Microemulsification of essential oils for the development of antimicrobial and mosquito repellent functional coatings for textiles

To develop an essential oil (EO)‐loaded textile coating using an environmentally friendly microemulsion technique to achieve both antimicrobial and mosquito repellent functionalities.


Introduction
Consumer demand for functional textiles has increased in recent years. Antimicrobial textiles are of particular interest due to an increase in awareness of deleterious effects of microorganisms on textiles (Riaz and Ashraf 2020). The moisture content and nutrient availability in textiles offer a favorable environment for the growth of bacteria and fungi, leading to the formation of odor and deterioration of the textiles, in addition to potentially contributing to the spread of pathogenic microorganisms (Sterndorff et al. 2020;Sanders et al. 2021). Antimicrobial textile finishes offer a variety of potential applications including prevention of odor (Morais et al. 2016) or as treatment or prophylaxis for skin infections, such as tinea pedis, caused by the dermatophyte Trichophyton rubrum (Gupta and Versteeg 2019), and atopic dermatitis, which is associated with Staphylococcus aureus colonization (Srour et al. 2019).
Several currently available textile finishes are potentially toxic to humans and the environment. For example, triclosan accumulates within the aquatic environment, where it is toxic to aquatic organisms; triclosan is also potentially toxic to humans, with a number of studies demonstrating cell cytotoxicity and endocrine disrupting properties and may induce multidrug resistance in bacteria (Zheng et al. 2019). This has resulted in the need for effective antimicrobial finishes that are safe, biodegradable, environmentally non-toxic and limit the development of microbial resistance.
Natural products could offer an environmental-friendly and biodegradable alternative to currently employed biocides for antimicrobial textile finishes. Essential oils (EOs) are aromatic natural products, typically extracted from plant matter by distillation (Georgiev et al. 2019). EOs have been a subject of interest as alternative antimicrobial agents, with a body of research suggesting that they convey broad spectrum antimicrobial activity. For example, cinnamon bark inhibited E. coli, S. aureus, Pseudomonas aeruginosa and Acinetobacter baumannii with minimum inhibitory concentrations (MICs) ranging 0Á015-0Á125%, and all tests species except P. aeruginosa were inhibited by cinnamon leaf, clove, lemongrass, rosewood and thyme EOs with MICs ranging 0Á125-1Á0% (Elcocks et al. 2020).
EOs could potentially be incorporated into textiles to create antimicrobial fabrics, however EO compounds are often volatile, and sensitive to light and oxygen. The successful application of EOs onto textiles requires a formulation that protects the EOs from volatilization and degradation and controls its release rate to prevent unacceptable deterioration of the final product (Volić et al. 2020). Microemulsification of EOs may preserve the functional and physicochemical properties of the oil and allow for greater durability of the final product; Sayed et al. (2017) applied a nanoemulsion encapsulating neem EO on cotton fabric and reported a 71Á73 and 65Á69% reduction of S. aureus and E. coli after four washes. Biopolymers including chitosan and alginates are an attractive option for drug delivery systems due to their favorable biodegradable, biocompatible and mucoadhesive properties (Gómez-Guillén and Montero 2021). Previous research demonstrated the successful encapsulation of EOs within biopolymers and their use within antimicrobial textile finishes; cotton treated with alginate and gelatin-encapsulated lime EO was antimicrobial against Klebsiella pneumoniae, E. coli, Staphylococcus epidermidis and S. aureus according to the disc diffusion method (Julaeha et al. 2021).
Textile finishes based upon encapsulated EOs have also been shown to possess mosquito repellent activity. For example, Grancaric et al. (2020) reported 100% repellency of Aedes aegypti mosquitos by cotton fabric treated with microencapsulated immortelle EO. Mosquitos are important vectors for several diseases, for example the female A. aegypti mosquitoes are vectors for the transmission of yellow fever, dengue fever and Zika fever viruses. Current commercially available mosquito repellants such as N,N-diethyl-3-methylbenzamide (DEET) have shown potentially serious adverse skin reactions (Azeem et al. 2019), providing a rationale for the development of novel mosquito repellents.
Therefore, the aim of this study is to develop a functional antimicrobial cum mosquito-repellent coating for textiles based on EO-loaded microemulsions using a novel green polyelectrolyte micro-assembly technique. It is envisioned that the EO-loaded microemulsion selfassembly prepared in this study will provide a tool for controlled (sustained) delivery of a combination of EOs with synergistic antimicrobial activity against the skin associated pathogens including S. aureus, P. aeruginosa, S. epidermidis, E. coli and T. rubrum as well as potential mosquito repellency effects. These provide unique advancements in science of the application of EOs over the existing knowledge in literature.

Antimicrobial activity of EOs
Screening for antimicrobial activity Trichophyton rubrum total spore suspensions were prepared by scraping spores from a pre-cultured agar plate with 1 ml of 0Á01% polysorbate 80 solution (Fisher Scientific, Loughborough, UK). The resulting suspension was filtered through five layers of muslin cloth and washed thrice (2000 g, 5 min) in sterile distilled water. The total spore count was adjusted to 10 7 colony forming units (CFU) per ml using a hemocytometer.
A method adapted from Fisher and Phillips (2006) was used. Aliquots (50 μl) of T. rubrum spore suspension (10 7 CFU per ml) or overnight cultures of S. aureus, P. aeruginosa, E. coli, and S. epidermidis (10 8 CFU per ml) were spread on BHI agar plates before filter paper discs (20 mm, Whatmann, Maidstone, UK) were placed on the surface and impregnated with 25 μl EO. Plates were incubated at 37°C for 18 h (bacteria) or 30°C for 7 days for T. rubrum before zones of inhibition (ZoIs) were measured. Controls were plates without oil impregnated filter discs.

Minimum inhibitory concentrations
MICs were determined using a method adapted from the International Standards Office (2006) broth microdilution method. Doubling dilutions of EOs (final concentrations 0Á1-40 μl ml −1 ) were prepared for litsea, lemon and rosemary EO in BHI or SD broth supplemented with 10% dimethyl sulfoxide (DMSO; Fisher Scientific). Aliquots (100 μl) of the EO suspension were mixed with an equal volume of T. rubrum spore suspension (10 7 CFU per ml) or overnight cultures of S. aureus, P. aeruginosa, E. coli or S. epidermidis (10 8 CFU per ml) in 96 well plates. A control of 10% v/v DMSO only was included.
For bacteria, the MIC was determined by measuring optical density (595 nm) before and after 24 h incubation at 37°C using a SpectraMax Plus 384 microplate reader with SoftMax Pro version 6.4 software (Molecular Devices, San Jose, CA). For T. rubrum, the MIC was determined by visually inspecting wells for growth after incubation at 30°C for 7 days.

Fractional inhibitory concentrations
A method adapted from Owen et al. (2017) was used. Serial dilutions of rosemary, lemon and litsea EOs were prepared in BHI broth supplemented with 10% DMSO (bacteria) or 10% polysorbate 80 (T. rubrum spores) before inoculation with the microbial test species. Double and triple combinations of EOs were prepared in 96 well plates to yield a matrix of varying concentrations of each EO (0-4Á5 μl ml −1 litsea EO, 0-20 μl ml −1 lemon EO and 0-45 μl ml −1 rosemary EO) and mixed by pipetting. The MICs of each combination was determined as described above and used to calculate the fractional inhibitory concentration (FIC) and FIC index (FICI) according to Eqn 1: where FICI ≤0Á5 indicates a synergistic effect, whilst 0Á5 ≤FICI ≤4 indicates no interaction and FICI >4 indicates an antagonistic effect (Odds 2003).
Gas chromatography-mass spectroscopy of Litsea and lemon EOs The major components of litsea and lemon EOs were determined using a Bruker (Billerica, MA) 450-RC gas chromatograph (GC) equipped with a Rxi-5ms (Restek, Bellefonte, PA) column (30 m × 0Á25 mm i.d. × 0Á25 µm film thickness) and coupled with a 300-MS SQ signal electron impact mass spectrometer (MS). EOs were diluted 1 : 100 in n-hexane (Fisher Scientific), filtered through a 0Á45 µm polyethylene (PET) filter (Sigma-Aldrich) and 1 μl was injected into the GC (injection temperature 280°C; split ratio 1 : 100) using a Bruker (Billerica, MA) CP8400 autosampler. Helium was used as a carrier gas at 1Á5 ml min −1 . Oven temperature was held at 60°C for 5 min, followed by a 4°C min −1 ramp to 220°C before an 11°C min −1 ramp to 250°C, held for 15 min. MS was conducted in positive mode with a source temperature of 230°C, ionizing energy of −70 eV, CID gas pressure of 1Á5 mTorr and a detector voltage of 1000 V. Mass spectra were acquired over a mass range of 50-350 m/z. EO components citral and limonene were identified by comparing with retention time and mass of analytical grade standard reference compounds (citral (99%), (R)-limonene and (S)-limonene; Sigma Aldrich). Calibration curves were prepared for citral and limonene between 0Á78 and 200 mmol l −1 (R 2 = 0Á99, data not shown).

Microemulsion preparation
Oil in water (o/w) microemulsions of litsea-lemon EO blend (1 : 2 ratio) were prepared by mixing the EO blend (30% v/v final concentration) with sodium alginate solution (Sigma Aldrich; 0Á1% w/v final concentration) for 5 min before adding chitosan solution (Sigma Aldrich; 1Á0% w/v final concentration) and homogenizing for 5 min at 8000 rev min −1 (IKA Ultra-Turrax ® disperser, Staufen, Germany). Calcium chloride solution (Fisher Scientific; 0Á1% final concentration) was added dropwise under further homogenization at 8000 rev min −1 for 1 h. All microemulsions were cured by storing them for 24 h at room temperature for elastic recovery, and at 40°C for the same period for consolidation of the crosslinked polyelectrolyte micro-assembly respectively, prior to further analysis. The pH was measured using a Mettler Toledo (Columbus, OH) FG2-Kit pH Meter (target pH <5).
The percentage incorporation efficiency (%IE) of the EOs in the microemulsions was calculated from Eqn 2.
where M ME is the amount of the limonene and citral exhaustively extracted from the microemulsion and M EO the initial amount of limonene and citral extracted in the neat EOs. M ME and M ME were determined from the area under the curve and peak height of the GC-MS spectra within the calibration standards of this study. All measurements were an average of six determinations.
Microemulsion particle size Particle size and particle size distribution of the litsealemon EO microemulsion were determined by dynamic light scattering (DLS) using a NanoBrook Omni particle sizer (Brookhaven Instruments, Holtsville, NY). The microemulsion was diluted 1 : 100 in distilled water and measurements performed in triplicate five times at 25°C. Span value for the distribution of particle sizes (polydispersity index; PDI) was determined according to Eqn 3: where d(90), d(50) and d(10) are the particle diameters at 90, 50 and 10% cumulative volume, respectively (Campelo et al. 2017).

Microemulsion physical stability
The flocculation/creaming stability of the microemulsion was assessed by storing 5 ml emulsion in measuring cylinders at room temperature for 5 weeks, with daily observation and recording of the volume of creaming. The Creaming Index (CI) was estimated according to Eqn 4: where H c is the height of clear layer below the sample and H t the total emulsion height.

Microemulsion long-term physical stability
The accelerated stability of the microemulsion was assessed by centrifuging at 3549 g for 2 h. The emulsions were observed every 5 min for separation of the internal phase and the CI under stress conditions was characterized. All samples were analysed in triplicate.

Microemulsion chemical stability
The concentration of citral within the microemulsion was assessed at zero and 28 days by shaking 1 ml emulsion in 5 ml of n-hexane (Fisher Scientific) for 5 min in a 50 ml separating funnel before GC-MS analysis as described above.
Treatment of polyester and cotton fabric with the Litsealemon EO microemulsion Knitted bleached cotton and knitted polyester fabric samples were scoured with 2 g l −1 non-ionic surfactant Ultravon PL (Ciba Specialty Chemicals, Basel, Switzerland) for 30 min at 60°C, followed by a hot and cold-water rinse to remove the surfactant. The fabric was sterilized at 160°C for 2 h before soaking for 15 min in the microemulsion. Soaked fabric samples were passed through a laboratory pad (Ernst Benz, Eysins, Switzerland) at 35 kg cm −2 pressure and 1 m min −1 ; re-soaked and padded again. All investigations were conducted using freshly treated fabric dried for 24 h at room temperature, unless otherwise specified.

Distribution of major EO components on Litsea-lemon EO microemulsion treated fabric
Microemulsion-treated polyester and cotton fabric samples (1, 2 and 16 cm 2 ; 24 h post-treatment) were subject to solvent extraction in n-hexane; the citral and limonene content was then quantified using GC-MS as described above. Identical treated polyester and cotton fabric samples were also stored at 4°C for 7 days prior to determining the citral and limonene content.

Antimicrobial activity of the litsea-lemon EO microemulsion
Screening for antimicrobial Activity The antimicrobial activity of the litsea-lemon EO microemulsion, the microemulsion components (10% litsea EO, 20% lemon EO, 1% chitosan solution, 1% sodium alginate) and major chemical components of litsea and lemon EO (citral, R-limonene and S-limonene) was assessed against S. aureus, S. epidermidis and E. coli using the disc diffusion screening method described above, however 6 mm filter paper discs were used. histidine (Sigma Aldrich), 30 g l −1 polysorbate 80 (Fisher Scientific), 3 g l −1 asolectin from soybean (Sigma Aldrich) and 5 g l −1 sodium thiosulfate (Fisher Scientific) was used in time kill assays to prevent antimicrobial carryover during enumeration. The neutralizer was validated as nontoxic and effective using the BS EN 1276:2009 Annex C neutralizer toxicity and dilution-neutralization validation tests (British Standards Institute 2009). For time kill assays, the microemulsion was solubilized in an equal volume of DMSO and a 200 μl aliquot mixed with 9Á7 ml sterile distilled water. The test solution was inoculated with S. aureus, S. epidermidis or E. coli (10 7 -10 8 CFU per ml) and incubated at 37°C (bacteria) or 30°C (T. rubrum). Aliquots (1 ml) of the test solution were taken at 0 and 5 min (bacteria) or 0, 5, 10, 20, 30, 40, 50 and 60 min (T. rubrum) and diluted in 9 ml neutralizer. The test mixture was left in contact with the neutralizer for 5 min before spread plating and enumeration. The inoculum of each microorganism was evaluated without the microemulsion as the respective negative control.

Qualitative antimicrobial efficacy test of litsea-lemon EO microemulsion-treated textile
The antimicrobial and antifungal activity of microemulsion-treated polyester and cotton was determined against E. coli and S. epidermidis type and clinical isolates, S. aureus, MRSA and T. rubrum using adapted BS EN ISO 20645:2004method (British Standards Institution 2004. The antimicrobial activity was also determined against treated and untreated cotton and polyester following washing at 40°C in a standard domestic wash cycle (Indesit IWSD61251 Eco machine) using 24Á08 g of ECE non-phosphate reference detergent (A) with 4Á4 g of sodium perborate tetrahydrate (SDC Enterprises, Bradford, UK) and drying for 24 h at room temperature. Aliquots of 5 ml molten BHI or SD agar containing 10 5 CFU per ml E. coli and S. epidermidis type and clinical isolates, S. aureus, MRSA or T. rubrum were overlayed onto 10 ml solid agar and allowed to set. Circular (25 mm) textile samples were pressed onto the surface of the agar and ZoIs were determined after incubation for 18-24 h at 37°C for bacteria and for 7 days at 30°C for fungi. Untreated textile was included as a control.
Quantitative antimicrobial efficacy test of Litsea-lemon EO microemulsion-treated textile The antibacterial activity of microemulsion-treated polyester and cotton against E. coli, S. aureus and S. epidermidis was quantified using a method adapted from BS EN ISO 20743:2013(British Standards Institution 2013 and antifungal activity determined against T. rubrum spores using a method adapted from BS ISO 13629-2:2014 (British Standards Institution 2014).
Treated fabric samples (0Á40 AE 0Á05 g) were inoculated with 0Á1 ml of microbial culture (10 7 -10 8 CFU per ml). Immediately after inoculation, triplicate samples were vortexed in 10 ml neutralizer for 5 × 1 min cycles before spread plating on to BHI agar (bacteria) or SD agar (T. rubrum spores) for enumeration. A further three inoculated textile samples were incubated at 37°C for 18-24 h (bacteria) or 30°C for 48 h (T. rubrum spores) before neutralization and spread plating for enumeration. Control of untreated polyester and cotton were included. The percentage reduction (R) of viable microorganisms was calculated by comparing the number of surviving microorganisms on untreated and treated polycotton at 0 and 24 h or 48 h.

Aedes aegypti mosquito repellency of Litsea-Lemon EO microemulsion treated textile
Female A. aegypti mosquitoes (n = 10; 12-h starved) were released through the base leg of an Olfactometer (Ross Lifescience, Pimpri-Chinchwad, India; airflow 0Á20 AE 0Á05 m s −1 ; 0Á40 AE 10 m s −1 ) using aspirators and allowed to acclimatize in a holding port for 15 min without treatment (Rutledge et al. 2015). The following two treated fabrics were tested: microemulsion treated cotton samples and cotton samples impregnated with neat litsealemon EO blend (1 : 2). Untreated cotton was included as a control. After acclimatization, 100 cm 2 of the treated fabric was place in the test port of the olfactometer and 100 cm 2 untreated fabric place in the control port.
The Olfactometer control and test trapping ports were opened to allow the mosquitos to migrate to either the test or control chamber. After 30 s, the base leg holding port was closed and the number of mosquitos that migrated towards the control or test chamber was recorded every minute for 3 min. Physically injured mosquitoes and/or those incapable of flying or walking were not recorded in the results. Tests were conducted in triplicate.
The percentage of mosquitos repelled from the treatment was calculated according to Eqn 5: where MC is the number of mosquitos in the control port and MT the number of mosquitos in the treatment port.

Statistical analysis
The significance of differences (P ≤ 0Á05) between group means were determined by one-way analysis of variances (IBM, Armonk, NY). If assumptions of normality and variances of homogeneity were violated according to the Kolmogorov-Smirnov and Levene's test, Welch tests were performed. All investigations were carried out in triplicate on at least two separate occasions (n = 6), unless stated otherwise.

Antimicrobial activity of EOs
All EOs displayed antimicrobial activity against S. aureus, S. epidermidis and T. rubrum according to the disc diffusion method, with ZoIs ranging 22-90 mm (Table 1). Escherichia coli was inhibited by all tested EOs except citronella and peppermint EOs and all EOs displayed antimicrobial activity against P. aeruginosa with the exception of bitter orange, sweet orange, rosewood and wild thyme EOs. Bergamot, lemon, litsea and rosemary EOs inhibited all test species, with ZoIs ranging 20-90 mm; lemon, litsea and rosemary EOs generally had larger ZoIs than bergamot and were taken forward for further study (Table 1). Litsea EO had the greatest antimicrobial activity against all test species, with MICs ranging 0Á6 μl ml −1 against S. epidermidis to 10 μl ml −1 against P. aeruginosa (Table 2). Lemon EO exhibited the weakest antimicrobial activity with MICs ranging from 10 μl ml −1 against T. rubrum to >40 μl ml −1 against P. aeruginosa. Pseudomonas aeruginosa was the least susceptible microorganism with generally higher MICs than the remaining test species (Table 2).
The triple combination showed synergism against E. coli (FICI = 0Á50) yet was indifferent against S. aureus, S. epidermidis and P. aeruginosa (FICI = 0Á66-2Á03 and therefore did not further reduce the EO MICs compared to the double combinations. A double combination of litsea and lemon EO was synergistic against four out of five organisms tested, with the lowest FICI of 0Á09 among the double combinations and so was taken forward for further investigation. The minimum concentrations of litsea and lemon EO to inhibit all test species in combination were 2Á5 and 5Á0 μl ml −1 respectively, representing a litsea : lemon ratio of 1 : 2, which was tested hereafter.

GC-MS of litsea and lemon EOs
GC-MS analysis demonstrated that litsea EO produced 3 major peaks corresponding to limonene (retention time (RT) 9Á33 min, (m/z): [M] + 136Á30) and the E/Z isomers of citral with RTs of 17Á70 and 18Á84 min, (m/z): [M] + 152Á20. Limonene was also a major component of lemon EO, where a large peak was detected at an RT of 9Á59 min. E/Z isomers of citral appeared as more minor components with RTs of 17Á55 and 18Á61 min.

Microemulsion characterization
The globule size of litsea-lemon EO microemulsion was 1Á556 AE 0Á142 µm and the span value was 0Á500 AE 0Á02 µ m. The microemulsion was stable for 14 days, with an average CI of 12Á67%, where after the CI increased to  44Á6% in 28 days and 50% in 42 days (Fig. 1a). However, microemulsions produced a CI over 60% within 3 cycles (15 min) of centrifugation (Fig. 1b), indicating that the long-term stability of the microemulsion is low. The percentage incorporation efficiency of the active compounds in the EOs is reasonably high (86Á5 AE 3Á44%) however large fluctuations in concentrations of citral present in the microemulsion were observed over 28 days storage at 15 or 40°C indicating inconsistent distribution of the active ingredients of the EOs within the chitosan-alginate assembly in the microemulsion (Fig. 1c). The concentration of citral present when stored at 15°C significantly decreased (P ≤ 0Á05) from 136Á81 to 0Á02 mmol l −1 , while when stored at 40°C there was an increase in citral concentration after 28 days from 114Á33 to 254Á23 mmol l −1 (Fig. 1c).

Treatment of polyester and cotton fabric with the Litsealemon EO microemulsion
The liquor pickup (LPU) of the microemulsion was 112Á89 AE 1Á59% for cotton and 97Á45 AE 5Á97% for polyester during the padding process. The amount of citral found in the EO microemulsion-coated polyester and cotton decreased significantly (P ≤ 0Á05) over time, for example from 21Á03 to 16Á03 mmol l −1 on 16 cm 2 polyester (Fig. 2a) and 18Á41 to 12Á03 mmol l −1 on 16 cm 2 cotton (Fig. 2b). The concentration of limonene was significantly lower than citral, however there was also a significant (P ≤ 0Á05) reduction in citral within the fabrics over 7 days storage (Fig. 2a,b).

Antimicrobial activity of the litsea-lemon EO microemulsion
Screening for antimicrobial activity The disc diffusion method indicated that the microemulsion inhibited E. coli, S. aureus and S. epidermidis, with ZoIs of 22Á73-38Á00 mm (Fig. 3a). All individual components of the microemulsion possessed antimicrobial activity except 1% w/v sodium alginate (Fig. 3a). The EO blend alone produced similar ZoIs to the microemulsion, ranging 24Á04-34Á94 mm (Fig. 3a).

Time-kill assay
The microemulsion (1Á0% v/v in water) showed rapid antimicrobial activity against S. epidermidis, S. aureus and E. coli, with a complete reduction of all bacterial test species (7Á31-7Á54 log 10 CFU per ml) within 5 min (Fig. 3b). A complete reduction (7Á13 log 10 CFU per ml) of T. rubrum was achieved within 2 h (Fig. 3b).

Qualitative antimicrobial efficacy test of Litsea-lemon EO microemulsion treated textile
The microemulsion-treated cotton and polyester inhibited the growth of E. coli, S. aureus, S. epidermidis, and T. rubrum according to the qualitative antimicrobial textile efficacy test (Fig. 4a). Cotton and polyester (24 h post-treatment) produced ZoIs against S. epidermidis clinical isolate (13Á29-15Á09 mm) and MRSA (7Á65-17Á64 mm), and although ZoIs were not observed for E. coli and S. aureus, growth of microorganisms in direct contact with the textile was inhibited. S. epidermidis type strain was not inhibited by polyester 24 h post-treatment. Polyester and cotton (7 days posttreatment) inhibited all test species, with ZoIs ranging from 0Á53 mm against E. coli clinical isolate to 65Á74 mm against T. rubrum (Fig. 4a). Microemulsion-treated cotton and polyester samples did not retain their antimicrobial activity following washing at 40°C against all test species (ZoIs = 0Á00 AE 0Á00 mm) except for cotton against T. rubrum (ZoI = 0Á52 AE 0Á24 mm).

Repellency of Litsea-lemon EO blend and microemulsiontreated cotton against A. aegypti mosquitoes
The mean repellency of cotton samples treated with the microemulsion (24 h post-treatment) against A.

Discussion
Antimicrobial textile finishings have a potential application for wound dressings or sportswear to control bacterial and fungal contamination. Natural products could offer an environmentally friendly and biodegradable alternative to currently employed biocides for antimicrobial textile finishes. The aim of this study was to develop an antimicrobial textile coating loaded with EOs using a novel green polyelectrolyte micro-assembly process.
Here, 10 EOs were screened for antimicrobial activity against two Gram-positive bacteria (S. aureus and S. epidermidis), two Gram-negative bacteria (E. coli and P. aeruginosa) and a dermatophyte (T. rubrum) associated with skin conditions. All EOs tested demonstrated antimicrobial activity against S. aureus, S. epidermidis and T. rubrum while bergamot, lemon, litsea and rosemary EOs inhibited all the tested species (Table 1). The broadspectrum antimicrobial activity of the EOs in this study are consistent with previous research, as outlined by a literature by Orchard and Van Vuuren (2017); it was reported that bergamot, lemon, litsea and rosemary EOs possess antimicrobial activity against S. aureus, while rosemary EO also inhibited E. coli and T. rubrum, lemon EO inhibited E. coli and P. aeruginosa and bergamot inhibited E. coli and T. rubrum (Orchard and Van Vuuren, 2017). Previous studies have reported synergistic interactions between EOs, resulting in reduced effective doses (Lee et al. 2020) which in turn may decrease the risk of toxicity of EOs towards the user due to potential concentration-dependent skin irritation of EOs (Lee et al. 2013). In this study, the majority of combinations were indifferent (Table 2), however litsea and lemon EOs were synergistic against S. aureus, E. coli and T. rubrum (FICIs = 0Á09-0Á49), reducing the MICs of the individual EOs as indices of enhanced antimicrobial potency. Triple combinations of the EOs in this study only showed synergism against E. coli but did not show any significant effect on the MIC values compared to the double combinations against S. aureus, S. epidermidis and P. aeruginosa (Table 2). In contrast, a triple combination of litsea, clove and rosewood EOs was more potent than double combinations in terms of MIC against the acne-vulgaris associated pathogen Cutibacterium acnes (Owen et al. 2017). Overall, the MICs of litsea and lemon EOs in a combination that inhibited all test species was 2Á5 and 5Á0 μl ml −1 respectively, representing a litsea : lemon ratio of 1 : 2, which was carried forward for further investigations.
EOs have limited applications due to their volatility (Volić et al. 2020). Microencapsulation has previously been used to improve the durability of natural products on fabrics (Julaeha et al. 2021). The litsea and lemon EO blend (1 : 2 ratio) identified in this study was microemulsified and stabilized with a chitosan-sodium alginate polyelectrolyte assembly within the microemulsion, showing moderate stability over 14 days and a CI of around 10% (Fig. 1a). Though flocculation/creaming of the microemulsion was observed within 24 h, this is reversible by shaking, allowing for treatment of fabrics despite creaming. The chemical stability of the microemulsions was monitored via the major components of litsea and lemon EOs, citral and limonene. The citral concentration within microemulsions varied after 28 days, with a significant increase observed at 40°C but a near complete decrease at 15°C (Fig. 1c). The initial high EO concentration at 15°C followed by a decrease is ascribed to a burst release or loss of the loosely bonded EOs at the surface of the chitosan-alginate self-assembly whereas higher temperatures (40°C) will breakdown the chitosan-alginate self-assembly leading to the release of higher concentrations of the EOs from the inner core of the self-assembly. It is evident that the release of the EO incorporated into the chitosan-alginate microemulsion can be controlled. Citral is reportedly prone to autooxidation (Bailly 2020), and storage conditions such as temperature, humidity and light can affect the concentration of EO compounds (Soltanbeigi 2020), whereas as shown in this study degradation of citral at 40°C was not evident. In order to prevent any signs of physical or chemical instability of the EOs, only fresh microemulsions were used during the treatment and analysis of cotton and polyester fabrics; indeed, a significant difference (P ≤ 0Á05) in limonene and citral concentrations were noted on the treated cotton at 24 h post-treatment compared to 1-week posttreatment (Fig. 2).
Quantification of citral and limonene within the treated fabric demonstrated that there is a direct relationship between the entrapment of the EOs (within the microemulsion) on the fabric and the fabric area (Fig. 2), suggesting that the EOs were evenly distributed across the treated textile. This in turn indicates that the treatment of the fabric will also be potentially successful at both a small and larger scale, making the method scalable.
The microemulsion showed significant antimicrobial activity; E. coli, S. aureus and S. epidermidis were reduced by 7 log 10 CFU per ml within 5 min by the microemulsion (Fig. 3b). T. rubrum was less susceptible to the microemulsion, with a complete inactivation (7 log 10 CFU per ml) after 120 min (Fig. 3b). Previous research has demonstrated the antimicrobial activity of EO emulsions, for example bergamot EO nanoemulsions reduced E. coli by up to 4Á5 log 10 CFU per ml after 5 h (Marchese et al. 2020). Textile challenge tests show that microemulsion-treated polyester and cotton also possessed good antimicrobial activity, with a complete reduction achieved by both treated fabrics after 24 h for all test bacteria (Fig. 4b) and 48 h for T. rubrum (Fig. 4c). In a similar study, Khodary et al. (2017) reported that a cotton wound dressing treated with microencapsulated geranium extract completely reduced E. coli and S. aureus after 4 h. No antimicrobial activity was retained for microemulsion treated textiles after washing at 40°C with domestic detergent (Fig. 4a), this can be attributed to the lack of cross-linking chemicals or fixation agents used in the application of the microemulsion to textiles in this study. Further research into the use of environmentally friendly fixation techniques to improve the durability of the finish are therefore warranted, which would improve the cost and sustainability of the treatment. The current treatment could nonetheless be suitable for single use applications such as wound care, however further studies on toxicity would need to be carried out to determine the human sensitivity to the micro-emulsified EO blend when used topically. In addition, there can be batch-tobatch variation in the chemical composition of EOs, and standardization of EO composition is required for successful application of novel EO based formulations (Baptista-Silva et al. 2020).
In common with many natural volatile oils this blend did demonstrate some repellent properties against mosquitoes, with the microemulsion indicating superiority to the EO alone. However, the longevity and usefulness of this property needs to be further demonstrated with laboratory and field work utilizing human volunteers.
In conclusion, natural products could be a potential source of environmentally friendly and effective antimicrobial fabric finishes. Micro-emulsified litsea and lemon EOs stabilized in a chitosan-sodium alginate polyelectrolyte assembly in this study showed significant antimicrobial activity against the skin associated microorganisms E. coli, S. aureus, S. epidermidis and T. rubrum, indicating that EOs are potentially useful as finishing agents for the creation of environmentally friendly functional antimicrobial textiles. Further research is required to improve the stability of micro-emulsified EOs and investigate their attachment on the textile fibers to enable their use in wider applications beyond single-use treatments.