Biocomposites of Silk-Elastin and Essential Oil from Mentha piperita Display Antibacterial Activity.

In this study, novel antimicrobial biocomposite films comprising a genetically engineered silk-elastin protein polymer (SELP) and essential oil from Mentha piperita (MPEO) have been fabricated and tested for the antibacterial performance. SELP/MPEO biocomposite films were prepared by solvent casting using water as the solvent and aqueous emulsions of MPEO at different concentrations. Emulsions of MPEO were investigated, showing that the mixing method, relative amount of surfactant, and the presence of SELP influence particle size and homogeneity. The aqueous emulsions of SELP/MPEO were characterized by a population of particles between 100 and 300 nm, depending on the MPEO concentration. The emulsified oil droplets at the highest concentration showed to be homogeneously distributed into the SELP matrix and demonstrated antibacterial activity against Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. Moreover, the antibacterial activity of the biocomposite films was retained after a period of storage for 7 days at 4 °C. The formulation of composites comprising natural active fillers and recombinant protein polymers opens opportunities to develop new green, functional biocomposite materials, paving the way for a new generation of multifunctional materials.

Introduction

Plant-extracted phytochemicals, such as essential oils (EOs), have attracted great attention in recent years as alternative approaches to traditional antibiotics and as preservatives in the food industry.[1−5] EOs are extracted from aromatic plants and comprise a complex mixture of low molecular weight volatile secondary metabolites that act synergistically.[6,7] These plant-derived compounds are widely used in the pharmaceutical, cosmetic, and food industries due to the extensive set of bioactivities that range from antimicrobial, insecticidal, analgesic, and anti-inflammatory to antioxidant and anticarcinogenic properties.[8−11]

The antimicrobial activity of EOs is of particular interest due to the broad spectrum that includes antiviral, antibacterial, and antifungal properties.[2,3,10,12] This places EOs as promising natural antimicrobial agents, especially on the light of the increasing emergence of antibiotic-resistant bacteria that demands new antibacterial alternatives. EOs are considered as green antimicrobials and classified as GRAS (generally recognized as safe) for both people and the environment.[2] Due to the great number of components, EOs do not seem to have specific cellular targets; however, most of the components are hydrophobic and the default mechanism of action involves disrupting the different layers of the cytoplasmic membrane, destabilizing the microbial membrane.[2,10,13] Also, once internalized, EO molecules may act on organelles and interfere with enzymatic activities or even by reversing the drug-resistant ability of bacteria by eliminating antibiotic resistance plasmids.[2,10,13] Therefore, EOs have the potential to become a promising tool to circumvent the bacterial resistance to antibiotics.

The remarkable properties of EOs have prompted its use as additives for the formulation of functional biocomposites, especially for food packaging and biomedical purposes.[4,5,8,14−22] Still, the growing demand for new biomaterials and biopolymers from renewable resources is pushing the limits of materials design, leading to the development of greener alternatives.[21,23−25] Among others, poly(lactic acid),[26] chitosan,[27−29] natural silk fibroin,[30−32] gelatin,[17] and starch[33] are biopolymers that have been incorporated with EOs to obtain materials possessing antimicrobial activity. However, these often require harsh chemicals/additives—or complex or additional steps—for polymer solubilization and processing.

Recombinant protein polymers (rPPs) such as silk-elastin-like proteins (SELPs) are highly interesting materials for the formulation of novel biocomposites due to the exceptional mechanical properties, biocompatibility, and biodegradability.[34−36] These protein polymers consist of repeats of silk and elastin blocks inspired by natural structural proteins—silk fibroin and mammalian tropoelastin—and are obtained by recombinant DNA technology with precisely defined composition and length.[35,37,38] The silk block imparts thermal and chemical stability,[35,39] whereas the elastin block increases water solubility and provides flexibility and resiliency.[35,39] Due to the presence of the elastin unit, SELPs are water-soluble, which allows us to implement eco-friendly processing methods, using only water as the solvent. Moreover, rPPs are produced in microbial cell factories through fermentation, meaning that production is not strictly dependent on natural or oil-based resources, representing an opportunity to develop more sustainable solutions.

In this work, we investigate the feasibility of using the EO of Mentha piperita (peppermint) as an antimicrobial additive for the formulation of bioactive SELP-based biocomposites using water as a dispersant and employing simple and non-harmful procedures. The SELP used in this work has been previously obtained by our group.[40] It demonstrated to be non-cytotoxic to normal human skin cell lines[41−43] with potential for wound healing applications,[41,43] and a high versatility of processing into fiber mats,[41] films,[36] and composite materials.[42−44] The essential oil from M. piperita (MPEO) is classified as GRAS[45] and is extensively used in personal healthcare products possessing antimicrobial activity against different pathogens, antioxidant and anti-inflammatory properties[46−48] as well as the ability to accelerate wound healing in infection models.[49,50]

The combination of genetically engineered protein polymers with natural functional constituents such as EOs furthers expands the potential range of applications to develop new green biocomposites. Also, the ease of processing facilitates the employment of green processes to obtain different structures. To the best of our knowledge, this is the first time that an essential oil is used as a bioactive additive for the formulation of recombinant protein polymer biocomposites. The SELP/MPEO biocomposites demonstrated to inhibit the growth of bacteria and might be applied in the preparation of antibacterial films/coatings, circumventing the need to use conventional antibiotics and find application in, for instance, as active wound dressings.

Results and Discussion

Antimicrobial Activity of EOs

The antimicrobial activity of the essential oil from M. piperita was determined against four different bacteria using two different methods—broth microdilution and agar diffusion (Table ). In the broth microdilution assay, the minimum inhibitory concentration (MIC) was determined as the lowest concentration of MPEO able to inhibit the visible growth of the testing bacteria. In the disk diffusion test, the MIC was determined as the minimum concentration at which the presence of an inhibition halo was observed. Because of the hydrophobic nature of essential oils that can prevent a uniform diffusion through the growth medium, Tween 80 (T80) was used as an oil-in-water surfactant in the diluted samples.

Table 1

Minimum Inhibitory Concentration of Essential Oil fromM. piperita, Determined from Broth Microdilution and Agar Diffusion Testsa

	broth microdilution (mg/mL)	agar diffusion (mg/mL)
Escherichia coli	14	114
Bacillus subtilis	7	454
Pseudomonas aeruginosa	>454	454
Staphylococcus aureus	14	227

Values are rounded to the nearest integer.

The MIC values obtained by the microdilution method indicate that MPEO is able to inhibit the growth of bacteria at concentrations lower than 14 mg/mL, except for P. aeruginosa in which no MIC was found within the range of tested concentrations (Table ). The increased resistance of P. aeruginosa to MPEO could be a consequence of an outer membrane characterized by low permeability and the constitutive expression of several efflux pumps with wide subtract specificity.[6,51] The diffusion assays revealed a similar trend, with MPEO showing the presence of an inhibition halo although at much higher concentrations than those of the broth microdilution assay (Figure S2). The differences between the microdilution and the disk diffusion assays are most probably explained by the experimental procedure as the essential oil is more exposed to volatilization or oxidation processes in the diffusion test.

Characterization of Emulsions

Polysorbate 80, conventionally termed as Tween 80 (T80), is an amphipathic molecule with the ability to form micelles with a hydrophobic core and hydrophilic shell, considering that the critical micelle concentration is met.[52] Emulsions of MPEO in aqueous solutions were prepared with T80 as the surfactant due to the fast formation of micelles within a nanosecond time scale.[52] Experiments involved analysis of particle size by DLS using aqueous solutions of MPEO and T80 at a concentration of 1% (v/v) in relation to the amount of essential oil (2.5 μL; v/vEO) or to the volume of the working solution (30 μL; v/vsolution) followed by mixing with vortex or sonication. The use of the high-energy emulsification method by sonication resulted in the formation of opaque solutions suggesting the formation of a colloidal suspension; on the other hand, mechanical mixing by vortexing resulted in the formation of much more clear solutions (Figure S3). Analysis of emulsions by DLS demonstrated that the mixing method was not the only factor influencing particle size but also the amount of surfactant in solution (Figure ). The average droplet/particle diameter (Z-average) and polydispersity index (PdI) of the emulsions are represented in Figure A and Figure B, respectively, and summarized in Table . For all samples, the use of sonication resulted in a population of smaller and more homogeneous droplets than vortexing, with an average particle size between 79 and 327 nm and PdI values lower than, approximately, 0.5. On the other hand, samples mixed by vortexing revealed higher Z-average values and, in some cases, within the micron scale with PdI values greater than 0.6 (Figure A). The difference in particle size is associated with the use of low energy or high energy methods for emulsification.[53,54] Comparing the amount of surfactant, the presence of 30 μL of T80 resulted in particles with lower size in all samples (Figure A). This effect was even more noticeable in the samples mixed by vortexing showing a dramatic decrease in Z-average values from the micron to the nanoscale. The decrease in particle size with increased T80 content was also evident in emulsions produced by sonication, resulting in particles with lower Z-average and PdI values (Table ). Since micelle formation is a very fast event, we also evaluated if addition of T80 directly to the MPEO would infer any changes in particle size. For this, we devised two different formulations prior to vortexing/sonication: formulation A, in which T80 was first added to the MPEO and then transferred to the working solution, and formulation B, in which all components were added into the working solution. Comparing both formulations, the main differences were found for the samples with 30 μL of T80. Formulation B resulted in larger particles with higher polydispersity for samples submitted to vortexing and sonication.

Figure 1

DLS analysis of aqueous emulsions containing 25 μL of MPEO and 1% T80 expressed as (A) average particle size and (B) polydispersity index (PdI). A, emulsions in which T80 was first added to MPEO; B, emulsions in which all components were added to the final solution; [0.25], emulsions with 1% T80 (0.25 μL), calculated in relation to the volume of MPEO; [30], emulsions with 1% T80 (30 μL), calculated in relation to the volume of working solution.

Table 2

Mean Particle Size (Z-Average) and Polydispersity Index (PdI) Values of Emulsions Prepared from Aqueous Solutions of MPEO (25 μL) and T80a

	Z-average (nm)	PdI
A[30] vortex	203.3 ± 12.49	0.684 ± 0.072
B[30] vortex	504.3 ± 113.7	0.827 ± 0.107
A[0.25] vortex	5959 ± 1059	0.848 ± 0.261
B[0.25] vortex	5448 ± 1003	0.727 ± 0.222
A[30] sonication	145.1 ± 2.461	0.238 ± 0.010
B[30] sonication	79.23 ± 5.605	0.546 ± 0.026
A[0.25] sonication	278.1 ± 5.118	0.245 ± 0.017
B[0.25] sonication	327.3 ± 6.912	0.320 ± 0.044

A, emulsions in which T80 was first added to MPEO and then transferred to the working solution (water, final volume of 3 mL); B, emulsions in which all components were added to the working solution; [30], emulsions with 1 % T80 (30 μL), calculated in relation to the volume of working solution; [0.25], emulsions with 1 % T80 (0.25 μL), calculated in relation to the volume of MPEO.

To evaluate the effect of the protein polymer in particle size, 30 μL of Tween 80 (v/vsolution) was added to the essential oil and then transferred to the working solution consisting of 3% (w/v) SELP in water followed by sonication. The Z-average and PdI values obtained for solutions comprising different concentrations of MPEO are represented in Figure and summarized in Table . Aqueous solutions consisting of only SELP 3% (w/v) showed Z-average values of 79.1 nm with a PdI of 1.0, suggesting the absence of self-assembled structures and most likely attributed to the non-organized polypeptide chain. Compared with the solutions without SELP (A[30] in Table ; MPEO in Table ), the presence of the protein polymer (SELP/25MPEO in Table ) resulted in a slight decrease in the particle mean size from 145 to 113 nm, accompanied by an increase in the PdI from 0.24 to 0.41. At higher concentrations of MPEO (samples with 150, 200, and 250 v/mSELP % of MPEO), the particles demonstrated greater Z-average values with values above 200 nm and PdI between 0.46 and 0.31, suggesting a concentration-dependent interaction between the SELP and the T80/EO complexes.

Figure 2

DLS analysis of emulsions obtained by sonication (final volume of 3 mL) using 3% (w/v) SELP aqueous solutions with T80 and different concentrations of MPEO, expressed as (A) average particle size and (B) polydispersity index (PdI). SELP, aqueous solution of SELP; MPEO, aqueous solution with 25 μL of MPEO and 30 μL of T80; SELP/25MPEO, SELP/150MPEO, SELP/200MPEO, and SELP/250MPEO, aqueous solution of SELP with 30 μL of T80 and 25/150/200/250% v/mSELP, respectively.

Table 3

Mean Particle Size (Z-Average) and Polydispersity Index (PdI) Values of Emulsions Obtained by Sonication (Final Volume of 3 mL) Prepared from 3% (w/v) SELP Aqueous Solutions with T80 (30 μL) and Different Concentrations of MPEOa

	Z-average (nm)	PdI
SELP	79.11 ± 4.39	1.0
MPEO	145.1 ± 2.46	0.238 ± 0.01
SELP/25MPEO	113.2 ± 3.89	0.410 ± 0.029
SELP/150MPEO	277.2 ± 8.01	0.457 ± 0.019
SELP/200MPEO	245.2 ± 6.52	0.352 ± 0.028
SELP/250MPEO	295.3 ± 7.74	0.308 ± 0.021

SELP, aqueous solution of SELP; MPEO, aqueous solution of 25 μL of MPEO and 30 μL of T80, values from “A[30] sonication” in Table . Samples 150MPEO, 200MPEO, and 250MPEO correspond to the percentage of essential oil in relation to the mass of SELP (v/mSELP).

Morphological analysis and distribution of the SELP/MPEO complexes were assessed by STEM using SELP/MPEO solutions with 25 and 200% MPEO (SELP/25MPEO and SELP/200MPEO, respectively) (Figure ). Particle size revealed to be in agreement with DLS results, showing similar size and trend. At a concentration of 25%, the presence of sparingly distributed particles with a size of around 100 nm was observed. However, at an increased concentration of 200% MPEO, there was a dramatic increase in particle size with the presence of randomly distributed spherical droplets.

Figure 3

STEM micrographs of SELP/MPEO solutions with (A) 25% and (B) 200% MPEO.

Film Production and Characterization

After solvent casting, the biocomposite films demonstrated to be easily handled, rigid to the touch, and more opaque than pristine SELP films, which can be attributed to the presence of T80 and MPEO (Figure ). At concentrations above 150%, the opacity was increasingly evident (Figure B), suggesting an accumulation of emulsified micrometric oil droplets, since film opacity increases with the oil content.[31] At the highest concentration of 250%, the films showed a uniform opaque-white surface, maintaining a strong mint aroma.

Figure 4

Visual aspect of SELP/EO biocomposite films. (A) films without MPEO; (B) films with different MPEO contents. All films have Ø 13 mm.

To obtain a further insight into the microstructure of the SELP/MPEO biocomposite films, SEM analysis was carried out with samples incorporating different amounts of MPEO (Figure ). Micrographs show marked differences between the control (pristine SELP, without essential oil) and the SELP/MPEO films. The control revealed a smooth and uniform surface, which was expected for a homogeneous material.[36] Opposingly, the biocomposites showed a distinct irregular surface, with roughness increasing with the MPEO concentration. At a concentration of 200% MPEO, the samples appeared to be heterogeneous with an apparent agglomeration of structures, suggesting a heterogeneous distribution and accumulation of emulsified droplets at the film surface. At an increased concentration of 250%, the micrographs revealed a homogeneous structure, indicating a good dispersion of the emulsified droplets.

Figure 5

SEM micrographs of SELP/MPEO biocomposite films.

To confirm the presence of the essential oil in the SELP/MPEO film samples, the produced biocomposite films were characterized by FTIR and compared with pristine SELP materials (Figure ). The infrared spectrum of M. piperita essential oil (Figure A) presents a major absorption peak characteristic of chemical bonds from carboxylic acids, alcohols, ethers, and esters, which represent the chemical families of the main compounds (Table ). The most prominent contributions are attributed to the vibrations of OH stretching (3420 cm–1), asymmetric/symmetric stretching of CH2 and CH3 groups (2954, 2921, and 2870 cm–1), C=O stretching (1710 cm–1), CH2 and CH3 bending (1456, 1368, and 1245 cm–1), and C–O stretching in the cyclohexane ring (1045 and 1025 cm–1). Additional absorption bands within the fingerprinting region at 920 and 844 cm–1 may be related with the vibrations of the vinyl group due to the high content of menthol and menthone.[55] To assess the contributions of the surfactant into the infrared spectrum, pristine SELP films and films prepared with T80 were compared and are depicted in Figure B. The spectrum of films containing T80 presents the characteristic amide I (mainly the C=O stretching vibration) and amide II (N–H bending and C–N stretching vibrations) bands of proteins at 1619 and 1526 cm–1, respectively.[44,56] Tween 80 is mainly composed of aliphatic ester chains, terminal hydroxyl groups, and an aliphatic chain. The strong absorptions at 2922 and 2857 cm–1 are assigned to the assymetric and symmetric stretching vibrations of methylene (CH2), respectively.[57,58] The absorption peaks at 1733 and 1095 cm–1 are attributed to the C=O and C–O–C stretching vibrations of the ester group,[57,58] and the peak at 946 cm–1 is assigned to the vibration of C–O–C in the ether bond from aliphatic esters.[58] FTIR analysis of the SELP/MPEO biocomposites revealed that only films with concentrations above 150% demonstrated the presence of absorption bands attributed to the MPEO (Figure C). The infrared spectrum of the biocomposites shows the characteristic amide I and amide II bands of proteins and additional bands at 1368, 1045, 1025, 920, and 844 cm–1 attributed to the presence of MPEO.

Figure 6

ATR-FTIR spectra of (A) essential oil from M. piperita, (B) SELP films, and (C) SELP/MPEO films containing 150, 200, and 250% of M. piperita. Orange squares in panel (C) refer to the major contributions of amide I (1619 cm–1) and amide II (1526 cm–1) in SELP-59-A. Black circles in panel (C) indicate contributions from MPEO at 1368, 1045, 1025, 920, and 844 cm–1.

Table 4

Main IR Band Assignments of M. piperita Essential Oil[55,59−61]a

frequency (cm–1)	band assignment
3420	ν(OH)
2954	νas(CH3)
2921	νas(CH2)
2870	νs(CH2)
1710	ν(C=O)
1456	δs(CH2) and δ(CH3)
1368	δs(CH3)
1245	ν(C–O) and δ(CH2)
1045	ν(C–O)
1025	ν(C–O)

as, asymmetric; s, symmetric; ν, stretching; δ, in-plane bending; γ, out-of-plane bending.

Antibacterial Activity of SELP/MPEO Biocomposite Films

Antibacterial activity was evaluated against four bacterial strains by assessing the capability of the biocomposites to inhibit bacterial growth. Figure shows representative growth inhibition halos for SELP/MPEO films with inhibition zone diameters summarized in Table . The antibacterial activity demonstrated to be dependent of the MPEO content with growth inhibition results, indicating that the inhibition of bacterial growth is only achieved at samples with a concentration higher than 150%. Below this concentration, no growth inhibition zones were observed for all microbial strains (Figure S4). As expected, no growth inhibition zone was observed for the SELP sample without incorporation of essential oil (negative control for growth inhibition). Except for P. aeruginosa, in which no or residual growth inhibition halos were found, the biocomposites demonstrated to inhibit the growth of the remaining bacteria, showing clear inhibition areas increasing with concentration (Figure A and Table ). These results are in agreement with the MIC values found for pure MPEO as P. aeruginosa demonstrated to be resistant to the essential oil (Table ). Comparing the microorganisms, B. subtilis was the most susceptible strain followed by E. coli and S. aureus (Table ). The diameter of the inhibition zone for the lowest (150%) up to the higher concentration (250%) of MPEO ranged from 0.88 to 4.39 mm for E. coli, 1.84 to 8.09 mm for B. subtilis, and 0.59 to 5.15 mm for S. aureus. This demonstrates an increase of more than 5-fold between the biocomposites films with the lowest and the highest MPEO content.

Figure 7

Inhibition halos resulting from agar diffusion assays performed using SELP/MPEO biocomposite films (Ø 13 mm) against (A) E. coli, (B) B. subtilis, (C) S. aureus, and (D) P. aeruginosa. KAN, disks impregnated with 30 μg of kanamycin (positive control for growth inhibition); C, SELP films without incorporation of essential oil (negative control for growth inhibition).

Table 5

Inhibition Areas for SELP/MPEO Biocomposites against E. coli, B. subtilis, S. aureus, and P. aeruginosaa

	growth inhibition area (mm)
	E. coli	B. subtilis	S. aureus	P. aeruginosa
Kan	14.1 ± 1.2	13.8 ± 2.0	20.4 ± 0.7	5.3 ± 0.7
SELP/150MPEO	0.9 ± 0.8	1.8 ± 1.3	0.6 ± 0.6	0.00
SELP/200MPEO	2.6 ± 0.9	4.5 ± 2.8	3.3 ± 1.1	0.06 ± 0.1
SELP/250MPEO	4.4 ± 2.8	8.1 ± 2.1	5.1 ± 1.1	0.2 ± 0.2
SELP/250MPEO (4 °C)	2.5 ± 1.3	2.8 ± 1.2	6.5 ± 2.3	0.00
SELP/250MPEO (RoomT)	2.1 ± 0.9	0.3 ± 0.3	5.4 ± 1.6	0.00
SELP/250MPEO (RoomT in desiccator)	0.3 ± 0.4	1.2 ± 1.1	1.7 ± 2.1	0.00

The capacity of the SELP/250MPEO films to retain antimicrobial activity was evaluated after 7 days of storage at 4 °C, at room temperature (RoomT), or at RT in a desiccator. Disks impregnated with kanamycin (Kan) were used as positive control for growth inhibition.

To evaluate the antibacterial stability of the produced materials during short-term storage, the biocomposites with 250% of essential oil (SELP/250MPEO) were stored for 7 days at 4 °C, at room temperature (RT), or in a desiccator containing silica gel at RT followed by assessment of growth inhibition against the four bacterial strains (Figure ; for representative agar diffusion assays, see Figure S5). These conditions were defined based on the possible rate of MPEO evaporation—from low (4 °C) to high (desiccator with silica at RT). In all cases, the biocomposites demonstrated to retain the antibacterial activity after 7 days of storage, although with decreased antibacterial performance (Figure ). Comparing the different storage conditions, the film samples stored at 4 °C demonstrated better performance showing growth inhibition zones of 2.5, 2.84, and 6.5 mm for E. coli, B. subtilis, and S. aureus, respectively (Table ). Overall, the reduction in antibacterial performance was more evident for the samples stored in the desiccator, which can be attributed to the lower relative humidity, resulting in a higher evaporation rate of the volatile compounds.[62]

Figure 8

(A) Inhibition halo diameters obtained in agar diffusion assays using SELP/MPEO biocomposite films with 150, 200, and 250% of essential oil. (B) Inhibition halos diameters for SELP/MPEO films incorporating 250% of essential oil after short-term storage for 7 days at 4 °C (“4 °C”), room temperature (“RoomT”), and at room temperature in a desiccator containing silica (“RoomT desiccator”). Fresh-film: as-produced films. Kanamycin (30 μg) was used as positive control for growth inhibition. Bars represent mean ± SD; ns, nonsignificant; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Conclusions

In this work, antimicrobial biocomposites of a genetically engineered SELP incorporating essential oil emulsions from M. piperita were successfully obtained from aqueous solutions. Submicroemulsions were prepared by vortexing or sonication, demonstrating that the mixing method exerts a strong effect on particle size and population homogeneity. The use of sonication proved to be an adequate method, resulting in the formation of relatively monodisperse spherical droplets of approximately 145 nm. Compared with samples prepared in water, the presence of SELP during sonication demonstrated to slightly decrease the mean droplet size to around 113 nm, whereas the particle size showed to increase with an MPEO concentration of up to 295 nm at the highest concentration. SEM analysis of biocomposite films with 250% MPEO revealed a uniform distribution of emulsified droplets and demonstrated to inhibit the growth of B. subtilis, E. coli, and S. aureus, even after short-term storage at 4 °C. This work opens opportunities to develop new active sustainable materials for instance and active wound dressings and contributes to advance the development and application of genetically engineered protein polymers as a new class of emerging advanced materials.

Experimental Section

Materials

Essential oil of M. piperita (MPEO; ρ = 0.908 g/cm3) was obtained from Plena Natura (www.plena-natura.pt). Composition details were provided by the manufacturer: 42.07% (v/v) menthol, 24.50% (v/v) menthone, 5.01% (v/v) menthyl acetate, 4.94% (v/v) 1,8-cineole, 4.25% (v/v) isomenthone, 2.27% (v/v) limonene, and other components (Table S1). After opening, the EO was kept in the original dark glass flask, sealed, and stored at 4 °C in the dark to minimize oxidation and chemical alterations. Recombinant SELP-59-A (SELP, MW = 56.6 kDa) was biologically produced in E. coli by auto-induction and purified as described elsewhere.[40] SELP-59-A consists of nine tandem repetitions of sequence S5E9, where S is the silk block with sequence GAGAGS and E is the elastin block with sequence VPAVG.

Determination of Emulsion Parameters

To determine the best conditions for emulsions, initial experiments involved a combinatorial study of the essential oil dispersion in aqueous solution containing polysorbate 80 (Tween 80, T80, Sigma-Aldrich) as the emulsifier and 25 μL of MPEO while testing different conditions (Table ). Based on previous works, a solution of T80 1% (v/v) was used.[63−65] For the preparation of SELP solutions, pure lyophilized SELP-59-A was dissolved in ice-cold water at a concentration of 3% (w/v; 90 mg of SELP, 3 mL of deionized water). A detailed experimental design can be found in Figure S1.

Table 6

Experimental Conditions Used for Determination of Emulsion Parameters

(i) working solution	deionized water
	3% (w/v) SELP solution
(ii) 1% (v/v) T80	in relation to the volume of essential oil (v/vEO)
	in relation to the total volume of the working solution (v/vsolution)
(iii) formulation order	T80 first added to MPEO and then transferred to the working solution (formulation A)
	T80 and MPEO directly added to the working solution (formulation B)
(iv) emulsification method	vortex
	sonication

In a typical experiment, working solutions of 3 mL were prepared with 25 μL of MPEO and T80 in deionized water (dH2O) or in 3% (w/v) SELP solution. The amount of T80 at a defined concentration of 1% (v/v) was calculated in relation to the volume of essential oil (v/vEO; 0.25 μL) or to the volume of working solution (v/vsolution; 30 μL). Two mixing conditions were investigated: (i) T80 was first added to MPEO, mixed, and transferred to the working solution followed by emulsification (formulation A) and (ii) MPEO and T80 were directly added to the working solution followed by emulsification (formulation B). Emulsification was carried out by mechanical mixing by vortexing (VWR, DVX-2500; 10 min, 2500 rpm, room temperature) or by sonication (Sonic, UltrasonicProcessor GEX 400; Ø 3 mm probe, 25% amplitude, three cycles of 25 s, on ice).

Dynamic Light Scattering Analysis of Emulsions

After emulsification, samples were analyzed by dynamic light scattering (DLS) in a Zetasizer Nano ZS (Malvern) to determine the homogeneity and particle size. Measurements were performed at 25 °C with 1 min equilibration time and a backscatter angle of 173° using four clear side cells (10 mm pathway length). Each analysis included five readings, and the number of runs per reading was defined automatically by the equipment. Data analysis was performed using Zetasizer Software (Malvern).

Preparation of Biocomposite Films

Biocomposite films of SELP/MPEO were prepared by solvent casting. The concentration of MPEO was determined in relation to the amount of SELP-59-A (v/mSELP, μL/mg). Casting solutions (3 mL) were obtained by dissolving the purified protein polymer in deionized water to final concentrations of 3% (w/v) SELP, 1% (v/v) of Tween 80, and 12.5, 25, 50, 150, 200, and 250% of MPEO (v/mSELP %; corresponding to 12, 25, 50, 99, 149, 198, and 248 μL). Solutions were emulsified via sonication (Sonic, UltrasonicProcessor GEX 400; Ø 3 mm probe, 25% amplitude, three cycles of 25 s while keeping the samples on ice), poured into polytetrafluoroethylene (PTFE) molds (Ø 13 mm), and evaporated at room temperature under extraction. After complete evaporation, the resulting SELP/MPEO biocomposites were carefully peeled off and used for subsequent analysis. In some cases, for evaluation of storage conditions, films were kept at 4 °C until use.

Fourier Transform Infrared (FTIR) Analysis

The attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectra of films and pure MPEO were acquired at room temperature from 4000 to 450 cm–1 with a Bruker Alpha spectrometer (Bruker Optics) in attenuated total reflection mode (ATR), after 64 scans with a resolution of 4 cm–1.

Scanning Electron Microscopy Analysis

Casting solutions of SELP/MPEO were analyzed by field emission scanning electron microscopy (FE-SEM). Cu–C grids were immersed in the casting solutions and analyzed in an ultrahigh-resolution FE-SEM_EDS/EBSD (Nova 200 NanoSEM, Fei; Pegasus X4M, EDAX) with an acceleration voltage of 17.5 kV and a scanning transmitted electron microscopy (STEM) detector. For scanning electron microscopy (SEM) analysis of the SELP/MPEO biocomposites, film samples were coated with a gold layer using a sputter coater and visualized in a JSM-6010 LV (JEOL, Japan).

Measurement of Antimicrobial Activity

Antibacterial activity of liquid MPEO solutions and SELP/MPEO biocomposites was evaluated against clinically relevant bacteria—E. coli HB 101 (Gram-negative), P. aeruginosa ATCC 10145 (Gram-negative), B. subtilis 48886 (Gram-positive), and S. aureus ATCC 6538 (Gram-positive)—by determination of the minimum inhibitory concentration (MIC) via the broth microdilution assay and agar diffusion test following CLSI/EUCAST recommendations.

The minimum inhibitory concentration (MIC) of MPEO was determined as previously described by Wiegand et al.[66] Briefly, a bacterial inoculum was prepared in 10 mL of sterile Muller Hinton broth (MHB), incubated overnight at 37 °C with agitation, and used to prepare bacterial suspensions with 1 × 106 CFU/mL. Stock solutions of MPEO in MHB supplemented with 0.5% (v/v) T80 were used for the preparation of two-fold serial dilutions (50 μL) within the range of 455–0.028 mg/mL in sterile 96-well microplates. The percentage of T80 was defined based on literature values to enhance the solubilization of the oil.[64,67] Bacterial suspensions (50 μL) were then added to each well to achieve a final biomass of 5 × 105 CFU/mL and incubated at 37 °C for 18 h. Each plate included a set of controls: 100 μL of bacterial suspension (5 × 105 CFU/mL) in MHB and 100 μL of MHB + 0.5% (v/v) T80 as positive and negative controls for growth, respectively. The MIC was determined as the lowest concentration of essential oil that inhibited the visible growth of the testing bacteria (here, assessed as the optical density at 600 nm, OD600). Three independent replicates were performed.

Inhibition of bacterial growth by agar diffusion was assessed by measuring the diameters of growth inhibitory zones (zone of inhibition) with different concentrations of MPEO, obtained by sequential two-fold dilutions ranging from 100 to 3.125% (corresponding to 908–28 mg/mL, respectively). Overnight cultures of the testing bacteria were diluted in Muller Hinton agar (MHA; 0.8 wt % agar) supplemented with 0.5% (v/v) T80 to a final cell density of 1 × 106 CFU/mL (5 × 105 CFU/mL in the case of B. subtilis) and layered on MHA (1.5 wt % agar) plates supplemented with 0.5% (v/v) T80. Sterile susceptibility discs (Ø 6 mm, Oxoid Fisher Scientific) were placed in contact with the top-layer surface, impregnated with 15 μL of pure EO and two-fold dilutions of MPEO in sterile water supplemented with 0.5% (v/v) T80, and incubated overnight at 37 °C. Sterile water supplemented with 0.5% (v/v) Tween 80 and 30 μg of kanamycin (Formedium) were used as negative and positive controls for growth inhibition, respectively. After overnight incubation at 37 °C, results were recorded and analyzed using ImageJ image processing software.[68] Three independent replicates were performed.

For the SELP/MPEO biocomposites, the antimicrobial activity was assessed by the agar diffusion test using films incorporating different percentages of MPEO. Top agar plates were prepared as described above: bacterial cell suspensions diluted in MHA (0.8 wt % agar) supplemented with 0.5% (v/v) T80 were layered on MHA (1.5 wt % agar) plates. Biocomposite films containing different percentages of MPEO (v/mcopolymer) were placed in contact with the top-layer surface and incubated overnight at 37 °C. Kanamycin disks (30 μg, Formedium) and SELP films without MPEO were used as positive and negative controls for growth inhibition, respectively. After the incubation period, the diameters of growth inhibitory zones were evaluated using ImageJ image processing software. Three independent replicates were performed.

Statistical Analysis

One-way analysis of variance (ANOVA) with Dunnett’s post-test was carried out with GraphPad Prism 8 software to compare the means of different data sets within each experiment. A value of P < 0.05 was considered statistically significant. All experiments were performed in triplicate.