NANOEMULSIONS FOR INTERNAL HUMECTATION OF MYCELIUM-BASED TEXTILES

Abstract

Described herein are oil-in-water (o/w) nanoemulsions for internal humectation of mycelium-based textiles using fatty acids of vegetable origin and/or their synthetic derivatives and non-ionic surfactants. These nanoemulsions may be used as fatliquoring solutions in materials based on fungal mycelium, bacterial recombinant proteins, plant-based textiles, and/or other materials used as animal leather substitutes, including its potential use as environmentally friendly (e.g., green) and cost-effective fatliquoring alternative for animal leather. Also described are methods and apparatuses for scaling up, recovering, and reusing nanoemulsions. The nanoemulsion fatliquoring compositions and methods described herein may reduce frictional forces, improve tensile strength and elongation at break, introduce softness, and protect mycelium-based textile material against cracking.

Claims

1. A mycotextile comprising: one or more crosslinked mycelium layers having a plurality of nanoparticles within the one or more crosslinked mycelium layers, wherein the plurality of nanoparticles are functionalized to crosslink chitin/chitosan within hyphae of the one or more crosslinked mycelium layers; and an oil-in-water nanoemulsion coating the hyphae of one or more crosslinked mycelium layers.

2. The mycotextile of claim 1, wherein the oil-in-water nanoemulsion comprises a vegetable-based oil phase surrounded by a nonionic surfactant.

3. The mycotextile of claim 1, wherein the ratio of nonionic surfactant to vegetable-based oil is between 1:2 and 1:15.

4. The mycotextile of claim 1, wherein the vegetable-based oil phase comprises more than 85% saturated fatty acids.

5. The mycotextile of claim 1, wherein the vegetable-based oil phase comprises coconut oil.

6. The mycotextile of claim 1, wherein the vegetable-based oil phase comprises one or more of coconut oil, soybean oil, canola oil, corn oil, avocado oil, and olive oil.

7. The mycotextile of claim 1, wherein the vegetable-based oil phase comprises one or more synthetic derivatives of vegetable oils.

8. The mycotextile of claim 1, wherein the vegetable-based oil phase comprises cetearyl ethylhexanoate; octanoic acid, 1,3-propanediol ester; 2,2-[oxybis(methylene)]bis [2-(hydroxymethyl)-1,3-propanediol] decanoate.

9. The mycotextile of claim 1, wherein the vegetable-based oil phase comprises one or more natural waxes.

10. The mycotextile of claim 1, wherein the vegetable-based oil phase comprises beeswax and/or lanonil waxes.

11. The mycotextile of claim 1, wherein the nonionic surfactant comprises one or more of polyoxyethylene, a polyoxyethylene derivative, polyoxypropylene, a polyoxypropylene derivative, sorbitan anhydride, a sorbitan anhydride derivative, an alkanol amide, and a nonionic fat.

12. The mycotextile of claim 1, wherein the nonionic surfactant comprises one or more of polysorbate 20, polyoxyethylene (20) sorbitan monolaurate, glyceryl monooleate, polysorbate 80, sorbitan monolaurate, polysorbate 80, polyoxyethylene (20) sorbitan monooleate, hydrogenated and ethoxylated castor oil, and glyceryl monostearate.

13. The mycotextile of claim 1, wherein the nonionic comprises a hydrogenated castor oil.

14. The mycotextile of claim 1, wherein the nanoemulsion droplets have a mean droplet diameter of 20-35 nm.

15. The mycotextile of claim 1, wherein the oil-in-water nanoemulsion comprises an oil-based fragrance.

16. A mycotextile comprising: A support scaffold layer; a first crosslinked mycelium layer extending adjacent to the first side of the support scaffold layer, and a second crosslinked mycelium layer extending adjacent to a second side of the support scaffold layer; a plurality of nanoparticles within the first and second crosslinked mycelium layers, wherein the plurality of nanoparticles are functionalized to crosslink chitin/chitosan within hyphae of the first crosslinked mycelium layer and the second crosslinked mycelium layer; and an oil-in-water nanoemulsion coating the hyphae of the first and second crosslinked mycelium layers.

17. An oil-in-water nanoemulsion composition for fatliquoring a textile material, the composition comprising: between 0.1% and 5% of a vegetable-based oil phase; and between 1% and 10% of a nonionic surfactant, wherein the nanoemulsion comprises droplets of the vegetable-based oil surrounded by the nonionic surfactant and dispersed within a continuous water phase.

18. An oil-in-water nanoemulsion composition for fatliquoring a textile material, the composition comprising: a vegetable-based oil comprising 85% or more saturated fatty acids; and a nonionic surfactant, wherein the surfactant to oil (O:S) ratio is between 1:2 and 1:15, and the composition comprises between 0.1% and 5% of vegetable-based oil, further wherein the nanoemulsion comprises droplets of the vegetable-based oil surrounded by the nonionic surfactant and dispersed within a continuous water phase.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] A better understanding of the features and advantages of the methods and apparatuses described herein will be obtained by reference to the following detailed description that sets forth illustrative embodiments, and the accompanying drawings of which:

[0033] FIGS. 1A-1D illustrate different types of emulsions.

[0034] FIG. 2 schematically illustrates one example of the interface between the aqueous and oily phases through a layer of surfactant molecules where the polar head of the molecule is located towards the outside of the drop (directed towards the continuous aqueous phase), and the non-polar tails are distributed within the drop.

[0035] FIG. 3 illustrates examples of emulsions according to droplet size and shape, stability, synthesis method, and expected polydispersity.

[0036] FIG. 4 is a graph showing a representative particle size profile vs. volume distribution for the sample in Example 1.

[0037] FIG. 5 is a graph showing a representative particle size profile vs. volume distribution for the sample in Example 8.

[0038] FIG. 6 illustrates the mean droplet size of several nanoemulsion systems using tween 80 as a surfactant.

[0039] FIG. 7 graphically illustrates the representative z potential profile for the sample in Example 8.

[0040] FIG. 8 shows a representative particle size profile vs. volume distribution for the sample in example 13.

[0041] FIG. 9 illustrates the mean droplet size of several nanoemulsion systems using a PEG-based nonionic surfactant.

[0042] FIG. 10 shows a representative z potential profile for the sample in Example 13.

[0043] FIG. 11 illustrates different emulsions including (left, A) an emulsion prepared in Example 1, and a nanoemulsion prepared in Example 13 (middle, B), as compared with distilled water (right, C).

[0044] FIG. 12 is a graph showing tensile strength (TS, MPa) and elongation at break (E, %) of the mycotextile after the application of several nanoemulsion formulations.

[0045] FIG. 13 Average droplet size distribution of two nanoemulsions encapsulating orange oil, both starting with a concentration of 55% surfactant, 12.5% coconut oil, 12.5% orange oil, and 20% water. The tall and sharp peak corresponds to the formulation using a first nonionic surfactant and the wider and bimodal distribution corresponds to the formulation using T80.

[0046] FIG. 14 Average droplet size distribution of two nanoemulsions encapsulating lemon oil, both starting with a concentration of 55% surfactant, 12.5% coconut oil, 12.5% lemon oil, and 20% water. The sharp peak on the left side corresponds to the formulation using a PEG-based nonionic surfactant and the wider distribution at the right corresponds to the formulation using T80.

[0047] FIGS. 15A-15D illustrates examples of scanning electron microscope images of the nanoemulsion application for the humectation of fungal hyphae in different mycotextiles. FIG. 14A shows a control: hyphae without treatment. FIG. 14B shows humectation using the formula of Example 1. FIG. 14C shows humectation using the formula of Example 8. FIG. 14D shows humectation using the formula of Example 13. The scale bar in all the images corresponds to 10 m.

[0048] FIGS. 16A-16D show scanning electron microscope images of the humectated samples reinforced with spherical inorganic nanoparticles. FIG. 15A shows control: hyphae without treatment, FIG. 15B shows humectation using the formula of example 1, FIG. 15C shows humectation using the formula of Example 8, FIG. 15D shows humectation using the formula of Example 13. The scale bar in all the images corresponds to 10 m.

[0049] FIG. 17 illustrates an infrared spectrum of the mycelium treated with the formulation of Example 1 (emulsion T80+S80) compared to the mycelium without treatment (control) and the oil phase (coconut oil).

[0050] FIG. 18 shows infrared spectra of the mycelium treated with the formulation of Example 8 (nanoemulsion T80) compared to the mycelium without treatment (control) and the oil phase (coconut oil).

[0051] FIG. 19 is a graph showing infrared spectra of the mycelium treated with the formulation of Example 13 (Eu=PEG-based nonionic surfactant) compared to the mycelium without treatment (control) and the oil phase (coconut oil).

[0052] FIG. 20 show four infrared spectra collected for sample A to calculate the oil content.

[0053] FIG. 21 shows the effect of the velocity of water addition on the particle size distribution during the scaling-up (Example 14). The taller curve illustrates the addition in one step (1), while the shorter curve shows the addition in two steps (2).

[0054] FIG. 22 is a graph showing the effect of the scaling-up in the particle size distribution: 500 g (curve 1) versus 15000 g (curve 2).

[0055] FIG. 23A-23B schematically illustrate methods of the scaling up approaches to apply the nanoemulsion formulations using a spraying strategy (FIG. 23A) or an immersion strategy (FIG. 23B).

[0056] FIG. 24A-24B show graphs of the oil content calculated at the top (FIG. 24A) and the bottom (FIG. 24B) of the mycelium layer of the mycotextile using both scaling-up approaches.

[0057] FIGS. 25A-25C illustrate the recovery of the nanoemulsion using microfiltration and the addition of sodium hypochlorite for reuse.

DETAILED DESCRIPTION

[0058] Described herein are nanoemulsions of oily phases in water using non-ionic surfactants that are stable over longer periods of time to improve the flexibility and moisture retention in materials of biotechnological origin, and in particular mycotextiles that may be used as substitutes for animal leather. The nanoemulsions described herein may have a droplet size of between about 20 and 500 nm, which is a thousand times smaller than droplets of a conventional emulsion. These nanoemulsions may be deposited in an oily phase through a fibrous and/or porous material more efficient than conventional emulsions, resulting in greater availability of the oil in the mycotextile, as well as in the stability (shelf life) of the solution, which would allow its reuse and lower scaling costs.

[0059] The vegetable oil nanoemulsions (in water) described herein may be formed using a biocompatible surfactant and may be used as a fatliquoring agent.

Formulations

[0060] Nanoemulsions that may be used are described herein. Example protocols to prepare the different nanoemulsions, macroemulsions, and microemulsions for comparison are described below. These examples are not intended to be limiting but illustrate testing compositions used herein.

Preparation of 2 L of Macroemulsion:

[0061] In a 250 mL beaker, 20 g of coconut oil, 12 grams of Tween 80, and 28 grams of Span 80 are weighed, heated to 80 C., and homogenized with manual agitation. In a 5 L capacity flask, weigh 1940 grams of distilled water and heat it to 80 C. Once the required temperatures are reached, the water flask is placed under mechanical agitation of 1200 RPM. The homogeneous mixture of coconut oil with both surfactants is added gradually (in thread) and continuously under agitation. Once the whole volume is added, it is kept stirring for 10 min. At the end of the 10 min, the agitation is decreased to 700 RPM, and it is left agitating for 10 min more, observing a white and homogeneous solution. It is taken to an Ultraturrax homogenizer for 30 seconds at 10000 RPM. Once the agitation process is completed, the emulsion is transferred to a 2 L container which should be stored at a temperature between 3 and 35 C.

Preparation of 500 g of Direct Nanoemulsion:

[0062] Weigh 12.5 grams of coconut oil and 58.35 grams of Tween 80 into a 250 ml beaker. Heated to 60 C. under magnetic stirring at 600 RPM. Weighed 429.15 grams of distilled water into another 1 Liter beaker and heated to 60 C. Once the temperature is reached in both cases, the water is added drop by drop over the oil and surfactant mixture, under constant agitation at 600 RPM and maintaining the temperature at 60 C. Once the addition is finished, the agitation is maintained for 5 min. This mixture can be kept at room temperature.

Preparation of 100 g of Concentrated Sample to Obtain Nanoemulsion by Indirect Method (Phase Inversion by Composition or PIC):

[0063] Weigh 15 grams of coconut oil and 70 grams of tween 80 in a glass beaker of 2 L capacity and proceed to heat, under mechanical agitation, on a hotplate, ensuring a temperature of 60 C. 15 grams of distilled water are placed in a 250 ml beaker and heated to 60 C. on a hotplate. Once the reaction temperature is reached, it proceeds to incorporate by dripping the water of the glass into the mixture, raising the agitation to 600 RPM. Once the incorporation of the water into the mixture is completed, the agitation is maintained for 20 min at the same conditions and speed.

Phase Inversion and Dilution to Obtain 500 g of Nanoemulsion by Indirect Route (PIC):

[0064] Approach 1: Approximation 1 is performed by slowly incorporating the dilution water (in the form of drops). For this purpose, 1400 grams of distilled water at 85 C. are added to 100 grams of the concentrated sample at 85 C., drop by drop, under a mechanical agitation of 1000 RPM.

[0065] Approximation 2 was realized by the addition of water in two stages. The first of the total mass (approximately 470 g) is slowly added and controlled, and the remaining (approximately 930 g) is at three times the rate.

Examples

[0066] Example 1. A mixture of tween 80 with span 80 in a ratio (3:7) was made for conventional emulsions containing 1% oil phase (O:S=1:2). For this preparation, the corresponding proportions of both surfactants are mixed in the oil phase and brought to 80 C. Then, the aqueous phase is also brought to the same temperature. Both solutions are mixed with a magnetic stirrer at 1200 rpm for 10 minutes, adding the water to the oily phase as a thread, and then it is lowered to 700 rpm for 10 min. Finally, it is taken to an Ultraturrax type homogenizer at 10000 RPM for 30 sec. The resulting mixture has a milky appearance and low viscosity with a tendency to cream after hours of rest. The droplet size distribution analysis was carried out in a Malvern Mastersizer 2000 model using the general purpose-normal sensitivity model and, as optical parameters, a refractive index of 1.47 and zero absorbance. The average size obtained for this mixture is 9.79 m with a distribution d(0.1) of 1.46 m, d(0.5) of 6.24 m, and d(0.9) of 10.03 m. This distribution justifies the creaming effect observed in this type of mixture. This formulation's typical droplet size distribution profile is shown in FIG. 4.

[0067] Example 2. A preparation was made with tween 80 by the direct method using a 1:4.67 surfactant oil ratio for emulsions containing a 2.5% oil phase. A translucent low-viscosity mixture is obtained, suggesting a microemulsion is obtained.

[0068] Example 3. This system was made directly by mixing at 70 C. the different components in such a way as to obtain a final concentration of 15% Tween 80, 1% coconut oil, and 84% water. It instantly has a cloudy appearance with a slightly milky appearance, with creaming after 24 hours.

[0069] Example 4. Preparation of liquid crystals or bicontinuous microemulsions of tween 80 for indirect nanoemulsions (PIC method) starting from 75% tween 80, 5% coconut oil, and 20% water. This sample presents a transparent appearance and a viscous consistency maintained for up to 24 h.

[0070] Example 5. Preparation of liquid crystals or bicontinuous microemulsions of tween 80 for indirect nanoemulsions (PIC method) starting from 70% tween 80, 15% coconut oil, and 15% water, presenting a cloudy appearance and a viscous consistency. After 24 hours, the viscosity increased.

[0071] Example 6. In this system, 1 g of the preparation of example 4 was taken, and 4 g of water was added dropwise, resulting in a concentration of 15% Tween 80, 1% coconut oil, and 84% water. Initially, it has a transparent, fluid appearance; after 24 hours, the system maintains its appearance. The droplet size distribution analysis was carried out in a Malvern equipment model Zetasizer Nano ZS presenting an average size of 10.37 nm with a polydispersity index (PDI) of 0.268.

[0072] Example 7. A nanoemulsion was made by indirect method 8.75% of Tween 80, 1.875% of coconut oil, and 89.375% of water starting from the preparation of example 5. This means that an amount of the concentrated sample must be prepared and homogenized at a given temperature. In this case, an increase in fluidity is observed at 60 C. and above. Subsequently, at the same temperature, water is added by dripping. The system was prepared by taking 1.5 g of the concentrate of 70% Tween 80 and 15% coconut oil and diluted with 10.5 g of water dropwise. It exhibited a translucent appearance with low viscosity.

[0073] Example 8. From a sample prepared as the example 5, a sample containing 4.67% Tween 80, 1% coconut oil, and 94.33% water was prepared. A quantity of the concentrated sample is prepared, homogenized at a temperature of 70 C., and then, at that temperature, water is added to induce phase transition and subsequent dropwise dilution. FIG. 5 shows a typical droplet size distribution profile obtained with this method, which indicates a mean droplet size of 24.05 nm with a PDI of 0.089. FIG. 6 compares the droplet sizes of different examples with tween 80. The first two bars on the left correspond to the results obtained by scaling the preparation to 500 g using two different temperatures (70 and 80 C.). It is observed that by increasing the temperature by 10 C., the average droplet size decreases from 121.70 nm to 66.18 nm. Then, as the S/O ratio increases, as occurs in the comparison between the third and fifth bars (from left to right), a decrease in the average droplet size from 20.54 nm to 10.37 nm is observed. This confirms that the higher the % surfactant (e.g., 15:1 ratio), the larger the surface area of the micelle and, thus, the smaller the droplet size. Furthermore, increasing the oil phase concentration from 1% to 2% while keeping the S/O ratio fixed shows a slight increase in the average droplet size from 20.54 nm to 24.23 nm. The results also indicated that the mixture containing a lower percentage of surfactant (e.g., 4:1 ratio) presents a higher polydispersity index, thus concluding that the surfactant tween 80 may present more variations as a homogenization function.

[0074] On the other hand, as shown in FIG. 7, the zeta potential of the system is low, which is attributed to the nonionic nature of the surfactant. Although it could be concluded that these low values detract from the system's stability, we have observed that the nanoemulsions present very high kinetic stability thanks to the steric stabilization conferred by the ethoxylated chains of the nonionic surfactant.

[0075] Example 9. Formulations of liquid crystals or bicontinuous microemulsions of nonionic surfactant for indirect nanoemulsions (PIC method) with composition 65% nonionic surfactant, 20% coconut oil, 15% water or 70% nonionic surfactant, 15% coconut oil, 15% water. We started by making the water-surfactant-coconut oil samples based on the nonionic surfactant, to proceed in a second stage to dilute them with deionized water and thus prepare the nanoemulsions using the indirect method of phase inversion by concentration (PIC). They were briefly vortexed and briefly subjected to a temperature of 80 C. to homogenize; several cycles of this treatment were carried out until homogeneous samples were obtained.

[0076] Example 10. A composition of 8.125% nonionic surfactant, 2.5% coconut oil and 89.38% water was prepared by PIC. It started from one of the samples of example 9. A quantity of the concentrated sample with 65% oil phase is prepared and homogenized at a temperature of 60-85 C. Subsequently, at that temperature, water is added little by little (dripping). This process is necessary to be carried out at 60-85 C. so that the concentrated systems flow a little more, mainly at the beginning of the process, since we are starting from a system that is very viscous at room temperature. They were subjected to agitation with a magnet and vortex. The resulting mixture is translucent and fluid.

[0077] Example 11. Starting from a concentrated sample of composition: 65% nonionic surfactant, 20% coconut oil, and 15% water, a quantity of the concentrated sample is prepared and homogenized at a temperature of 60-70 C. Subsequently, at that temperature, water is added to dilute, and the water is added little by little (dripping) to reach a final composition of 3.25% nonionic surfactant, 1% coconut oil, and 95.75% water. This process needs to be carried out at 60-70 C. to allow the concentrated systems to flow a little more, mainly at the beginning of the process, since we are starting from a very viscous system at room temperature. They were subjected to agitation with a magnetic plate and vortex.

[0078] Example 12. A composition of 11.66% nonionic surfactant, 2.5% coconut oil, and 85.84% water is prepared. It starts from a concentrated sample of example 9, where the ratio of the oily to aqueous phase is 1. A quantity of the concentrated sample is prepared, homogenized at a temperature of 60-70 C., and then, at that temperature, water is added to dilute, and the water is added little by little (dripping). This process must be carried out at 60-85 C. so that the concentrated systems flow a little more, mainly at the beginning of the process, since the starting point is a medium viscous system at room temperature. They are subjected to agitation with a magnetic agitator and vortex.

[0079] Example 13. A composition of 4.67% nonionic surfactant, 1% coconut oil, and 94.33% water was prepared. It started from a concentrated composition sample: 70% nonionic surfactant, 15% coconut oil, and 15% water. A quantity of the concentrated sample is prepared, homogenized at a temperature of 60-85 C., and then, at that temperature, water is added to dilute, and the water is added little by little (dripping). This process must be carried out at 60-85 C. so that the concentrated systems flow a little more, mainly at the beginning of the process, since the starting point is a medium viscous system at room temperature. They were subjected to agitation with a magnet and vortex. Freshly prepared, it takes on a transparent, translucent appearance, bluish in color, very low viscosity, and without evidence of creaming. After one month at rest, the appearance remains the same, which confirms the stability of these preparations guaranteeing long shelf life. FIG. 8 shows a typical droplet size distribution profile obtained with this approach indicating an average droplet size of 21.730.34 nm with a PDI of 0.1190.014. FIG. 9 compares different preparations made with nonionic surfactant. The system prepared with PEG-based nonionic surfactant shows very slight variations in the average droplet size. For example, comparing the first bar (29.40 nm) with the last bar (21.73 nm), a slight decrease in droplet size (approximately 26%) is observed when the surfactant/oil ratio is increased 1.5 times, unlike the system with tween 80 in which the change of this ratio leads to more significant changes in the average droplet size. The change in the concentration of the oil phase, keeping the surfactant/oil ratio fixed, barely causes an increase from 23.99 nm to 24.88 nm (approximately 4%), as can be seen by comparing bars 2 and 3 of the graph, suggesting that this system supports higher concentrations of the oil phase without drastically changing the droplet size and stability of the system. Finally, scaling from 20 g to 500 g causes a slight increase from 21.73 nm to 24.05 nm, as observed in the last two bars of the graph. Likewise, FIG. 10 shows a representative Z-potential profile for a preparation with 1% coconut oil and a surfactant-to-oil ratio equal to 4.7. As in the example with tween 80, the low zeta potential of the system is attributed to the nonionic nature of the surfactant at different scaling factors. However, the high stability observed is attributed to the steric stabilization conferred by the ethoxylated chains of the PEG-based nonionic surfactant. These observations suggest that the preparations with this surfactant are much more robust, admitting changes in the scaling-up process that allow optimizing mixing and preparation times, among others. Using FIG. 11 as reference, comparing the appearance of the nanoemulsion prepared with nonionic surfactant with that of a macroemulsion (for example, that of example 1) with distilled water corroborates that physically its characteristic coincides with that expected for a nanoemulsion, i.e., it is translucent (not transparent). The bluish color confirms the presence of droplets of sizes less than 100 nm.

[0080] Example 14. A composition consisting of 23.35% nonionic surfactant, 5% coconut oil, and 71.65% water was prepared by the PIC method starting from one of the samples of example 9. A mass of the concentrated sample was homogenized at a temperature ranging from 60 to 90 C. Subsequently, at the same temperature, water was dripped until the final concentration of the oil phase reached 5%. The resulting mixture is translucent and less fluid than the obtained in examples from 10 to 13 thus working temperature should be as high as possible.

[0081] Example 15. A sample prepared as example 13 replacing the oil phase by cetearyl ethylhexanoate (DUB LIQUIDE 85 by Stearinerie Dubois) presents similar optical characteristics and stability.

[0082] Example 16. A sample prepared as example 13 replacing the oil phase by octanoic acid, 1,3-propanediol ester (Propanediol dicaprylate, DUB ZENOAT by Stearinerie Dubois) presents similar optical characteristics and stability.

[0083] Example 17. Lanolin nanoemulsion formulations started from bicontinuous microemulsions of nonionic surfactant with compositions of 50% nonionic surfactant, 25% lanolin, 25% water, or 50% nonionic surfactant, 12.5% coconut oil, 12.5% lanolin and 25% water. The concentrated samples were prepared under continuous stirring at temperatures ranging from 70 to 75 C. We started by making the water-surfactant-oil concentrated samples based on the nonionic surfactant (e.g., a PEG-based nonionic surfactant) to proceed in a second stage to dilute them with deionized water and thus prepare the nanoemulsions using the indirect method of phase inversion by concentration (PIC). This concentrate initially presented a slightly viscous appearance at the preparation temperature but after a few minutes. At room temperature, it behaves very viscous and milky in appearance. Therefore, the dilution must be done immediately after obtaining the concentrated mixture or it must be heated to a temperature between 7 and 75 C. before the PIC method.

[0084] Example 18. The PIC method prepared a composition consisting of 4% nonionic surfactant, 2% lanolin, and 94% water starting from one of the samples of example 15. This system appeared fluid but cloudy, with a milky appearance. After 1 hour, the nanoemulsion began to appear cloudier until it creamed.

[0085] Example 19. A composition with a 4:2 surfactant to oil ratio consisting of 4% nonionic surfactant, 1% lanolin, 1% coconut oil and 94% water was prepared by PIC method starting from one of the samples of example 15. This system presented a fluid and transparent appearance when first prepared, the same optical characteristics remain after several hours at room temperature and no creaming was observed in this system, suggesting that an oil-wax mixture is more efficient to emulsify the lanolin wax. The transparent appearance may indicate the presence of a microemulsion.

[0086] The improvement due to the use of these mixtures as fatliquoring agents in treating mycotextiles and/or leather-equivalent materials from biotechnological sources can be evidenced by increased flexibility and resistance to internal moisture loss (wetting). FIG. 12 shows the tensile strength values (MPa) and the elongation at break (E %) of different samples subjected to the greasing process with the formulations presented herein. As can be seen, all the fatliquored samples compared to the control (a sample harvested and dried without subjecting it to any other process), have an improvement in elongation, that ranges between 19 and 34%; while the tensile strength improves between 44 and 166%. In the case of the combination of vegetable oil with a fat (lanolin) there was no important difference regarding the material only treated with the vegetable oil. However, when combining the vegetable oil with two synthetic oils, a greater effect on the elongation was observed. The highest increase in tensile strength was achieved with the mixture between coconut oil and cetearyl ethylhexanoate.

[0087] Example 20. In other approaches including nanoemulsion encapsulating essential oils, T80 was used as a surfactant starting from a concentrated mixture consisting of 70% T80, 15% of orange or lemon essential oils, and 15% water. Both nanoemulsions were made at 80 C. by the PIC method slowly adding the necessary amount of water to achieve a final concentration of 4.66% T80, 1% AC, and 94.3% water. As observed in Table 3, using orange oil the average particle size result in 36.82 nm while using lemon oil the average particle size resulted in 158.50 nm. Comparing these values with the one obtained in Example 8 (24.05 nm) using only coconut oil with an equivalent composition, the droplet size increased significantly. The different type of oil affects the droplet size of the nanoemulsion and hence, the appearance of the nanoemulsion.

[0088] Example 21. Likewise, the nanoencapsulation at 80 C. of the essential oils using nonionic surfactant causes an increase in the droplet size (Table 3), and it occurs at a lower surfactant-to-oil ratio (3.25:1). For these formulations the concentrate mixture should be 65% PEG-based nonionic surfactant, 20% essential oil and 15% water. By diluting up to a final composition of 3.25% PEG-based nonionic surfactant, 1% essential oil and 94% water, the particle size for the orange oil is 112.20 nm and 98.67 for the lemon oil. In this case, the difference between both systems is less. However, the optical appearance of the lemon oil is closer to the expected for these systems. These first examples indicate that both essential oils could be nanoencapsulated.

[0089] Example 22. When mixed with other oil such as coconut oil to increase the solubility in the oil phase, a dramatic decrease in the particle size was observed. In this example, T80 was used as an emulsifier, and the PIC method started from a concentrated mixture of 55% T80, 12.5% coconut oil, 12.5% essential oil and 20% water under sitting at 80 C. According to Table 3, it is possible to achieve average droplet sizes of 49.22 nm for the orange oil and 76.72 nm for the lemon oil.

[0090] Example 23. By changing the emulsifier of Example 20 by nonionic surfactant and keeping constant the starting concentration and mixing conditions the obtained droplet sizes diminished dramatically to 69.98 nm for the orange oils and to 27.33 nm for the lemon oil. In both cases, the PDI also diminishes, confirming that a nonionic surfactant is more appropriate to achieve the desired properties of the nanoemulsions.

[0091] Comparing the distribution of the particle size in FIG. 13 for the nanoencapsulated orange oil it is observed a tall and sharp peak that corresponds to the formulation using PEG-based nonionic surfactant and a wider and bimodal distribution corresponding to the formulation using T80.

[0092] Similarly, as shown in FIG. 14 the average droplet size distribution of two nanoemulsions encapsulating lemon oil shows a sharp peak in the left corresponding to the formulation using a PEG-based nonionic surfactant and a wider distribution at the right corresponding to the formulation with T80.

TABLE-US-00003 TABLE 3 Optical characteristics, average droplet size, and polydispertion index (PDI)of nanoemulsions containing essential oils. Average Nanoemulsion Optical droplet size formulation appearance (nm) PDI 4.66% T80, 1% orange oil, Transparent 36.82 0.200 94.3% water 4.66% T80, 1% lemon oil, Translucid 158.50 0.296 94% water 3.25% Eu, 1% orange oil, Opaque (white) 112.20 0.661 95.75% water 3.25% Eu, 1% lemon oil, Transparent 98.67 0.244 95.75% water 4.66% T80, 1% orange oil, Translucid 49.22 0.220 1% coconut oil, 94.3% water 4.66% T80, 1% lemon oil, Opaque (white) 76.72 0.199 1% coconut oil, 94.3% water 4% Eu, 1% orange oil, Translucid 69.98 0.148 1% coconut oil, 94% water 4% Eu, 1% lemon oil, Translucid 27.33 0.101 1% coconut oil, 94% water Eu = PEG-based nonionic surfactant

[0093] Example 24. Using the formulation and procedures of Example 21, three different types of fragrances (e.g., MoodScentz Givaudan fragrances) were nano encapsulated. These are examples of fragrances that blend psychological and neuroscientific measurement techniques that may provide emotional benefits. By combining this technology with mycotextiles (mycoleather), the customer's final experience is enhanced.

[0094] FIG. 15 shows the effect of incorporating these formulations on the hyphae's morphology conforming the mycelium layer before the crosslinking process (which in this case is assisted with nanoparticles as presented in the U.S. Pat. No. 11,993,068B2). One of the effects is the swelling of the fibers due to the penetration of the 20 nm nanodroplets in the case of nanoemulsions. Images in FIGS. 15C and 15D are compared with the control (FIG. 15A). This effect can be seen in several material areas. In the case of the macroemulsion (FIG. 15B), this effect is less marked due to the larger droplet size. In the latter case, the effect is only superficial, i.e., due to Van de Waals-type interactions, the fatty acids interact with the polysaccharides and proteins on the surface of the fungal cell wall. After cross-linking assisted by nanoparticles, it can be observed in FIG. 16B-16D that a greater interconnection of the mycelium occurs after the process in all cases, as compared to the control in FIG. 16A. In the case of the application with the nano emulsions, a more homogenous effect is possible because the wetting process occurs throughout the mycelium layer internal, whereas, when dosing the oil through a macroemulsion, the fats remain on the surface of the fibers, and the effect is more disordered.

[0095] FIGS. 17 to 19 shows the infrared spectra of the mycelium treated with the formulations compared to the control representing the untreated mycelium and the oil phase corresponding to the example to demonstrate its incorporation into the material. In all examples, it can be seen in the spectrum of the treated material, either with a macroemulsion or with a nanoemulsion of coconut oil, a band at 1738 cm.sup.1 corresponding to the carboxylic groups (stretching of CO bonds) within the fatty acids of the oil, as confirmed by the spectrum of the pure oil.

[0096] The quantification of the oil content in the material is carried out by calculating the ratio between the intensity of this CO band with an internal standard. The internal standard is a signal that does not change in any sample and is not affected by the procedure. In this case, the stretching of COC bond in the carbohydrates conforming the cell wall of the fungus, located around 1038 cm.sup.1, is a good internal standard, as it does not change either the intensity or the position after adding the nanoemulsion. Moreover, to ensure that the oil was deposited throughout the thickness of the mycelium layer of the fabric, the mycelium layer was tested in both sides: at the top of the layer (front surface) and, at the bottom of the layer (back surface). To make a proper comparison, each sample should be compared with the control sample, which is the material before the incorporation of the oil. This strategy generates four FTIR spectra required for the quantification of the oil content for each analyzed sample, as observed in the FIG. 20 (in this case named as sample A). For clarity, the position of both bands in 1738 and, in 1038 cm.sup.1 are pointed together with the absorbance value (which is the intensity of the band).

[0097] The collection of a typical spectrum could be done in an infrared spectrophotometer, equipped with an attenuated total reflectance accessory to analyze directly the samples with comparable thickness (e.g., 1 mm). The recording conditions could be 32 scans, and 4 cm.sup.1 resolution in a spectral range of 800 to 1800 cm.sup.1. the obtained spectrum should be baseline-corrected and normalized to the maximum absorbance, in this case is the one located in 1038 cm.sup.1.

[0098] The absorbance values extracted from the spectra in FIG. 20 are listed in table 4. Likewise, the formula used for the calculation and the corrected values of the oil content in the top and in the bottom of the mycelium layer in sample A are compared in the table. As observed, in the top of the mycelium layer the calculated oil content is 31% and in the bottom of the mycelium layer is 27%, indicating that the oil content is homogenous in all the profile of the mycelium layer, ensuring the proper humectation of the mycotextile.

TABLE-US-00004 TABLE 4 Corrected oil content calculated with the absorbance of the CO band of the oil and the COC of the mycelium at the top of the mycelium layer. Absorbance Absorbance Corrected at at (A.sub.1733/ oil 1038 cm.sup.1 1733 cm.sup.1 A.sub.1038) * 100 content Sample name (u.a.) (u.a.) (%) (%) A/control top 1.0005 0.0778 7.8 NA A top 1.0010 0.3804 38.8 31.0 A/control bottom 1.0003 0.0482 4.8 NA A bottom 1.0005 0.3183 31.7 26.9

Scaling-Up

[0099] The industrialization of these formulations requires controlling parameters such as agitation, water addition, and temperature, mainly during the preparation of the concentrated mixture (liquid crystal or bicontinuous microemulsion). To obtain 15 Kg of the nanoemulsion of example 13, it is necessary to start with stirring at 2400 RPM and reach stirring at 3600 RPM while adding water. In this case, a higher temperature (e.g., 90 C.) is helpful to increase the system's fluidity. Once the system is entirely homogeneous (e.g., stirring for 30 minutes at 90 C.), the process of phase inversion and corresponding dilution is started. The agitation is increased up to 5000 RPM. The water is added, which can be according to two approaches: 1) slowly, in the form of droplet threads or, 2) in two parts, a first stage in the form of drops until the viscosity decreases and the phase inversion is just observed and, subsequently, the rest of the water until the desired final concentration is reached. In this case, it is recommended to use water near average boiling temperature (e.g., 95 C.). In FIG. 21, in the case of systems with PEG-based nonionic surfactant, the water addition rate has an essential effect. However, from a practical point of view, piecewise addition is more efficient in terms of preparation time. Likewise, comparing the droplet size distribution profile in a 500 g system versus a 15000 g system (FIG. 22, it can be confirmed that the system obtained with PEG-based nonionic surfactant is quite robust in the preparation as it does not show a significant change in the average droplet size. However, there is a very slight shift towards higher values in the droplet size distribution profile. Three replicates were prepared under these conditions to verify the reproducibility of the scaling-up process (Table 5). The mean value of the hydrodynamic radius (e.g., droplet size) was 18.32.0 nm, meaning that the deviation percentage is 1.1%. This result confirms the reproducibility of the scaling-up procedure proposed for this formulation. Likewise, the polydispersion index ranges 0.180.04 accounting for the homogeneity of the measurement.

TABLE-US-00005 TABLE 5 Replicates of 15 Kg of nanoemulsion using the PEG-based nonionic surfactant to verify the reproducibility of the scaling-up process. Hydrodynamic radius Polydispersion index Replicate (nm) (a.u.) 1 20.53 0.136 2 17.10 0.209 3 17.17 0.194 Mean value 18.3 0.180 Standard deviation 2.0 0.001

[0100] Taking into consideration the challenges regarding the scaling up not only for the preparation but also in the application on the mycotextiles (mycelium fabric), two different approximations were explored, both allowing in-line workflows: (i) a spray strategy vs (ii) an immersion strategy. FIGS. 23A-23B shows examples of both schemes for the scaling-up. In the case of the spraying (FIG. 23A) an automatic spray gun machine, as the same used for surface painting or in leather finishing, can be calibrated for this specific formulation. Here, a spraying gun carrousel is responsible for the uniform spray onto the surface. In step 1, the mat is pressed to a homogenous thickness to guarantee a homogenous penetration of the mixture. In step 2 the pressed mat is transported below the spraying gun carousel. This rotating device can be located between 15 to 150 cm above the mycotextile, applying the mixture at an atomization pressure around 0.3 to 0.9 bar, flow rate around 0.5 to 1000 g/min and, loading around 0.5 g/sqft to 50 g/sqft. The nozzle sizes may range from 0.2 to 2 mm. In the step 3, the fatliquored mycotextile is transported to a roller press to dewater the excess of solution using pressures around 20 to 100 Kg/cm.sup.2 and residence times around 5 to 60 seconds.

[0101] In the case of the immersion bath in FIG. 23B, a similar scheme could be adapted but replacing the strategy of the application; here, an immersion bath with the proper dimensions to contain the mycotextiles, either individually or in-line, is filled with the nanoemulsion formulation. For instance, a 10010010 cm tray can be filled with around 0.01 to 0.1 m.sup.3 of the liquid and the mycotextile is covered with at least 1 cm in depth of the liquid. In the step 1 the mycotextile is flattened, in the step 2 the mycotextile is immersed in the nanoemulsion bath during residence times between 1 to 10 min, in the step 3 the soaked material is transported to a roller press to dewater the excess of solution using pressures around 20 to 100 Kg/cm.sup.2 and residence times around 5 to 60 seconds.

[0102] FIG. 24A and FIG. 24B show a comparison of the oil content in several 100100 cm mycotextiles using both strategies (i.e., spraying and immersion). The mean value for each strategy is listed in Table 6. As observed, using the spraying strategy yields oil contents of 11 and 8% (top and bottom, respectively), while the immersion strategy yields oil contents of 24 and 28%. These results confirm that both strategies allow a uniform distribution of the oil throughout the thickness of the mycelium layer. The conditions analyzed in this case (Spraying: loading of 23 g/sqft of 1% nanoemulsion; Immersion: residence time of 10 minutes in a 1% nanoemulsion) induce a higher oil content by using the immersion strategy. By controlling these conditions, the oil content could be tailored for a targeted result of elongation and/or tensile strength of the final material.

TABLE-US-00006 TABLE 6 Mean oil content (%) in industrial scale applications (spray vs. immersion) in around 20 samples both at the top and the bottom of the mycelium layer Oil content (%) Mycelium layer Spray Immersion Top 11 3 24 6 Bottom 8 2 28 6

Recovery and Reuse

[0103] After its use in the immersion strategy, the nanoemulsion may take on a cloudy appearance with a specific yellowish coloration due to the remains of the fermentation medium. To facilitate its industrial application and extend its shelf life, this preparation can be treated to remove these contaminating agents. Three experiments were carried out to achieve recovery. First (1) filtration with activated carbon was used: for this, a column is filled with activated carbon, and the solution used is passed through the upper part of the column, allowing the liquid to pass through the carbon column by the effect of gravity to be collected at the bottom. The incorporation of finely divided material (activated carbon) into the liquid is observed. A second filtration using a column packed with cotton still yields a yellowish liquid, suggesting that the adsorption of the components causing the color was inefficient.

[0104] Second (2) vacuum filtration using cellulose acetate filters with pores of 1 m was used: in this case, a vacuum filtration system is used with a Buchner-type ceramic funnel connected to a Kitasato-type flask, which in turn is connected to a vacuum pump. A piece of cellulose acetate paper with pores of 1 m was placed in the funnel, covering the perforated section of the funnel. The solution was passed through this system, and a translucent, bluish-colored liquid was expected to be collected. However, with this system was impossible to eradicate the contaminants, and a slight turbidity and yellowish color were still observed.

[0105] Third (3) filtration with cellulose acetate filters of 0.45 m and subsequent addition of sodium hypochlorite: the suspended particulate material was removed using pores of 0.45 m. It is observed that the solution becomes translucent, indicating the removal of suspended particles larger than 0.45 m. However, due to the persistence of the yellowish color, sodium hypochlorite was added up to a final concentration of 0.02 wt. %, starting from a commercial chlorine solution for disinfection. The solution is repeatedly homogenized using vigorous manual agitation and allowed to stand for at least 10 min. Almost immediately, a considerable loss of the yellowish color begins to be observed, suggesting that this strategy is viable for recovering the excess nanoemulsion. DLS analysis confirmed that the strategy is suitable for nanoemulsion recovery since the filtered solution presented average droplet sizes of 23.80.6 nm with polydispersity indices of 0.3310.009. Whereas, upon addition of sodium hypochlorite, average droplet size and polydispersity index values were 23.10.2 nm and 0.1020.003, respectively. Three photographs can be seen in FIGS. 25A-25C showing the appearance of the nanoemulsion after its application on several mats by immersion strategy, in which the presence of turbidity is revealed, followed by a representative photograph of the same preparation after being filtered, and finally, a photograph of the same solution after the addition of sodium hypochlorite: confirming the characteristic translucid of the original liquid.

[0106] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Furthermore, it should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein and may be used to achieve the benefits described herein.

[0107] The various exemplary methods described and/or illustrated herein may also omit one or more of the steps described or illustrated herein or comprise additional steps in addition to those disclosed. Further, a step of any method as disclosed herein can be combined with any one or more steps of any other method as disclosed herein.

[0108] The processor, as described herein, can be configured to perform one or more steps of any method disclosed herein. Alternatively, or in combination, the processor can be configured to combine one or more steps of one or more methods as disclosed herein.

[0109] When a feature or element is herein referred to as being on another feature or element, it can be directly on the other feature or element, or intervening features and/or elements may also be present. In contrast, when a feature or element is referred to as being directly on another feature or element, no intervening features or elements are present. It will also be understood that when a feature or element is referred to as being connected, attached or coupled to another feature or element, it can be directly connected, attached or coupled to the other feature or element or intervening features or elements may be present. In contrast, when a feature or element is referred to as being directly connected, directly attached or directly coupled to another feature or element, there are no intervening features or elements present. Although described or shown concerning one embodiment, the features, and elements so described or shown can apply to other embodiments. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed of adjacent Another feature may have portions that overlap or underlie the adjacent feature.

[0110] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting to the invention. For example, as used herein, the singular forms a, an and the are intended to include the plural forms as well, unless the context indicates otherwise. It will be further understood that the terms comprises and/or comprising, when used in this specification, specify the presence of stated features, steps, operations, elements, and/or components but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof. As used herein, the term and/or includes any and all combinations of one or more of the associated listed items and may be abbreviated as /.

[0111] Spatially relative terms, such as under, below, lower, over, upper, and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is inverted, elements described as under or beneath other elements or features would then be oriented over the other elements or features. Thus, the exemplary term under can encompass both an orientation of over and under. The device may be otherwise oriented (rotated 90 degrees or at other orientations), and the spatially relative descriptors used herein are interpreted accordingly. Similarly, the terms upwardly, downwardly, vertical, horizontal and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.

[0112] Although the terms first and second may be used herein to describe various features/elements (including steps), these features/elements should not be limited by these terms unless the context indicates otherwise. These terms may be used to distinguish one feature/element from another feature/element. Thus, a first feature/element discussed below could be termed a second feature/element. Similarly, a second feature/element discussed below could be termed a first feature/element without departing from the present invention's teachings.

[0113] In general, any of the apparatuses and methods described herein should be understood to be inclusive, but all or a subset of the components and/or steps may alternatively be exclusive and may be expressed as consisting of or alternatively consisting essentially of the various components, steps, sub-components or sub-steps.

[0114] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word about or approximately, even if the term does not expressly appear. The phrase about or approximately may be used when describing magnitude and/or position to indicate that the value and/or position described is within a reasonable expected range of values and/or positions. For example, a numeric value may have a value that is +/0.1% of the stated value (or range of values), +/1% of the stated value (or range of values), +/2% of the stated value (or range of values), +/5% of the stated value (or range of values), +/10% of the stated value (or range of values), etc. Any numerical values given herein should also be understood to include about or approximately that value unless the context indicates otherwise. For example, if the value 10 is disclosed, then about 10 is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that when a value is disclosed that is less than or equal to the value, greater than or equal to the value and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value X is disclosed the less than or equal to X as well as greater than or equal to X (e.g., where X is a numerical value) is also disclosed. It is also understood that the throughout the application, data is provided in several different formats and that this data represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point 10 and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0115] Although various illustrative embodiments are described above, any of several changes may be made to various embodiments without departing from the scope of the invention as described by the claims. Optional features of various device and system embodiments may be included in some embodiments and not in others. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims.

[0116] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived therefrom, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term invention merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.