FIELD OF THE INVENTION

The invention relates to the field of fungal production of dyes and pigments and applying said fungal production of dyes for dyeing substrates. More specifically, the invention relates to an isolated fungus belonging to the species Fusarium solani, a dye produced by said fungus, a method for dyeing a substrate using said fungus, a method for dyeing a substrate using the dye produced by said fungus, a production method of said dye, and also further applications of said dye as an antimicrobial substance.

BACKGROUND OF THE INVENTION

The textile industry is one of the largest global industrial polluters and has one of the largest water footprints. The dyeing process is one of the main culprits of polluting rivers and lakes, posing a working hazard for textile workers and eventually the end consumers. Estimations reveal the use of 79 billion cubic meters of water within the global textile and clothing industry in 2015 corresponding to one third of EU's whole economy need in 2017.

Slama HB et al (2021 ) review synthetic dyes for the textile industry, their discharge impact and treatment methods. Dyes are used for colorization of different types of substrates such as textile fibers, paper, cosmetics, but also for food and pharmaceutical products. The textile industry alone accounts for ~75% of the global dye market and involves around ten thousand different dyes. Textile industries produce fibers to form yarn, which is converted to fabric. Different types of dyeing processes are used for dyeing textile materials such as coating the textile uniformly with the dye, printing of a dye in a specific area of the textile material, bleaching, and finishing comprising crosslinking, softening, and waterproofing the textile material. Two main categories of dyes are known: natural dyes which are derived mainly from plants, and synthetic dyes which are artificially synthesized from chemical compounds. Synthetic dyes are further classified into cellulose fiber dyes such as reactive dyes, direct dyes, indigo dyes, and sulfur dyes; protein fiber dyes such as azo dyes, anthraquinone dyes, triarylmethane dyes, and phthalocyanine dyes, and synthetic fiber dyes such as disperse dyes and basic dyes. As described by Slama HB et al (2021 ), synthetic dyes are mainly derived from petrochemical compounds and are commercialized in liquid, powder, pastes, or granule forms. The majority of these synthetic dyes cause harmful impacts when discharged in non-treated or partially treated forms in the environment and cause multi-contamination effects on air, soil, plants, and water resources, but they also cause severe human diseases.

Furthermore, the production of synthetic dyes from petrochemical compounds has a substantial impact on the environment because of the extensive and expansive environmental impact of the petrochemical industry.

The production of pigments and dyes by microorganisms as well as the subsequent method of dyeing are the promising alternatives for a “greener” and sustainable dyeing industry.

Kristensen SB et al. (2021 ) describe that the Fusarium solani strain 77-13-4 0E:fsr6 G418R by Nielsen M.R. et al. (2019) produces pigments such as aurofusarin, bikaverin and fusarubins under selected cultivation conditions. Nielsen M.R. et al. (2019) describe a vector system for targeted integration and overexpression of genes in Fusarium solani, wherein the Zn(ll)2Cyse transcriptional factor fsr6 controlling mycelial pigmentation was cloned and overexpressed. Thereby, Nielsen et al. (2019) targeted and activated the fusarubin (PKS3:fsr) gene cluster.

Menezes Bruna S. et al. (2020) describe pigment production by Fusarium solani BRM054066.

Molelekoa Tumisi Beiri J. et al. (2021 ) describe the production of pigments by filamentous fungi cultured of agro-industrial by-products using submerged and solid- state fermentation methods.

Rathna Janarthanam et al., (2016) describe the production of naphthoquinones and phenolics by Fusarium solani PSC-R of Palk Bay origin.

Venil CK et al. (2020) describe fungal pigments as potential coloring compounds for textile dyeing. However, the authors also point out that there is a necessity to explore novel pigments producing fungi in order to meet the existing demand for natural pigments. Thus, there is an urgent need for new biological and sustainable production systems for dyes and for biological and sustainable dyeing of substrates such as textiles. SUMMARY OF THE INVENTION

It is the objective of the present invention to provide a biological system for the sustainable production of dyes and pigments and for a sustainable colorization method of substrates.

The objective is solved by subject matter of the present invention.

It has been surprisingly shown that a specific isolated fungus belonging to the species Fusarium solani is able to produce a red dye which can be used for changing the color of different substrates. This isolated fungus lacks an artificial targeted activation of a gene cluster involved in dye production e.g., the isolated fungus of the invention lacks an artificial activation of the PKS3:fsr gene cluster. Furthermore, it has been surprisingly shown that the substrate can be colorized directly by incubating the substrate with the fungus or by incubating the isolated dye with the substrate. Even more surprisingly, it has been shown that the fungal dye is converted to a dark dye or pigment if FeCh is added to the fungal dye and that said dark pigment can also be used for the colorization of a substrate.

According to the invention there is provided an isolated fungus belonging to the species Fusarium solani deposited under the number DSM 34187 at the Leibnitz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH on February 24, 2022.

According to the invention, said isolated Fusarium solani is specifically used for dyeing a substrate.

According to a further embodiment of the invention, a method for changing the color of a substrate is provided, comprising the sequential steps of: i. providing an inoculum of the isolated fungus Fusarium solani deposited under the number DSM 34187; ii. inoculating a cultivation medium with the inoculum; iii. optionally pre-cultivating the inoculated cultivation medium and optionally inactivating the fungus after pre-cultivating; iv. contacting the substrate with the cultivation medium of ii. or iii. until the desired color is obtained, and v. heating the substrate.

According to a specific embodiment, the cultivation medium comprises a carbohydrate source, specifically glucose, preferably in the range of 1 to 4% (m/v). Specifically, pre-cultivating in iii. and/or contacting in iv. is performed at a pH in the range of pH 4.8 to 6.7., specifically in the range of pH 5.0 to 6.5.

Specifically, pre-cultivating in iii. and/or contacting in iv. is performed at a temperature in the range of 15°C to 35°C, specifically in the range of 25°C to 28°C.

More specifically, pre-cultivating in iii. and/or contacting in iv. is performed under aerobic conditions.

Specifically, the method for changing the color of a substrate described herein further comprises contacting of the substrate with FeCh, wherein said contacting specifically leads to a decrease in lightness (L) according to the HSL color model.

According to a further embodiment of the invention, herein provided is a method for changing the color of a substrate comprising the method described herein, further comprising contacting of the substrate with FeCh. Specifically, contacting the substrate with FeCh leads to a decrease in lightness (L) according to the HSL color model.

According to a further embodiment, herein provided is also a method for producing a fungal dye comprising the sequential steps of: i. providing an inoculum of the isolated fungus Fusarium solani deposited under the number DSM 34187; ii. inoculating a cultivation medium with the inoculum; iii. cultivating the inoculated cultivation medium; iv. harvesting the biomass and/or the cultivation medium; and v. optionally extracting the dye from the harvested material of iv..

According to a specific embodiment, the cultivation medium comprises a carbohydrate source, preferably glucose, optionally further comprising peptone.

Specifically, cultivating in iii. is performed at a pH in the range of pH 5.0 ± 0.2 to 6.5 ± 0.2.

Specifically, cultivating in iii. is performed at a temperature in the range of 15°C to 35°C, preferably in the range of 25°C to 28°C.

In a specific embodiment, cultivating in iii. is performed under aerobic conditions.

More specifically, cultivating in iii. is performed at least until the development of a red/purple color is detected in the cultivation medium and/or in the biomass.

According to an embodiment, the cultivation medium is a liquid or a solid medium.

According to a specific embodiment, the method for producing a fungal dye described herein further comprises the addition of FeCh for producing a dark pigment, specifically said pigment has a lightness (L) in the range of 0 to 20%. Herein provided is also a method for extracting a fungal dye, said method comprising the sequential steps of: i. drying harvested biomass and/or harvested cultivation medium comprising the fungus Fusarium solani deposited under the number DSM 34187; ii. suspending the dried material from i. in a solvent, preferably in an alcohol, more preferably in ethanol; iii. evaporating the solvent of ii.; iv. resuspending the residuum of iii. in a solvent different from the solvent of ii., preferably in an ester, more preferably in ethyl acetate; v. evaporating the solvent of iv.; and vi. optionally resuspending the residuum of v..

According to the invention, the isolated Fusarium solani of the invention can be used to produce a dye or the dye can be extracted from said fungus as described herein. Said dye has a color ranging of RGB 170 ± 50, 10 ± 5, 39 ± 10, ranging from cherry red, dark red, brown red and Bordeaux red or any variations thereof.

The present invention also provides a method for changing the color of a substrate comprising the sequential steps of: i. contacting the substrate with the dye described herein until the desired color is obtained, and ii. heating the substrate.

Specifically, heating the substrate is specifically performed at a temperature in the range of 60°C to 121 °C, specifically performed for at least 20 minutes.

Provided herein is also a method for producing a dark pigment comprising the addition of FeCh to the dye described herein or comprising the addition of FeCh to the production method described above.

The present invention further provides a dark pigment produced by the method described herein, wherein said pigment has a lightness (L) in the range of 0 to 20%.

According to a further embodiment of the present invention, herein provided is also a method for changing the color of a substrate comprising the sequential steps of contacting the substrate with the pigment described herein until the desired color is obtained, and heating the substrate.

According to an embodiment of the invention, the substrate is a textile material selected from the group consisting of natural textile material, synthetic textile material, and combinations of natural and synthetic textile materials. Specifically, the natural textile material can be, but is not limited to cotton, silk, wool, abaca, coir, linen, hemp, wood, cashmere, mohair.

Specifically, the synthetic textile material can be, but is not limited to polyester, rayon, acrylic, polycarbonate, polyethylene, spandex, acetate, lyocell, modal.

For dying the substrate, the substrate can be heated at a temperature in the range of 60°C to 121 °C, specifically said heating lasts for at least 20 minutes.

According to yet a further embodiment, the dye produced by the inventive Fusarium solani deposited under the number DSM 34187 has antimicrobial activity.

Specifically encompassed herein is also a substrate having antimicrobial activity, wherein said substrate is dyed with the dye produced by the inventive Fusarium solani deposited under the number DSM 34187.

FIGURES

Figure 1 : Phase contrast microscopy of FS Ila at 40 x magnification (A), 100 x magnification (B) and phase contrast microscopy of FS Ila at 40 x magnification showing a microconidium with 3 septae in comparison (C).

Figure 2: Incubation of Fusarium solani on Sabouraud agar (A), Incubation of Fusarium solani in Sabouraud bouillon (side view: B, top view: C).

Figure 3: Extraction with ethyl acetate results in a red dye/pigment (left flask). Addition of FeCh triggers the formation of a dark and insoluble complex (right flask).

Figure 4: Multifibres (MF) dyed with FS I la. 1 — MF is incubated in a 72 hrs culture of FS Ila for 3 hrs at RT, followed by fixation of pigment at 60 °C for 25 minutes. 2 - Subsequent overnight incubation in FeCh * 6 H2O (0.1 M, 0.03 g mH) changes the colour via complex formation of pigment and FeCh. 3 - MF is incubated for 1 .5 hours in a pigment dye bath followed by a heat treatment at 60 °C for 25 minutes. After cooling down, a second incubation in FeCh solution results in a colour change to grey/black for cotton, cellulose and silk. 4 - MF is incubated for 1.5 hours in a highly concentrated pigment dye bath and heat treated at 60 °C for 25 minutes.

Figure 5: Colour measurement according to RGB system

Figure 6: Colour measurement according to HSL system

Figure 7: Comparison of the red dye of the invention (three images in the middle) to commercial red dyes/colours (left image showing bordeaux colour and right image showing Barbados cherry colour located on the outer sides of the figure).

Figure 8: Results of disc test on antimicrobial activity of the dye of the invention Figure 9: A/. lichenicola Ila (DSM 34187, left) versus N. solani DSM 62805 (right). 4 days growth on Sabouraud agar at 28°C. Both strains produced a red-brownish pigment with isolate Ila showing a higher intensity in colour or higher amount of pigment. Both reached a diameter of ~ 4 cm.

Figure 10: N. lichenicola Ila (DSM 34187, left) versus N. solani DSM 62805 (right). 45 days growth on Sabouraud agar at + 4°C. Aerial mycelium of isolate Ila shows increased pigmentation, however both released pigment into the agar layer.

Figure 11 : N. lichenicola Ila (DSM 34187, left) versus N. solani DSM 62805 (right). 48 hours growth in Sabouraud bouillon at 28°C. Both strains released red pigment into the surrounding medium but again isolate Ila shows a more intense redish colour.

Figure 12: N. lichenicola Ila (DSM 34187, left) versus N. solani DSM 62805 (right). 72 hours growth in Sabouraud bouillon at 28°C. Both strains released red pigment into the surrounding medium but again isolate Ila shows a more intense redish colour.

Figure 13: Phase contrast microscopy of N. lichenicola Ila (DSM 34187).

Figure 14: Phase contrast microscopy of N. solani DSM 62805.

Figure 15: Chromatogram of molecule identification

DETAILED DESCRIPTION

Unless indicated or defined otherwise, all terms used herein have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Sambrook et al, "Molecular Cloning: A Laboratory Manual" (4th Ed.), Vols. 1 -3, Cold Spring Harbor Laboratory Press (2012); Krebs et al., "Lewin's Genes XI", Jones & Bartlett Learning, (2017); Berg et al, “Stryer Biochemie” Springer Verlag, 2018; and Murphy & Weaver, "Janeway's Immunobiology" (9th Ed., or more recent editions), Taylor & Francis Inc, 2017.

The subject matter of the claims specifically refers to artificial products or methods employing or producing such artificial products, which may be variants of native (wildtype) products. Though there can be a certain degree of sequence identity to the native structure, it is well understood that the materials, methods and uses of the invention, e.g., specifically referring to isolated nucleic acid sequences, amino acid sequences, expression constructs, transformed host cells and modified proteins and enzymes, are “man-made” or synthetic, and are therefore not considered as a result of “laws of nature”. The terms “comprise”, “contain”, “have” and “include” as used herein can be used synonymously and shall be understood as an open definition, allowing further members or parts or elements. “Consisting” is considered as a closest definition without further elements of the consisting definition feature. Thus “comprising” is broader and contains the “consisting” definition.

The term “about” as used herein refers to the same value or a value differing by +/-5 % of the given value.

As used herein and in the claims, the singular form, for example “a”, “an” and “the” includes the plural, unless the context clearly dictates otherwise.

According to the invention there is provided an isolated fungus belonging to the species Fusarium solani, wherein said isolated fungus can be applied for the production of a dye applicable in substrate colorization and also for the direct colorization of a substrate. Currently commercially used dyes for substrate colorization are chemically synthesized. Biological dyes and dyeing procedures have a lower environmental impact.

The isolated fungus of the invention is deposited under the number DSM 34187, and is also termed Fusarium solani Ha (FS Ila) or Neocosmospora lichenicola herein. Both terms can be used interchangeably.

Fungi belonging to the species Fusarium solani are filamentous fungi in the Ascomycota division. Fusarium solani is a common soil fungus belonging to the division of Ascomycetes. More than 60 species of this filamentous fungus are combined within the Fusarium solani Species Complex (FSSC) (Coleman, 2016). Within the Fusarium solani species complex (FSSC), many species are known and taxonomically, phylogenetically, and morphologically described (Kristensen et al., 2021 ). According to Short et al (2013) FSSC is a diverse complex of many phylogenetically distinct species. No obvious differences seem to occur in the morphology of divers Fusarium solani species (Schroers H J et al. 2016; Chehri K et al, 2015; Matuo T and Snyder WC, 1972).

Surprisingly, the isolated Fusarium solani Ila of the invention produces a red dye in high yields and short time e.g., after overnight incubation. Using this dye or the isolated fungus, dyeing methods are enabled which allow direct penetration of the color and thus, the adsorption of the pigment/dye onto the textile fiber or material surface.

The term “changing the color” as used herein refers to a change in the color of a substrate. Thus, the visual appearance of the substrate changes.

Colors can be described using color models. The RGB color model is one method for describing a color. The RGB color model is an additive color model in which the red, green, and blue primary colors of light are added together in various ways to reproduce a broad array of colors. An alternative method for describing a color is the CYMK model or the HSL color model. HSL stands for hue, saturation, and lightness.

The term “substrate” as used herein refers to any material which can be a target of color changing. Thereby, the substrate may be a textile material but also a raw material which is converted into a textile material such yams or threads.

The term “textile material” as used herein refers to a material which is made by creating an interlocking bundle of yarns or threads, which are produced by spinning raw fibers into long and twisted lengths. The raw fibers may be of natural or synthetic sources. Textile materials are formed by weaving, knitting, crocheting, knotting, tatting, felting, bonding, or braiding these yarns together. The terms “textile material”, “textile”, and “fabric” may be used interchangeably herein.

According to a specific embodiment, the substrate is a textile material selected from the group consisting of natural textile materials, synthetic textile materials, and combinations of natural and synthetic textile materials.

Natural textile materials can be, but are not limited to cotton, silk, wool, abaca, coir, linen, hemp, wood, cashmere, and mohair, or any combinations thereof.

Specifically, synthetic textile materials can be, but are not limited to polyester, rayon, acrylic, polycarbonate, polyethylene, spandex, acetate, lyocell, and modal, or any combinations thereof.

The terms “dye” and “pigment” are used herein to distinguish the red dye produced by the fungus of the invention from the dark pigment produced by adding FeCh to the red dye. Thereby, the term “dye” is used for water-soluble red dye. The term “pigment” is used for the less water-soluble dark pigment.

According to one embodiment of the invention, a method for changing the color of a substrate is provided. Said method comprises the steps of providing an inoculum of the isolated fungus of the invention, inoculating a cultivation medium with the inoculum, optionally pre-cultivating the inoculated cultivation medium and optionally inactivating the fungus after pre-cultivating, contacting the substrate with the cultivation medium of one of the two previous steps until the desired color is obtained, and heating the substrate.

According to a specific embodiment, said method for changing the color of a substrate comprises the steps of providing an inoculum of the isolated fungus of the invention, inoculating a cultivation medium with the inoculum, contacting the substrate with the inoculated cultivation medium until the desired color is obtained, and heating the substrate.

According to a specific embodiment, said method for changing the color of a substrate comprises the steps of providing an inoculum of the isolated fungus of the invention, inoculating a cultivation medium with the inoculum, pre-cultivating the inoculated cultivation medium and optionally inactivating the fungus after pre-cultivating, contacting the substrate with the pre-cultivated cultivation medium until the desired color is obtained, and heating the substrate.

According to a specific embodiment, said method for changing the color of a substrate comprises the steps of providing an inoculum of the isolated fungus of the invention, inoculating a cultivation medium with the inoculum, pre-cultivating the inoculated cultivation medium and inactivating the fungus after pre-cultivating, contacting the substrate with the pre-cultivated cultivation medium until the desired color is obtained, and heating the substrate.

The term “inoculum” as used herein refers to a population of the fungus of the invention that is introduced in a cultivation medium or any suitable medium for growing the fungus.

According to a specific embodiment, the inoculum may be in solid or liquid form. The inoculum may be a fresh mycelium or a taken from a frozen culture collection such as in the form of a cryo culture. The inoculum may comprise a solid medium comprising a culture of the fungus of the invention e.g., the fungus grown on an agar plate. Said solid fungal inoculum may be pre-cultured for several days prior to inoculation. Specifically, said solid fungal inoculum may be pre-cultured for 1 to 30 days or even longer depending on the cultivation conditions of pre-culturing. Alternatively, the inoculum may comprise a liquid culture of the fungus of the invention. Said liquid inoculum may be pre-cultured for several days prior to inoculation. Specifically, said liquid inoculum may be pre-cultured for 1 to 30 days or even longer depending on the cultivation conditions of pre-culturing. Specifically, the concentration of the inoculum may vary depending on the form of the inoculum and on the pre-culturing of the inoculum.

The term “inoculating a cultivation medium” as used herein refers to the transfer of the inoculum into the cultivation medium.

According to a specific embodiment, the isolated fungus of the invention is cultivated in various methods described herein. The term “cultivation” and “pre- cultivating” is used herein for the growth under conditions outside of the natural environment of the fungus.

According to a specific embodiment, in the case the fungus was not inactivated prior to contacting the substrate with the cultivation medium, the fungus is cultivated also in the step of contacting. In other words, if the substrate is contacted with the cultivation medium comprising a viable fungus, the color of the substrate is changed simultaneously with the growth of the fungus. Thus, the fungal produced dye is directly used for changing the color of the substrate.

The term “contacting” as used herein refers to the step of bringing the substrate into contact with the dye or the fungus producing the dye. An example of contacting is the addition of the substrate directly into the liquid cultivation medium in which the fungus is cultivated or still present. Alternatively, if the isolated fungal dye is used for changing the color of the substrate, contacting is performed by adding the substrate into the solution of the dye.

According to a specific embodiment of the invention, inactivating the fungus may be performed by heating the fungal culture or the cultivation medium comprising the fungus to a temperature of about 121 °C for at least 20 minutes.

According to a specific embodiment, the substrate is removed from the cultivation medium after the step of contacting and prior to the heating step.

According to a specific embodiment, the step of heating the substrate is applied to fix the color.

According to specific embodiment, heating the substrate is performed at a temperature in the range of 60°C to 121 °C. Specifically, a temperature of 121 °C is used if the heating step is also used for the inactivation of the fungus. Specifically, heating the substrate is performed at a temperature in the range of 60°C to 65°C, 65°C to 70°C, 70°C to 75°C, 75°C to 80°C, 80°C to 85°C, 85°C to 90°C, 90°C to 95°C, 95°C to 100°C, 100°C to 105°C, 105°C to 110°C, 110°C to 115°C, 115°C to 120°C, 100°C to 121 °C, 115°C to 121 °C, or at a temperature of 121 °C. Specifically, heating the substrate is performed for at least 20 minutes up to several hours. Specifically, heating the substrate is performed for 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes of even longer. Specifically, heating the substrate is performed for 20 to 25, 20 to 30, 20 to 40, 20 to 50, or 20 to 60 minutes. Specifically, heating may be performed by any method used in the field for heating a substrate. For example, the substrate may be heated in an oven or dryer. According to one embodiment of the invention, the method for producing a fungal dye comprises the sequential steps of providing an inoculum of the isolated fungus of the invention, inoculating a cultivation medium with the inoculum, cultivating the inoculated cultivation medium, harvesting the biomass and/or the cultivation medium, and optionally extracting the dye from the harvested material.

According to a specific embodiment, the cultivation medium can be a liquid or a solid medium.

According to a specific embodiment, in the method for changing the color of a substrate using the fungus, the cultivation medium is a liquid medium.

According to a specific embodiment, in the method for producing the fungal dye, the cultivation medium can be a solid medium or a liquid medium.

In general, a solid cultivation medium comprises a substance for solidification of the cultivation medium such as agar but may comprise the same concentrations of other components as in the respective liquid medium.

According to a specific embodiment, the cultivation medium comprises a carbohydrate source.

According to a specific embodiment, the cultivation medium comprises glucose as carbohydrate source.

According to a specific embodiment, the cultivation medium comprises glucose in the range of 1 % to 4% (m/v). Specifically, the cultivation medium comprises 1 %, 2%, 3%, 4% (m/v) glucose.

According to a specific embodiment, the cultivation medium may comprise a different carbohydrate source than glucose or the combination of glucose with a different carbohydrate source. Specifically, the cultivation medium may comprise maltose instead of glucose, or a combination of maltose and glucose.

According to a specific embodiment, the cultivation medium comprises peptone.

According to a specific embodiment, the cultivation medium comprises 1 %, 5%, 10%, 15%, 20%, 1 to 5%, 1 to 10%, 5 to 10%, 10 to 15%, 5 to 15%, or 1 to 15% (m/v) peptone.

According to a specific embodiment, the cultivation medium may comprise any other component commonly used in cultivation media. Non-limiting examples of such components are buffers, salts, yeast extract, malt extract, amino acids, starch, soybean meal, potato extract, rice extract, casein, dextrin, and antibacterial agents. According to a specific embodiment, the cultivation medium is Sabouraud- Dextrose Agar.

According to a specific embodiment, the cultivation medium is Sabouraud- Dextrose bouillon.

According to a specific embodiment, the cultivation medium has a pH in the range of pH 5.0 ± 0.2 to 6.5 ± 0.2. Specifically, the pH of the cultivation medium is pH 5.0 ± 0.2, 5.1 ± 0.2, 5.2 ± 0.2, 5.3 ± 0.2, 5.4 ± 0.2, 5.5 ± 0.2, 5.6 ± 0.2, 5.7 ± 0.2, 5.8 ± 0.2, 5.9 ± 0.2, 6.0 ± 0.2, 6.1 ± 0.2, 6.2 ± 0.2, 6.3 ± 0.2, 6.4 ± 0.2, or 6.5 ± 0.2.

According to a specific embodiment, said pH of the cultivation medium is maintained during the pre-cultivation of the fungus, during the cultivation of the fungus, during contacting the substrate with the cultivation medium comprising the inoculated cultivation medium, and/or during contacting the substrate with the cultivation medium comprising the optionally pre-cultivated inoculated cultivation medium and comprising the optionally inactivated fungus. The pH can be maintained by any known method such as using a buffered cultivation medium and/or adding acids or bases. The pH may be controlled by a control unit of a bioreactor.

According to a specific embodiment, pre-cultivating and cultivating of the fungus is performed at a temperature in the range of 10°C to 35°C. Specifically, pre-cultivating and/or cultivating of the fungus is performed at a temperature in the range of 15°C to 35°C. Specifically, at a temperature in the range of 15°C to 30°C. Specifically, at a temperature in the range of 15°C to 28°C. Specifically, at a temperature in the range of 20°C to 28°C. Specifically, at a temperature in the range of 25°C to 28°C. Specifically, at a temperature of 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35°C.

According to a specific embodiment, contacting the substrate with the cultivation medium comprising the inoculated cultivation medium is performed at a temperature in the range of 10°C to 35°C. Specifically, at a temperature in the range of 15°C to 35°C. Specifically, at a temperature in the range of 15°C to 30°C. Specifically, at a temperature in the range of 15°C to 28°C. Specifically, at a temperature in the range of 20°C to 28°C. Specifically, at a temperature in the range of 25°C to 28°C. Specifically, at a temperature of 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35°C.

According to a specific embodiment, contacting the substrate with the cultivation medium comprising the optionally pre-cultivated inoculated cultivation medium and optionally inactivated fungus is performed at a temperature in the range of 10°C to 35°C. Specifically, at a temperature in the range of 15°C to 35°C. Specifically, at a temperature in the range of 15°C to 30°C. Specifically, at a temperature in the range of 15°C to 28°C. Specifically, at a temperature in the range of 20°C to 28°C. Specifically, at a temperature in the range of 25°C to 28°C. Specifically, at a temperature of 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35°C.

According to a specific embodiment, the steps of pre-cultivating, cultivating, and contacting are performed under aerobic conditions. Aerobic conditions are conditions in which free oxygen or dissolved oxygen is present.

According to a specific embodiment, harvesting of the biomass and/or the cultivation medium may be performed by separating the fungal biomass from the liquid cultivation medium by e.g., centrifugation and/or filtration.

According to a specific embodiment, harvesting may also be performed by removing the solid medium from its container e.g., from a petri dish. Thereby, the solid medium comprising the fungus may be cut into pieces.

According to a specific embodiment, the cultivation of the fungus, the precultivation of the fungus, the contacting of the substrate with the cultivation medium comprising the inoculated cultivation medium, and/or the contacting of the substrate with the cultivation medium comprising the optionally pre-cultivated inoculated cultivation medium and optionally inactivated fungus may be performed as submerged fermentation in a bioreactor. The bioreactor may be equipped with various systems and sensors for control and automation such as e.g., an oxygen supply system, a pH control system, and/or a mixing system.

According to another specific embodiment, for the production of the dye, the fungus can be cultivated on a solid medium such as via solid state fermentation. Thereby, the dye accumulates in the medium and/or in the mycelium of the fungus.

According to a specific embodiment, in the method for the producing a fungal dye, the step of cultivating is performed at least until the development of a red/purple color is detected in the cultivation medium and/or at the biomass. Thereby, the cultivation is performed until the desired intensity of color is produced by the fungus. The duration of this step may vary depending on the specific conditions of the cultivating step, also depending on the form and concentration of the used inoculum of the fungus.

According to another embodiment, the dye can be extracted from the harvested biomass and/or from the cultivation medium. In the extraction method of the invention, the final residuum may be resuspended. The final residuum may also be further processed for storage of the dye e.g., by production of a powder.

According to one embodiment, the color of the dye produced by the fungus of the invention is in the range of RGB 170 + 50, 10 ± 5, 39 ± 10 using the RGB color model.

According to one embodiment, the lightness (L) of the dye produced by the fungus is in the range of 25-45% according to the HSL color model.

According to a specific embodiment, Fusarium solani Ila produces a red pigment whose color darkens with prolonged growth to dark-red, brown-red and bordeaux red.

According to one embodiment of the invention, the fungal dye can be used for changing the color of a substrate. Specifically, the color of a substrate can be changed by the sequential steps of contacting a substrate with the fungal dye until the desired color is obtained, and heating the substrate. As described previously herein, the heating step is performed to fix the color and is performed as describes elsewhere herein. The resulting color of the textile depends on the duration of the contacting step and also on the concentration of the dye.

According to a specific embodiment, in the step of contacting the substrate with the fungal dye, the dye may be present in liquid or solid form. Specifically, the substrate may be dipped into a solution comprising the dye. More specifically, the substrate may be fully dipped into the solution comprising the dye or may be partly dipped into the said solution. Alternatively, a solution of the dye or the dye in powder form may be sprinkled over the substrate. The substrate may be pre-treated or pre-wetted before the application of the dye. The solution of the dye may be sprayed onto the substrate. Different additives may be added to the solution of the dye e.g., binders and/or thickeners may be added to the solution of the dye for the formation of a paste.

According to another embodiment of the invention, a dark pigment is produced by addition of FeCh to the dye of the invention. Specifically, said dark pigment has a lightness (L) in the range of 0 to 20%. More specifically, said dark pigment has a lightness (L) of O, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20%.

According to a specific embodiment, the lightness (L) of the dark pigment is reduced compared to the lightness (L) of the fungal red dye.

According to another embodiment of the invention, the color of a substrate previously dyed with the fungus of the invention as described herein or with the fungal dye as described can be further changed by addition of FeCh to the dyed substrate. Thereby, the color of the substrate changes towards a darker color. The resulting color of said further treated substrate may be dependent on the color intensity of the prior dyeing step and may also be dependent on the material of the substrate.

According to a specific embodiment, the lightness (L) of the color of the substrate is decreased compared to the color of the substrate prior to contacting with FeCh. In other words, the contacting of a dyed substrate of the invention with FeCh leads to a decrease in the lightness (L) according to the HSL color model. Said decrease in lightness (L) compared to the L-value prior to the application of FeCh may be by 10, 20, 30, 40, 50%, or even more.

According to a specific embodiment, the color of a substrate can be changed by contacting the substrate with FeCh, wherein said substrate was dyed by the fungus or by the fungal dye of the invention prior to contacting with FeCh.

According to a specific embodiment, contacting of a substrate pre-dyed with the dye of the invention with FeCh leads to the formation of a dark color.

According to another embodiment, the color of a substrate can be changed by a method comprising the sequential steps of contacting the substrate with the dark pigment of the invention until the desired color is obtained, and heating the substrate. Again, heating is performed to fix the color.

According to another embodiment of the invention, the fungal dye of the invention has antimicrobial activity.

The term “antimicrobial activity” refers to all active principles that inhibit the growth of bacteria, prevent the formation of microbial colonies, and may destroy microorganisms.

According to another embodiment, the substrate dyed with the dye of the invention has antimicrobial activity. Specifically, the growth of microorganisms on said substrate is inhibited.

According to a specific embodiment, a textile material dyed with the dye of the invention has antimicrobial activity. Specifically, the growth of microorganisms on or in close proximity to the textile material is inhibited.

The invention further comprises the following embodiments:

1 . An isolated fungus belonging to the species Fusarium solani deposited under the number DSM 34187.

2. Use of the isolated fungus of item 1 for dyeing a substrate. 3. A method for changing the color of a substrate comprising the sequential steps of: i. providing an inoculum of the isolated fungus of item 1 ; ii. inoculating a cultivation medium with the inoculum; iii. optionally pre-cultivating the inoculated cultivation medium and optionally inactivating the fungus after pre-cultivating; iv. contacting the substrate with the cultivation medium of ii. or iii. until the desired color is obtained, and v. heating the substrate.

4. The method of item 3, wherein the cultivation medium comprises a carbohydrate source.

5. The method of item 4, wherein the carbohydrate source is glucose, preferably in the range of 1 to 4% (m/v).

6. The method of any one of items 3 to 5, wherein pre-cultivating in iii. and/or contacting in iv. is performed at a pH in the range of pH 5.0 ± 0.2 to 6.5 ± 0.2.

7. The method of any one of items 3 to 6, wherein pre-cultivating in iii. and/or contacting in iv. is performed at a temperature in the range of 15°C to 35°C.

8. The method of any one of items 3 to 7, wherein pre-cultivating in iii. and/or contacting in iv. is performed under aerobic conditions.

9. A method for changing the color of a substrate comprising the method of any one of items 3 to 8 further comprising contacting of the substrate with FeCh.

10. The method of item 9, wherein the contacting leads to a decrease in lightness (L) according to the HSL color model.

11 . A method for producing a fungal dye comprising the sequential steps of i. providing an inoculum of the isolated fungus of item 1 ; ii. inoculating a cultivation medium with the inoculum; iii. cultivating the inoculated cultivation medium; iv. harvesting the biomass and/or the cultivation medium; and v. optionally extracting the dye from the harvested material of iv..

12. The method of item 11 , wherein the cultivation medium comprises a carbohydrate source, preferably glucose.

13. The method of item 11 or 12, wherein cultivating in iii. is performed at a pH in the range of pH 5.0 ± 0.2 to 6.5 ± 0.2. 14. The method of any one of items 11 to 13, wherein cultivating in iii. is performed at a temperature in the range of 15°C to 35°C, preferably in the range of 25°C to 28°C.

15. The method of any one of items 11 to 14, wherein cultivating in iii. is performed under aerobic conditions.

16. The method of any one of items 11 to 15, wherein cultivating in iii. is performed at least until the development of a red/purple color is detected in the cultivation medium and/or at the biomass.

17. The method of any one of items 11 to 16, wherein the cultivation medium is a liquid or a solid medium.

18. A method for extracting a fungal dye, said method comprising the sequential steps of: i. drying harvested biomass and/or harvested cultivation medium of a culture of the fungus of item 1 ; ii. suspending the dried material from i. in a solvent, preferably in an alcohol, more preferably in ethanol; iii. evaporating the solvent of ii.; iv. resuspending the residuum of iii. in a solvent different from the solvent of ii., preferably in an ester, more preferably in ethyl acetate; v. evaporating the solvent of iv.; and vi. optionally resuspending the residuum of v..

19. A dye produced by the method of any one of items 11 to 17 and/or extracted by the method of item 18, wherein its color is in the range of RGB 170 ± 50, 10 ± 5, 39 ±

10.

20. A method for changing the color of a substrate comprising the sequential steps of: i. contacting the substrate with the dye of item 19 until the desired color is obtained, and

11. heating the substrate.

21. A method for producing a dark pigment comprising the addition of FeCh to the dye of item 19 or comprising the addition of FeCh to the production method of any one of items 11 to 17.

22. A dark pigment produced by the method of item 21 , wherein said pigment has a lightness (L) in the range of 0 to 20%. 23. A method for changing the color of a substrate comprising the sequential steps of: i. contacting the substrate with the pigment of item 22 until the desired color is obtained, and ii. heating the substrate.

24. The use of item 2 or the method of any one of items 3 to 10, item 20, and item 23, wherein the substrate is a textile material selected from the group consisting of natural textile material, synthetic textile material, and combinations of natural and synthetic textile materials.

25. The method of item 24, wherein the natural textile material is cotton, silk, wool, abaca, coir, linen, hemp, wood, cashmere, mohair.

26. The method of item 24, wherein the synthetic textile material is polyester, rayon, acrylic, polycarbonate, polyethylene, spandex, acetate, lyocell, modal.

27. The method of any one of items 3 to 10, item 20, and items 23 to 26, wherein heating the substrate is performed at a temperature in the range of 60°C to 121 °C.

28. The method of any one of items 3 to 10, item 20, and items 23 to 27, wherein heating the substrate is performed for at least 20 minutes.

29. The dye of item 19, wherein said dye has antimicrobial activity.

30. A substrate having antimicrobial activity, wherein said substrate is dyed with the dye of item 19.

The examples described herein are illustrative of the present invention and are not intended to be limitations thereon. Many modifications and variations may be made to the techniques described and illustrated herein without departing from scope of the invention. Accordingly, it should be understood that the examples are illustrative only and are not limiting upon the scope of the invention.

EXAMPLES

Example 1 : Cultivation of Fusarium solani of the invention (FS Ila)

Cultivations of Fusarium solani (FS) Ila were done in Luria-Bertani, Sabouraud and wort broth, respectively. Microscopy reveals hyphal growth. The body is formed by filaments or hyphae (Figure 1 A, 1 B, 1 C), which follows typical mycelial growth pattern of fungi belonging to the phylum Ascomycetes. FS Ila is cultivated in a temperature range from 25°C to 28°C aerobically.

Example 2: Production of dye

Fusarium solani Ila is grown on Sabouraud 4 % Glucose agar (Carl Roth, X932.2) as well as in Sabouraud 2 % Glucose Bouillon (Carl Roth, AE23.1 ) at 28 °C. Using this complex medium, a dark red-brown dye (see Figures 2A, 2B and 2C) is formed after overnight incubation (~12 to 16 hours), which diffuses into the agar or into the surrounding medium and colors the mycelium red. In liquid medium, dye formation can take up to 48 hours.

Colonies of FS Ila appear Bordeaux red to dark violet on Sabouraud dextrose agar containing 40 g/l dextrose and 10 g/l peptone at a pH of 5.6. Colonies develop pigmentation after 24 hours of incubation at 25°C to 28°C. The pigment diffuses into the agar leading to a dark Bordeaux red colored agar - almost appearing black.

Cultivation in Sabouraud dextrose broth containing 20 g I’ ¹ dextrose and 10 g I’ ¹ peptone at a pH of 5.6 results in a Bordeaux red colored medium and Bordeaux red colored rounded up mycelium after 24 hours incubation at 28°C at 150 rpm in a baffled or non-baffled Erlenmeyer flask.

Figure 2A shows Fusarium solani incubated overnight on Sabouraud agar, Figures 2B and 2C show Fusarium solani incubated overnight in Sabouraud bouillon.

Example 3: Dye

Extraction of the dye with ethyl acetate results in a red dye (see Figure 3, left flask). Addition of FeCh triggers the formation of a dark and insoluble complex (see Figure 3, right flask).

Example 4: Extraction of dye

Extraction of this Bordeaux red pigment was done according to the following protocol:

- After an incubation period of 10 days, the colored agar/medium including the colored mycelium of FS II on the surface is reduced to small pieces and dried.

- Dried pieces are suspended in EtOH (+10 % dH2O) and heated up to approx. 80°C for 20 minutes. Alternatively, evaporation can be done at room temperature over a period of 3 days.

- The residuum is resuspended in dH2O, and a first extraction is done via addition of ethyl acetate in a separation funnel. After evaporation of ethyl acetate, a sticky, intensively red matter remains.

- This red matter is resuspendable in dH2O, ethyl acetate, acetic acid and ethanol. Example 5: Dyeing 3 ml of fresh medium are inoculated with fresh mycelium or directly from a cryo culture with an inoculation loop. After overnight incubation at 28°C (stirred), fresh 200 ml medium are inoculated with the starter culture from the day before. Blank textile samples are added immediately at cultivation start or are added after sufficient pigment formation. Depending on that, the dyeing time frame ranges from 2 hours to 72 hours resulting in different color intensity. Blank textile samples are incubated in a dye bath containing resuspended red pigment. The colorization starts immediately and intensity increases with time. Each dyeing is finalized with a heat treatment from 60°C to 90°C for 30 minutes which inactivates the fungus (up to 121°C) and fixates the pigment on to material fibers. Example 6: Dyeing The fungus is cultured in liquid complex medium. After a pre-culture, the cells are inoculated in fresh liquid medium and grow in the presence of the fabric to be dyed. The dyeing process is stopped as soon as color intensity is reached. Subsequent heat treatment (60°C for 20 minutes or 90°C for 20 minutes) fixates the pigment within the textile. Example 7: Dyeing Multifibers (MF) were incubated in a 72 hrs culture of FS IIa for 3 hrs at RT, followed by fixation of pigment at 60 °C for 25 minutes. The results of the dyeing process are shown in row 1 of Figure 4. Subsequent overnight incubation of the dyed substrate in FeCl3 * 6 H2O (0.1 M, 0.03 g ml-1) changes the colour via complex formation of pigment and FeCl3 (see row 2 of Figure 4). Example 8: Dyeing The fungus is grown until reaching a desired density as well as dye production, afterwards it is inactivated using heat (121 °C for 20 minutes). Only following inactivation of the fungus, the fabric is added to the culture and incubated until a desired color is reached. Heat treatment finalizes the dyeing procedure. Example 9: Dyeing MF were incubated for 1.5 hour in a pigment dye bath followed by a heat treatment at 60°C for 25 minutes. After cooling down, a second incubation in FeCl3 solution resulted in a colour change to grey/black for cotton, cellulose and silk. The results are shown in row 3 of Figure 4. Example 10: Dyeing

MF were incubated for 1.5 hours in a highly concentrated pigment dye bath and heat treated at 60°C for 25 minutes. The results are shown in row 4 of Figure 4.

Example 11 : Dyeing

Crude extract - partly soluble in H2O - is used for dyeing. Incubation at room temperature for approx. 3 hours results already in a dark red colorization of textile/material. Heat treatment at 90°C for 30 minutes increases intensity and saturation of the color.

Example 12: Dyeing

Additional to described dyeing procedures, the red dye of FS Ila forms a complex with FeCh resulting in a color change to a dark color which is almost black for coloring silk.

Example 13: Characterization of dyed substrates

The dyed substrates shown in Figure 4 have been characterized using a color analysis device (RGB-2000, Voltcraft). The color was analysed for the RGB color model (see Figure 5) and for the HSL color model (see Figure 6).

Example 14: Antimicrobial activity

Antimicrobial activity was tested using a disc test. Thereby, the antimicrobial activity was tested against Escherichia coli, Staphylococcus aureus, Pseudomonas sp. and Bacillus subtilis. Fusarium solan! shows antimicrobial activity against Staphylococcus aureus and Bacillus subtilis as well as against Escherichia coli to a weaker extent. The disc test was performed by incubating Staphylococcus aureus, Escherichia coli, and Bacillus subtilis at 37°C, for 18 hours on Muller-Hinton agar in the presence of discs prepared with 20 μL of the aqueous extract of the dye of the invention. The result of the antimicrobial activity is shown in Figure 8. The sensitivity of selected microorganisms to the extract of Fusarium solan! Ila was determined via measuring the diameter of zones of inhibition (ZOI) in mm. The results of the antimicrobial activity are also shown in the following Table 1 .

Table 1 Example 15: Sequencinq/Re-identification of DSM 34187

After DNA extraction, partial gene tef (translocation elongation factor alpha-1 ) was sequenced using specific primers for the identification of Fusarium sp. Assembled DNA sequences were loaded at GenBank, MycolD and Fusarium ID database.

Date of Sequencing: 31 .03.2022

Sequencing by: DSMZ, Braunschweig (GER)

Sequencing result:

Identification:

Neocosmospora Hchenicola (C. Massal.) Sand. -Den. & Crous, [MB#822901 ]

Neocosmospora Hchenicola belongs to the Fusarium solani species complex, FSSC. N. Hchenicola belongs to FSSC group 16b. Example 16: Morphology of DSM 34187

Strain DSM 62805 Neocosmospora solani (see reference L. Lombard, N.A. van der Merwe, J.Z. Groenewald, and P.W. Crous Studies in Mycology 80: 189-245) was used as a reference strain to the isolate Ila. First investigations of potential morphological differences were performed via growth and aerial mycelium observation as well as growth patterns in liquid culture. Both strains were cultivated on Sabouraud agar (4 % glucose) and Sabouraud bouillon (2 % glucose) at 28 °C. See Fig. 9 to Fig. 14.

Example 17: Identification of molecule

The crude extract of the red pigment - produced by isolate Ila - was dissolved in EtOH + 25% H2O at a concentration of 0.5 mg ml" ¹ and loaded into an HPLC column (see chromatogram in Fig. 15). The chromatogram shows peak A at a retention time of around 1.45-1.50 min at a mAU of around 29, peak B at a retention time of around 1 .65 min at a mAU of around 21 , peak C at a retention time of around 1 .70 min at a mAU of around 3, and peak D at a retention time of around 1 .85 min at a mAU of around 4.

Example 18: Identification of molecule

The substances identified from the extract of Fusarium Hchenicola Ila (DSM 34187) are shown in the following table 2:

Table 2 REFERENCES

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