as the amount of additive was raised. In the case of oregano EO, total inactivation was achieved at 1000 ppm, a fact that did not occur with lemon EO, which was far from this level. Moreover, the difference between using the maximum or minimum concentration of this additive was not more than 0.8 log reductions. So, lemon EO can be considered the additive with the least effect on the inactivation of S. epidermidis when combined with CO₂. Figure S1 shows the mean of the inactivation degree achieved with each essential oil.

With respect to the other additives used, clove EO, cinnamon EO, peppermint EO, and thymol, a different trend was observed. In these cases, the difference between 1000 and 500 ppm was null or very little. Furthermore, in the case of peppermint EO, total inactivation was achieved at both concentrations. However, in all of them, there was a significant (p ≤ 0.05, Figure S2) difference in microbial inactivation of about 0.5 log when decreasing the concentration to 200 ppm.

To better appreciate the antimicrobial power of the additives, Figure 3 shows a comparison of all of them at the same concentration of 200 ppm. In the case of peppermint EO, it was achieved at approximately 4 log reduction, which was even greater than any other additive at its highest concentration (as shown in Figure 3). Peppermint EO was followed in efficiency by clove EO, with a difference between the two of 1.3 logs. Another important aspect to highlight is that thymol and the EOs of oregano, lemon, and cinnamon, at 200 ppm, did not significantly improve the bacteria inactivation with respect to that achieved by CO₂ alone.

In previous studies, thyme essential oil stood out when it came to inactivating microorganisms of a similar structure to S. epidermidis, such as S. aureus [22]. In the present work, its major component in isolation (thymol) had a reliable performance but not as good as the clove or peppermint EOs with all their components. This confirms the well-known synergistic effect of the EOs’ compounds [14,15].

It can be concluded, therefore, that at a concentration of 200 ppm, the essential oils studied did not achieve significant (p < 0.05) microbial inactivation, except for peppermint EO, with about 2 logs more reduction than CO₂ alone.

3.1. The Influence of Moisture

The influence of moisture on the inactivation of S. epidermidis was investigated by adding water to CO₂ in amounts of 200, 500, and 1000 ppm. It is of practical interest if some materials arrive wet (e.g., body fluids) for hypothetical application in the healthcare environment. The results obtained are presented in Figure 4.

The presence of water increased the effectiveness of the inactivation with CO₂, almost 1.0 log reduction when increasing from 200 to 1000 ppm of water, and 2.5 log cycles when compared to the inactivation achieved with CO₂ alone. Previous studies have shown that the presence of water favors the antimicrobial power of the scCO₂ [12,19] since cell swelling occurs, improving its penetration through the membrane.

With these premises, peppermint essential oil (PEO) and water were combined in quantities of 200 ppm each in the following experiments. Tests were carried out with two different types of peppermint essential oil, and the results can be seen in Figure 5.

The degree of inactivation obtained by using the organic PEO and water increased. However, it seems to be an additive and not a synergetic effect. Using organic PEO as the additive, the degree of inactivation was 3.9 logs, and with the addition of water it reached 5.8 logs (total inactivation).

3.2. Influence of the Origin of the Peppermint Essential Oil

Significant differences (p < 0.05, Figure S3) were observed between the organic peppermint essential oil and the conventional type, also shown in Figure 5. While with the conventional oil, 3.2 logs were obtained, with the organic one, total inactivation was achieved (null colony plate count); both in the presence of 200 ppm of water. This result is attributed to differences in the composition of the two oils. Compared to the conventional oil, the organic PEO contains a higher proportion of racemic menthol and menthone, along with other volatile compounds such as eucalyptol, beta-caryophyllene, and the monoterpenes alpha- and beta-pinene, all of which are not present in the conventional PEO and are known for their antimicrobial properties [23]. The presence of these compounds and their potential synergy enhances the antimicrobial potential of the organic PEO, explaining its superiority against S. epidermidis. In contrast, the conventional oil, rich in L-menthol but with a lower amount of menthone, presents a lower diversity of secondary compounds, which could limit its effectiveness. Consequently, the synergism or additive effect of the phytochemicals present in the essential oils is confirmed.

In addition, the antibacterial capacity of essential oils sold under the same brand and by the same supplier may vary from batch to batch. This could be related to the growing conditions of the plants, the geographical area, the harvesting period and method, and the extraction process, which will determine the composition and therefore the activity [14,22].

3.3. Influence of the Disinfection Treatment on the Properties of Gauze

The sterile gauze is made up of intertwined warp and weft thread cotton fibers. Next is reported the analysis of the gauze when it was treated with scCO₂ mixed with 200 ppm of the peppermint essential oil in the presence of 200 ppm of water, as this was the minimum concentration for the total inactivation of S. epidermidis. It was evident that when applying the highest concentration of the essential oils (1000 ppm with respect to the scCO₂), the gauze retained part of it, causing a high sensory impact, both olfactory and visual, giving rise to a colored gauze.

3.3.1. Scanning Electron Microscopy (SEM) Analysis

SEM images of the samples are compiled in Figure 6. In the lower magnification image, a slight fraying can be observed in the treated gauze (Figure 6d) in comparison with the untreated one (Figure 6a). This could be due to the influence of scCO₂ and essential oils, but mainly to the depressurization. Similar conclusions were previously reached [24,25]. Nevertheless, the cotton fibers maintained their size and shape.

Although the mechanical properties of the gauze were not evaluated, a previous study demonstrated that the physicochemical properties of FFP3 masks, with a layer of cotton in their structure, were not altered after up to 10 sterilization cycles under the same scCO₂ applied conditions as those used in this project [26].

3.3.2. Differential Scanning Calorimetry (DSC) Analysis

Figure 7 plots the thermal analysis of the gauze. The DSC curve shows, at approximately 100 °C, an endothermic peak in both the treated and untreated samples, indicating the presence of adsorbed water. Then, from 140 °C until approximately 275 °C, DSC curves of the treated and untreated samples were similar, with no relevant thermal events, and with homogeneous heat flow in both cases. Consequently, treatment with supercritical CO₂ and peppermint EO in the presence of some moisture (200 ppm) did not affect the thermal properties of the gauze.

3.3.3. Fourier Transform Infrared (FTIR) Spectroscopy Analysis

Figure 8 compares the infrared spectrum of an untreated gauze, a gauze treated only with CO_2, and a gauze treated with CO₂ and the mixture of peppermint essential oil and water. The FTIR spectrum of the gauze shows a wide absorption band between 3600 cm⁻¹ and 3000 cm⁻¹, which corresponds to the stretching vibrations of the cellulosic OH groups and H₂O. The absorption bands between 3000 cm⁻¹ and 2800 cm⁻¹ are attributed to the C-H stretching. The bands at 1160 cm⁻¹ and 1108 cm⁻¹ correspond to asymmetric C-O-C bridges, and the bands at 1030 cm⁻¹ and 1000 cm⁻¹ are assigned to C-O stretching vibrations. Finally, the absorption band at approximately 900 cm⁻¹ is attributed to the asymmetric out-of-phase ring stretching at C₁-O-C₄ β-glucosidic bond. All these bands are also observed in the FTIR spectra of the treated gauzes as being very similar. In general, there is a coincidence on the typical absorption bands of cotton that has also been related by other authors [24,27,28].

Figure 8a also shows the infrared spectrum of the peppermint essential oil to assess whether this EO could be found in the treated gauzes, proving that none of the characteristic bands in the peppermint essential oil were found. It seems that the CO₂ removed all the essential oil, preventing it from remaining on the gauze fabric. At 40 °C and 10 MPa, the solubility of this essential oil is relatively high, in the order of 0.3–0.4% in mass fraction [12].

This aspect is especially important because a significant amount of peppermint EO present in the gauze could be harmful to the skin. Based on the safety data sheet of this oil, some of its constituents are categorized as level 2 for risk of skin irritation, so professional exposure limit values are established by the legislation: Alpha-Pinene and Beta-Pinene in 20 ppm, D-Limonene in 30 ppm; in all cases with exposure limit times of 8 h. Note that these components constitute less than 10% of the essential oil and that the amount of peppermint EO used in the experiments was small (200 ppm) so it would be difficult for this amount to pose a risk.

Moreover, there are already medicines containing peppermint essential oil on the market (toothpaste, mouthwash, topical gels, etc.). The amount of peppermint essential oil in these preparations varies depending on the formulation but is in general much greater than that used in this research, in semi-solid and oily preparations 5–20%, in aqueous preparations of ethanol 5–10%, and in nasal ointments 1–5% [29].

4. Conclusions

The scCO₂ with small amounts of peppermint essential oil (200–500 ppm) could be considered an effective disinfection method. After the treatment of cotton gauzes contaminated with S. epidermidis, up to 4 log reductions of the bacteria was achieved. In practice, this log reduction is higher than the biocontamination of the microorganism. Future work should be performed on other microorganisms typical of hospital infections, such as methicillin-resistant Staphylococcus aureus (MRSA) or multi-resistant Gram-negative bacteria. It is also necessary to test it on other materials in the hospital field, such as implants, catheters, instruments, etc., to be able to propose this technology as a general method of disinfection. The impact on the quality of the treated material must also be demonstrated in each case. In this example, there were no significant physical changes in the treated gauzes at the optimal concentration of 200 ppm. This is especially important in the event of necessary reuse, as recently occurred in the COVID-19 crisis with many sanitary materials.

An important advantage is that peppermint EO is quite soluble in supercritical CO₂ and, as demonstrated by infrared spectroscopy, it is unlikely that there would be remaining peppermint oil in the gauze. If there was any, it would lead to a low intensity and recognizable pleasant odor, which would not impair any toxicity or skin irritation.

Current disinfection techniques are based on the use of very toxic chemicals and are sometimes aggressive towards the treated product and the operators. In our proposal, in which CO₂ is valorized, the medium is innocuous and environmentally friendly. Therefore, this method of microbial inactivation could be well accepted in the healthcare field.

Limitations to the use of essential oils as scCO₂ additives include variability in chemical composition which could affect the reproducibility of results. Fortunately, the essential oil producers provide the composition of each batch and show, with harvest control, a continuity in the quality of each variety.