Production of pigmented fungal mycelium

Abstract

The present invention relates to a method for producing a composition comprising a fungal biomass, the composition being characterized in that it has a particular color, as well as to the compositions and supernatants obtainable according to the method of the invention, and to their use in the production of food products, in particular food products characterized by a particular color.

Description

[Technical field]

[0001] The present invention relates to a method for producing a composition comprising a fungal biomass, the composition being characterized in that it has a particular color, as well as to the compositions and supernatants obtainable according to the method of the invention, and to their use in the production of food products, in particular food products characterized by a particular color. [Background technology]

[0002] In recent years, the production of food from animals has attracted attention due to its unsustainability and growing concerns about animal welfare. In the context of climate change, many plant-based meat substitutes have emerged with the aim of reducing CO2 emissions and animal suffering. However, these products are currently produced from three major monocrops (soybean, pea, and rice), whose cultivation requires a lot of land and water, relies heavily on chemical agents (pesticides and fertilizers), and generates a lot of waste because only proteins isolated from these crops are used to produce meat substitutes. In addition, these isolates have a strong bitter taste and do not have a unique texture or inherent color typical of certain foods, and therefore their use in foods requires further processing steps and the addition of further ingredients, including but not limited to flavorings, conditioners, and / or colorants. Thus, plant-based substitutes are not necessarily healthy, and their production causes other environmental problems such as deforestation, significant reduction in biodiversity, soil contamination, and / or water pollution.

[0003] The production of food using fermentation processes addresses some of these shortcomings. It allows for better use of land, since fermenters can be scaled vertically, allowing for on-site food production in urban or rural areas. Moreover, less water is required per kilogram of product than plant proteins, and with the continuous development and improvement of filtration and processing techniques, this water can be reused in the process. Another advantage of fungal fermentation over the production of traditional plant isolates is included in the resulting raw material - fungal biomass, which naturally already has the desired fibrous texture and results in a balanced nutritional profile with complete protein as well as dietary fiber, vitamins and micronutrients that provide a healthy product for the consumer. In particular, the use of mycelium isolated from fruiting bodies from known edible mushrooms allows the production of a clean and tasty product with a very short list of ingredients, which also results in the typical mushroom umami taste specific to this group, which varies little between species (e.g. morels, truffles or button mushrooms).

[0004] Currently, there are no methods for producing colored fungal mycelium for use in food production. It is noted that fungal mycelium has been used previously for the degradation of synthetic dyes and for decolorization of industrial wastewater.

[0005] US Patent Application Publication No. 2021 / 0045410 discloses a method of forming a bound textured substrate, comprising inoculating a textured substrate with at least one mycelium-producing fungus and growing the mycelium-forming fungus to form a matrix of mycelium inside, outside, or inside and outside the textured substrate. Specific embodiments are disclosed in which any combination of color and flavor is added to the textured substrate, such that the color and flavor or any combination thereof is taken up by the fungus and becomes part of the mycelium during growth. The present invention differs from US Patent Application Publication No. 2021 / 0045410 in that the mycelium grown according to the present invention is not substrate bound or is bound to powder particles rather than to a textured substrate.

[0006] WO 2021 / 195175 discloses a particular method for producing colored fungal biomass, which includes the steps of fermentation and extrusion, where color is added after extrusion.

[0007] U.S. Pat. No. 1,105,8137 discloses several examples of food products having a white or tan color and generally teaches that color can be influenced by substrate ingredients, such as plant ingredients that may be combined with mycelium.

[0008] US Patent No. 11166477 discloses a method for obtaining a food product by mixing a mycelium-processed material with an edible material, in which the edible material may contain a coloring agent.

[0009] US Patent Application Publication No. 20090148558 discloses a process in which colorants can be added to the mycelium during the mixing process.

[0010] U.S. Patent No. 7,035,160 teaches that edible ingredients, such as colorants, can be added to edible (e.g., proteinaceous) substances, including fungal cells of the order Mucorales having reduced (or low) RNA content, suitable for use in foods. The document further teaches that the color can depend on the mushroom used.

[0011] Wang and Jiu (Water Science and Technology, volume 38, issues 4-5, 1998, pages 233-238) disclose specific aspects of the adsorption and degradation of synthetic dyes onto the mycelium of Trametes versicolor.

[0012] Kasinath et al. (Enzyme and Microbial Technology, volume 32, issue 1, 2 January 2003, pages 167-173) disclose specific aspects of decolorization of synthetic dyes by Irpex lacteus in liquid culture and in a packed bed bioreactor.

[0013] Chander et al. (Journal of Industrial Microbiology and Biotechnology, Volume 31, Issue 2, 1 February 2004, Pages 94-97) disclose specific aspects of the biological decolorization of several industrial dyes by white-rot fungi.

[0014] Chinese Patent No. 113583882 discloses a specific method for preparing fungal mycelium for use in replacing proteinaceous meat preparations, which involves performing liquid mixed fermentation on edible fungi and Monascus.

[0015] JP 10-75739 A discloses a specific method for producing a mineral-containing mushroom mycelium food material, in which liquid culture of the mushroom mycelium is carried out using a medium comprising food by-products containing mineral components. Summary of the Invention

[0016] Desirable are methods for producing a fungal biomass or a composition comprising a fungal biomass, wherein the biomass or composition is characterized by a particular color. Particularly desirable are methods in which the produced composition is characterized by a strong and / or durable color that is resistant to washing and / or bleaching. Even more particularly desirable are methods in which degradation by the mycelium of the components responsible for the particular color of the mycelium is avoided.

[0017] As those skilled in the art will understand, this method is difficult to carry out.The use of mycelium to treat industrial wastewater and thereby degrade dyes, such as synthetic dyes, contained therein is well known in the art.Therefore, one of the challenges is to overcome the inherent ability of fungal mycelium to degrade the coloring agents to which it is exposed.

[0018] It was therefore an objective technical problem of the present invention to provide an improved method for the production of coloured biomass.

[0019] The objective technical problem is solved by the embodiments described herein and characterized in the claims.

[0020] The inventors have shown that when an additive that produces the color of the composition is added to a growing (cultured) fungal biomass at a particular time during its growth / culture or for a particular residence time, the biomass is able to bind or assimilate the additive in such a way that it cannot be easily washed away. The inventors have further shown that the optimal time or optimal residence time may depend on the nature of the additive and the desired color to be achieved.

[0021] The present invention is summarized in the following embodiments.

[0022] In a first embodiment, the present invention relates to a method for producing a composition comprising a fungal biomass, the composition being characterized in that it has a particular color, the method comprising the steps of: (a) providing a growth medium; (b) providing at least one fungal strain; (c) culturing at least one fungal strain in a growth medium; (d) supplementing the growth medium with at least one additive that produces a color in the composition; (e) further culturing the at least one fungal strain in a growth medium supplemented with at least one additive that produces the color of the composition; (f) recovering a composition comprising a fungal biomass from the growth medium, the composition being characterized as having a particular color; The particular color is due to at least one additive replenished in step (d).

[0023] In a further embodiment, the present invention relates to a composition obtainable according to the process of the present invention.

[0024] In a still further embodiment, the present invention relates to a food product comprising the composition obtainable according to the method of the present invention.

[0025] In a still further embodiment, the present invention relates to the use of the composition obtainable according to the process of the present invention in the manufacture of a food product.

[0026] In a still further embodiment, the present invention relates to a supernatant obtainable according to the method of the present invention.

[0027] In a still further particular embodiment, the present invention relates to the use of the supernatant of the present invention in the manufacture of a food product. [Brief description of the drawings]

[0028] [Figure 1] Typical growth profile of a microorganism, where x1 represents the time point on day 0 of fermentation when cocoa and lycopene are added for batch fermentation, x2 represents the time point in the deceleration phase when astaxanthin and paprika are added for batch fermentation, and x3 represents the time point in the exponential phase when colorants are added at optimized concentrations for optimal residence time at steady state conditions for a fermentation operated in continuous mode (the biomass, preferably understood as fungal cells, remains in this phase at a constant concentration). [Diagram 2] A) Orange / red mycelial biomass obtained from astaxanthin solution added during fermentation. Average RGB (186, 70, 43). B) Light orange / red mycelial biomass obtained from astaxanthin added after fermentation. Average RGB (228, 182, 133). C) Stereo microscope image of orange / red mycelial biomass obtained from astaxanthin added during fermentation. [Figure 3-1] A) Dark brown mycelial biomass obtained from cocoa powder suspension added during fermentation. Average RGB (32, 27, 24). B) Light brown mycelial biomass obtained from cocoa powder suspension added after fermentation. Average RGB (76, 43, 25). C) Microscopic images of colored biomass showing both adsorption and absorption mechanisms. D) Mycelial biomass with cocoa powder added at day 0 (left, color: light brown - not uniform) versus the slowdown phase (right, color: dark brown - uniform). [Figure 3-2]A) Dark brown mycelial biomass obtained from cocoa powder suspension added during fermentation. Average RGB (32, 27, 24). B) Light brown mycelial biomass obtained from cocoa powder suspension added after fermentation. Average RGB (76, 43, 25). C) Microscopic images of colored biomass showing both adsorption and absorption mechanisms. D) Mycelial biomass with cocoa powder added at day 0 (left, color: light brown - not uniform) versus the slowdown phase (right, color: dark brown - uniform). [Figure 4] A) Light orange (left) and dark orange (right) mycelial biomass obtained from a paprika suspension added during fermentation. Average RGB: left (151, 99, 59), right (119, 55, 33). B) Microscopic images of the colored biomass showing both adsorption and absorption mechanisms. [Diagram 5] A) Biomass was made into 4 patties of 50 g each and treated according to the conditions indicated (0.5 patties fried immediately after cooking in the two different conditions, 0.5 patties kept in the refrigerator, one patty kept in the refrigerator for 20 days from which 0.5 patties were fried and the other not). B)-C) Comparison of fried and non-fried patties made from biomass colored after (B) and during (C, darker) fermentation. Average RGB: B: from left to right: (167, 99, 49), (212, 143, 112), (155, 86, 59), (212, 143, 112), C: from left to right: (112, 58, 46), (187, 93, 69), (135, 50, 27), (187, 93, 69). [Figure 6]Comparison of deep-fried patties at day 0 or day 21 under different cooking conditions (1st column: 65°C for 15 min, 2nd column: 80°C for 15 min). The coloring performance and browning effect of the patties made from biomass colored during fermentation (bottom panel) was significantly better than that of the patties made from biomass colored after fermentation on day 0 (top panel). Both showed improved browning effect. Average RGB: top row left to right: (167, 99, 49), (155, 86, 59), 2nd row left to right: (145, 72, 40), (143, 55, 27), 3rd row left to right: (112, 58, 46), (135, 50, 27), bottom row left to right: (110, 54, 48), (105, 57, 53). [Figure 7] Biomass with a natural whitish tint. Average RGB(228, 226, 226). [Figure 8] Exemplary experimental procedures for the methods of the invention. [Figure 9] Mycelial biomass with 0.9 g / L astaxanthin (left, color: light orange, faint red) versus 1.8 g / L astaxanthin (right, color: red) added during the deceleration phase. [Figure 10] Mycelium biomass with 0.9 g / L astaxanthin (left, color: light orange, faint red) vs. 1.8 g / L astaxanthin (right, color: red) after 24 hours of soaking in tap water. [Figure 11] Example of water washing after 2 hours, 4 hours, and 6 hours for a sample containing 0.9 g / L astaxanthin. [Figure 12] Mycelial biomass supplemented with astaxanthin during the lag phase (X1), exponential phase (X3), and deceleration phase (X2) (colors from left to right: brown / red with white dots, pale orange / pink, red). [Figure 13] Water sample after heating biomass for 80 min. [Figure 14] Biomass samples stained with 0.9 g / L astaxanthin after fermentation at 25° C.—biomass before washing and after 2, 3, and 6 hours from left to right, respectively. [Figure 15]Biomass samples stained with 0.9 g / L astaxanthin after fermentation at 4° C.—biomass before washing and after 2, 4, and 6 hours from left to right, respectively. [Figure 16] Mycelial biomass supplemented with 0.9 g / L astaxanthin at time 0 in a 1 L fermenter (left, color: whitish / light brown) versus a 100 mL flask (right, color: brown / red with white dots). [Figure 17] Mycelial biomass was harvested in a 1 L fermenter and supplemented with 0.9 g / L astaxanthin during the deceleration phase (X2). [Figure 18] An internal view of the 1 L fermenter supplemented with 0.9 g / L astaxanthin during the deceleration phase (X2). [Figure 19] Mycelial biomass with 1 g / L lycopene added at day 0 (left, color: brown / red with white dots) versus the deceleration phase (right, color: orange / red). [Figure 20] Mycelial biomass from co-fermentation with Rhodotorula sp. [Figure 21] Trends in CO2 fixed by Haematococcus pluvialis microalgae. [Figure 22] Light brown mycelial biomass with average RGB of (185, 159, 136) stained with a lignocellulosic extract from wheat bran, the extract having a dark black / brown color with average RGB of (23, 23, 22) For the illustrative example here, the extract was added at X1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present invention is described below, it being understood that all combinations of features are envisaged.

[0030] In a first embodiment, the present invention relates to a method for producing a composition comprising a fungal biomass, the composition being characterized by having a particular color, where having a particular color as referred to herein is to be understood as the property of a material to absorb light of a particular wavelength.

[0031] Preferably, the specific colors referred to herein are colors that correspond to the colors of specific foods. Several systems for describing colors are known to those skilled in the art. Particularly useful is the use of the RGB color classification, which is based on an additive color model in which the red, green, and blue primary colors of light are added together in various ways to reproduce a wide range of colors. In the RGB model, each of the components preferably exists as an integer value between 0 (i.e., not present) and 255 (i.e., fully present). Typical colors of common types of foods are represented on the RGB scale as follows:

[0032] Red meat: R component: 100 to 255, preferably 150 to 255 G component: 0 to 100, preferably 0 to 60 B component: 0 to 100, preferably 0 to 60

[0033] White meat: R component: 150 to 255, preferably 200 to 255 G component 100 to 255, preferably 150 to 255 B component 100 to 255, preferably 150 to 255

[0034] Red fish (salmon and tuna): R component: 150 to 255, preferably 200 to 255 G component: 0 to 200, preferably 0 to 150 B component: 0 to 150, preferably 0 to 100

[0035] White fish: R component: 100 to 255, preferably 150 to 255 G component 100 to 255, preferably 150 to 255 B component 100 to 255, preferably 150 to 255

[0036] cheese: R component: 100 to 255, preferably 150 to 255 G component 100 to 255, preferably 150 to 255 B component: 0 to 250, preferably 0 to 200

[0037] yogurt: R component: 100 to 255, preferably 150 to 255 G component: 50 to 255, preferably 50 to 200 B component: 50 to 255, preferably 100 to 255

[0038] chocolate: R component: 0 to 255, preferably 0 to 200, most preferably 50 to 200 G component: 0 to 200, preferably 0 to 150, most preferably 50 to 150 B component: 0 to 200, preferably 0 to 150, most preferably 0 to 100

[0039] A method for producing a composition comprising a fungal biomass, the composition being characterized in that it has a particular color, the method comprising: (a) providing a growth medium; (b) providing at least one fungal strain; (c) culturing the at least one fungal strain in the growth medium; (d) supplementing the growth medium with at least one additive that produces color in the composition; (e) further culturing the at least one fungal strain in the growth medium supplemented with the at least one additive that produces a color in the composition; (f) recovering a composition comprising a fungal biomass from the growth medium, wherein the composition is characterized as having a particular color.

[0040] It should be understood that in the method of the present invention, the particular color of the composition results from at least one additive supplemented in step (d) of the method.

[0041] In the first step (a) of the method, a growth medium is provided. The growth of fungal biomass is known to those skilled in the art who can select a medium that supports the growth of a particular microorganism by providing appropriate growth conditions. Thus, the medium is not particularly limited.

[0042] As understood herein, the medium provided in step (a) may further comprise additives selected from vitamin B12, vitamin B6, vitamin B2, vitamin B3 (also called niacin), riboflavin, thiamine, vitamin A, vitamin E, omega-3 fatty acids, vitamin D2, folic acid, iodized salts (NaCl, further comprising iodized salts in an amount of up to 5% w / w), and minerals (e.g., salts containing calcium, iron, and / or potassium, etc.). Preferably, the medium provided in step (a) further comprises vitamin B12 and / or omega-3 fatty acids. Such further additives may be added to enhance the nutritional value and taste of the potentially obtained novel food product.

[0043] In step (b) of the method of the present invention, at least one fungal strain is provided. Preferably, the at least one fungal strain is a single fungal strain, in other words, one fungal strain. However, the term at least one fungal strain may also refer to two or more fungal strains, for example, two, three or four fungal strains.

[0044] Preferably, at least one fungal strain is an edible fungal strain. Preferably, an edible fungus is understood to be a fungus that can be consumed by a living organism, preferably by a human, thereby providing nutrition and not causing any negative effects to said organism, preferably a human.

[0045] More preferably, the at least one fungal strain is selected from Basidiomycota and Ascomycota.

[0046] Even more preferably, the at least one fungal strain is selected from Pezizomycotina and Agaricomycotina.

[0047] Even more preferably, the at least one fungal strain is selected from Peziomycetes, Agaricomycetes, and Sordariomycetes.

[0048] Even more preferably, the at least one fungal strain is selected from Pezizales, Boletales, Cantharellales, Agaricales, Polyporales, Russulales, Auriculariales, Sordoriales and Hypocreales.

[0049] Even more preferably, the at least one fungal strain is selected from Morchellaceae, Tuberaceae, Pleurotaceae, Agariaceae, Marasmiaceae, Cantharellaceae, Hydnaceae, Boletaceae, Meripillaceae, Polyporaceae, Strophariaceae, Lyophyllaceae, Tricholomataceae, Omphalotaceae, Physalacriaceae, Schizophyllaceae, Sclerodermataceae, Ganodermataceae, Sparassidaceae, Hericiaceae, Bondarzewiaceae, Cordycipitaceae, Auriculariaceae, Sordoriaceae, Nectriaceae and Fistulinaceae.

[0050] Even more preferably, the at least one fungal strain is P. pulmonarius P. ostreatus, P. citrinopileatus or P. salmoneostramineus, or the at least one fungal strain is M. esculenta, M. angusteps, M. deliciosa or M. rufobrunnea.

[0051] Most preferably, the at least one fungal strain is P. pulmonarius.

[0052] In another embodiment, the at least one fungal strain selected from Agaricales may be a fungal strain selected from Pleurotaceae. Even more preferably, the at least one fungal strain of the present invention is a fungal strain selected from Pleurotus pulmonarius, Pleurotus ostreatus, Pleurotus citrinopileatus, Pleurotus florida, Pleurotus eunosmus, Pleurotus columbinus, Pleurotus ferulae, Pleurotus salmoneo-stramineus, Pleurotus Sapidus, and Pleurotus salmoneostramineus, even more preferably selected from Pleurotus pulmonarius and Pleurotus ostreatus, and most preferably Pleurotus pulmonarius.

[0053] In another embodiment, the fungal strain selected from the Morchellaceae is Morchella esculenta, Morchella angusticeps, Morchella deliciosa, Morchella sceptrifomtis, Morchella steppicola, Morchella puncripes, Morchella rufobrunnea, Morchella importuna, Morchella Jaurentinaa, or Morchella purpumscens, preferably Morchella esculenta, Morchella angusticeps, or Morchella deliciosa.

[0054] Preferably, the at least one fungal strain is selected from Pleurotus pulmonarius, Pleurotus florida, Pleurotus citrinopileatus, Pleurotus salmoneostramineus, Morchella esculenta, Morchella angusticeps, Morchella deliciosa, and Morchella rufobrunnea.

[0055] Most preferably, the at least one fungal strain is selected from Pleurotus pulmonarius and Morchella rufobrunnea.

[0056] As understood herein, when a fungal strain is referred to by reciting a particular fungal species, it is preferably understood to encompass any fungal strain belonging to that species.

[0057] In step (c) of the method of the invention, at least one fungal strain is cultivated in a growth medium.

[0058] Typically, a constant temperature is maintained throughout the process, which may be selected for optimal growth of a particular fungal strain, as known to those skilled in the art. For example, for P. ostreatus, the cultivation is preferably carried out at a temperature of 25-30° C. More preferably, the cultivation is carried out at a pH of 3.0-8.5. As will be appreciated by those skilled in the art, the choice of pH may depend on the fungal strain being cultivated, or on potential contaminating strains that are excluded from the cultivation. More preferably, the cultivation step is carried out for a time period of 12-240 hours.

[0059] In step (c) of the method of the invention, the cultivation is preferably carried out as submerged fermentation.

[0060] Thus, the submerged fermentation in the method of the invention can be operated as a batch process, a fed-batch process or a continuous process. These three main fermentation methods are known to those skilled in the art and differ in the outflow and inflow of materials from and into the fermentation vessel.

[0061] Batch processes are characterized by the absence of an inflow of material into the fermentation vessel. In a batch process, all nutrients are provided at the beginning of the culture without further addition in the subsequent bioprocess. No additional nutrients are added during the entire bioprocess, except for gas, acid and base. The bioprocess then continues until the nutrients are consumed. This strategy is suitable for rapid experiments such as strain characterization or nutrient medium optimization. The drawback of this convenient method is that the yield of biomass and products is limited. Usually, the microorganisms are not in logarithmic growth phase for a long time, since the carbon source and / or oxygen transfer are the limiting factors. After the end of a bioprocess carried out in batch mode, only the biomass or the medium is recovered and appropriately processed to obtain the desired product. From the bioreactor's point of view, the process is repeatedly interrupted by cleaning and sterilization steps, and the biomass is simply produced in stages.

[0062] In fed-batch processes, substrates, nutrients and other substances can be added to the fermentation vessel to, among other things, extend the possible cultivation time or increase the yield. The advantage of feeding during cultivation is that it allows to achieve a higher overall production. Under certain growth conditions, the microorganisms and / or cells constantly double and thus follow a logarithmic growth curve. Therefore, in certain embodiments, the feed rate can be increased logarithmically as well. Generally, the substrate is pumped from a feed bottle into the cultivation vessel, for example through silicone tubing. The user can manually set the feed at any time (linearly, logarithmically, pulsed) or add nutrients when certain conditions are met, such as when a certain biomass concentration is reached or when nutrients are depleted. Fed-batch processes offer a wide range of control strategies and are also suitable for highly specialized applications. However, fed-batch processes can increase processing times and potentially lead to inhibition due to the accumulation of toxic by-products.

[0063] Preferably, in the method of the invention, the submerged fermentation is operated as a continuous process. After a batch growth phase, an equilibrium is established with respect to certain components (also called steady state). Under these conditions, the same amount of fresh culture medium is added as is removed (chemostat). These bioprocesses are called continuous cultures and are particularly suitable when excess nutrients would result in inhibition, for example due to acid or ethanol accumulation or excessive heating. It is understood that after reaching a steady state, the continuous mode always operates in the logarithmic growth phase, where the cells are maintained at a constant concentration. The transient state is the state before a certain steady state condition is reached in the continuous mode, and is in fact similar to the start of the batch mode operation. Other advantages of this method include reduced product inhibition and improved space-time yield. When the medium is removed, the cells are harvested, which is why the inflow and outflow rates must be less than the doubling time of the microorganism. Alternatively, the cells can be retained in a wide variety of ways, called perfusion (for example in spin filters). In a continuous process, the space-time yield of the bioreactor can be further improved compared to that of a fed-batch process. However, long culture periods also increase the risk of contamination and long-term changes in the culture.The three most common types of continuous culture are chemostat (the rate of addition of a single growth-limiting substrate controls cell growth), turbidostat (an indirect measurement of cell number, i.e., turbidity or optical density, which requires additional sensors, is driven by real-time feedback to control the addition and removal of liquid), and perfusion (this type of continuous bioprocessing mode is based on either retaining the cells in the bioreactor or recycling the cells back to the bioreactor, providing fresh medium and removing cell-free supernatant at the same rate).

[0064] In one embodiment of the invention, in the method of the invention the submerged fermentation is not operated as a continuous process.

[0065] In step (d) of the method of the invention for producing a composition comprising a fungal biomass, characterized in that the composition has a particular color, the growth medium is supplemented with at least one additive that produces the color of the composition, which is described in detail below.

[0066] According to the inventors, the timing of the supplementation in step (d) relative to steps (c) and (e) is important for the method for producing a composition comprising a fungal biomass of the present invention, characterized in that the composition has a particular color. In particular, according to the inventors, the timing of the supplementation in step (d) relative to steps (c) and (e) is important for the color that characterizes the composition produced in the method for producing a composition comprising a fungal biomass of the present invention. Without being bound by theory, the timing is important, for example, to avoid degradation of at least one additive that causes the color of the composition by the mycelium.

[0067] Preferably, the supplementation in step (d) is carried out in the lag, acceleration, logarithmic, deceleration or stationary phase of biomass growth. Different phases of growth are known to those skilled in the art. When the amount of biomass, which can be measured as the concentration of cells present in the culture or the total mass of mycelium formed in the culture, is plotted as its logarithm against the culture time, as shown in FIG. 1, the very limited initial phase of growth observed is called the lag phase. It should be understood that the lag phase encompasses the start of the cultivation of the fungal biomass. As shown in FIG. 1, the lag phase is followed by an acceleration phase. The acceleration phase is followed by a logarithmic phase, which is shown as a linear curve in the graph of the logarithm of the amount of biomass (which may also be referred to as the logarithmic increase of biomass) plotted against the culture time. As known to those skilled in the art, in the logarithmic phase, the amount of biomass is exponentially increasing. Due to the limitations of the culture conditions (e.g. reaching too high a cell concentration or exhausting the nutrients available in the medium), the exponential growth rate in the culture cannot be maintained indefinitely in batch fermentation. Thus, the exponential phase is followed by a deceleration phase, during which growth slows down due to nutrient depletion, preferably carbon source depletion. Thus, one exemplary carbon source is glucose. Preferably, the deceleration phase is defined herein as the point in time when at least 80% by weight of the nutrient (e.g., carbon source, e.g., glucose) is depleted, more preferably at least 90% by weight of the nutrient (e.g., carbon source, e.g., glucose) is depleted, even more preferably at least 95% by weight of the nutrient (e.g., carbon source, e.g., glucose) is depleted. The skilled person is in a position to determine whether deceleration has started based on the determination of the concentration of the carbon source, in particular glucose, by withdrawing a sample from the reactor / fermenter / flask and measuring the content of glucose (or any other suitable carbon source in question) by using, for example, an HPLC-based method. Alternatively, the depletion rate of another limiting nutrient (e.g., nitrogen source) can also be monitored to determine whether the process is in a deceleration phase. For example, such a nutrient may be the nutrient (e.g., nitrogen source) that will be completely consumed first.As shown in FIG. 1, the deceleration phase is followed by a stationary phase during which the amount of biomass remains constant (in other words, the growth rate is equal to the rate of biomass dying).

[0068] Preferably, the supplementation in step (d) is carried out during the deceleration or exponential phase of biomass growth. It should be understood that if growth is carried out as a batch fermentation, step (d) is preferably carried out during the deceleration phase. It should be understood that if growth is carried out as a continuous process, step (d) is preferably carried out during the exponential phase.

[0069] Even more preferably, the supplementation in step (d) is carried out during the slowdown phase of biomass growth (in the case of batch fermentation). As understood herein, supplementation in step (d) during the slowdown phase of biomass growth can avoid oxidative damage to the additives added in step (d) and therefore can avoid the use of antioxidants in the fungal culture.

[0070] When the supplementation in step (d) is carried out during a period of slowdown in biomass growth, the at least one additive that produces color in the composition is a carotenoid, preferably selected from astaxanthin and lycopene, more preferably astaxanthin.

[0071] However, in certain embodiments, the supplementation in step (d) is carried out at the start of biomass growth or prior to biomass growth.

[0072] As understood herein, the considerations at the time of step (d) above preferably apply to embodiments in which the at least one fungal strain is not cultured in continuous mode.

[0073] The color-producing additives of the composition can be sterilized prior to their addition. Methods of sterilizing such ingredients, including sterile filtration or sanitization, particularly those that avoid the use of heat, are known in the art and can thus be practiced by one of ordinary skill in the art.

[0074] As will be further appreciated by those skilled in the art, the at least one additive causing the color of the composition may already be added to the growing biomass at the seeding stage, i.e. before the cultivation of step (c), which is preferably carried out in a large-scale fermentation or bioreactor. This is particularly preferred when the at least one additive supplemented in step (d) comprises a powder (described below).

[0075] It should be understood herein that the specific color of the composition produced in the method of the present invention is due to at least one additive supplemented in step (d). In other words, when the method of the present invention is carried out without the addition of additives, i.e. without step (d), the composition obtained accordingly is not characterized by said color. In other words, when the culture steps (c) and (e) are integrated into a single culture step and the supplementation of step (d) is not carried out, the composition is not characterized by said color.

[0076] In one embodiment of the present invention, the at least one additive that produces the color of the composition comprises a material that can form a dispersed phase when added to the growth medium. Dispersed phase is understood herein as a system in which particles (e.g., droplets) of one material are dispersed in a continuous phase of another material. Preferably, the at least one additive that produces the color of the composition comprises a powder or a liquid that can form a dispersed phase when added to the growth medium. More preferably, the at least one additive that produces the color of the composition comprises a powder. As understood herein, preferably, the powder is capable of forming a dispersed phase when supplemented to the growth medium in step (d) or when continuously supplemented at a given concentration in case of a continuous fermentation process. It should be understood herein that preferably, the continuous addition / supplementation of the powder allows the concentration to be maintained at a desired level during the cultivation. In addition, adding a powder (e.g., cocoa powder) during the deceleration phase results in a better and more uniform coloring and dispersion compared to adding the color at day 0. FIG. 3D shows mycelium biomass with cocoa powder added at day 0 (left, color: light brown - not uniform) versus added during the deceleration phase (right, color: dark brown - uniform). As understood herein, uniform coloration and distribution preferably refers to a situation where each of the multiple samples recovered from the biomass exhibits the same (or substantially the same) color characteristics as each other, for example, when the color of each sample is represented by the RGB scale, as demonstrated herein. In other words, uniform coloration relates to a situation where a person skilled in the art, when comparing the color characteristics of different samples recovered from the same biomass, comes to the conclusion that these samples have the same color characteristics. It can therefore be said that the color distribution is uniform. Therefore, adding cocoa powder during the deceleration phase results in a better color distribution.

[0077] Preferably, the powder is not completely soluble in the growth medium.

[0078] Thus, preferably, in the method of the invention for producing a composition comprising a fungal biomass, characterized in that the composition has a particular color, the at least one additive causing the color of the composition comprises a powder. As preferably understood herein, a powder is a solid in the form of particles, with an average particle size of less than 1000 micrometers, preferably less than 500 micrometers, more preferably less than 250 micrometers. The measurement of the particle size distribution can be performed by dynamic light scattering. This is based on the observation that the angle of a laser diffracted by a particle corresponds to the size of the particle. In a composite sample containing particles of different sizes, light diffraction results in a particular scattering pattern. By analyzing such a pattern, the exact particle size distribution of the sample can be estimated. In this specification, the diameter of the dispersed phase particles is preferably defined as d 50 Therefore, unless otherwise stated, d 50 Preferably, refers to the volume median particle size measured by DLS. Note that a person skilled in the art can determine the volume median particle size based on the results of DLS measurements.

[0079] Preferably, the powders discussed herein are selected from cacao, beet powder, duckweed powder, spirulina powder, paprika powder, turmeric powder, blueberry powder, strawberry powder, berry pigment powder, heme powder, lycopene powder, betanin powder, alfalfa powder, saffron powder, mint powder, and annatto extract. As understood herein, combinations of two or more of the powders listed herein may be used within the scope of the present invention.

[0080] As preferably understood herein, cocoa powder preferably relates to waste cocoa powder containing melanoidins.

[0081] As preferably understood herein, paprika powder, also called paprika extract, preferably contains capsanthin and / or capsorubin.

[0082] As preferably referred to herein, blueberry powder, also called blueberry extract, contains anthocyanins.

[0083] As preferably referred to herein, beetroot extract, also called beetroot powder, contains betanin.

[0084] As preferably referred to herein, tomato paste, which may also be referred to as lycopene powder, contains lycopene.

[0085] More preferably, the powder is cocoa powder, duckweed powder, spirulina powder, paprika powder, turmeric powder, heme powder or beet powder, or a combination thereof.

[0086] Even more preferably, the powder is cocoa powder, paprika powder, or beet powder.

[0087] Most preferably, the powder is cocoa powder.

[0088] The powders used herein are preferably edible, in other words, they can be consumed by humans without causing any adverse effects.

[0089] Powder as understood herein may also include lignocellulosic materials, particularly lignocellulosic materials derived from industrial and / or agricultural by-streams.

[0090] Lignocellulosic materials are preferably defined herein as materials comprising dry plant matter. Preferably, the lignocellulosic materials comprise cellulose, hemicellulose and lignin. Preferably, the at least one lignocellulosic material is at least one industrial and / or agricultural by-stream as defined herein. More preferably, the lignocellulosic materials are preferably solid or processed into a powder before use. As understood herein, the lignocellulosic materials are preferably characterized by a particular color. Thus, the lignocellulosic materials are preferably selected such that, upon its addition in step (d) of the method of the present invention, the resulting composition is characterized by a particular color.

[0091] Examples of lignocellulosic materials include distillers' grains, cereal bran, cotton, cottonseed hulls, bagasse, cocoa shells, cacao, cocoa pods, sunflower, peanut, hazelnut, palm oil, cotton and oilseed pomace from olives, husks and shells from nuts, grass and leaf waste, wood chips, coffee grounds, coffee husks, coffee silverskin, rapeseed and by-products from the soybean industry such as soybean pulp ("okara").

[0092] The industrial and / or agricultural sidestreams referred to herein are not particularly limited and may be any industrial and / or agricultural sidestreams known to those skilled in the art. Preferably, at least one industrial and / or agricultural sidestream refers to one industrial and / or agricultural sidestream. Preferably, the industrial and / or agricultural sidestream is a solid sidestream. As defined herein, the term solid sidestream relates to any sidestream that cannot be handled as a liquid, e.g., cannot be pumped, in contrast to liquid sidestreams that cannot be handled as a liquid, e.g., can flow without the application of mechanical force, e.g., molasses or stillage. Non-limiting examples of solid sidestreams are given below. More preferably, the solid by-stream is selected from brewer's grains, cereal bran, cotton, cottonseed husks, bagasse, cocoa shells, cocoa, cocoa pods, sunflower, peanuts, hazelnuts, palm oil, cotton and oil press cake from olives, hard shells and husks from nuts, grass and leaf waste, wood chips, coffee grounds, coffee husks, coffee silver skins, rapeseed and / or by-products from the soybean industry such as soybean pulp ("okara"). Even more preferably, the solid by-stream is brewer's grains. The inventors have surprisingly found that when the growth medium is supplemented with a powder, which is dispersed in the growth medium, the growing mycelium adheres to said particles of powder dispersed in the growth medium during step (e). In other words, the growing mycelium captures the particles of powder dispersed in the growth medium. As a result, the mycelium is present in the growth medium in the form of a composition comprising mycelium bound to the particles of powder dispersed in the growth medium. It should be understood that the particles bound by or entrapped by the mycelium give rise to the color of the composition obtained according to the method of the present invention.

[0093] Examples 2-3 show that when an additive that is a powder is added during cultivation, the color is more intense than when the same additive is added after growth of the fungal biomass is complete.

[0094] Therefore, preferably, the composition comprising fungal biomass comprises mycelium bound to the particles of powder dispersed in the growth medium. It should be understood that preferably, the growing mycelium captures the particles of powder dispersed in the growth medium. The growing mycelium can also be referred to as active mycelium. In contrast, the stationary mycelium, in other words the stationary phase mycelium, is less likely to actively capture the particles of powder.

[0095] The inventors further experimentally tested the washing off (i.e. removal by washing) of the particles from the mycelium to which they are bound. Preferably, in the composition obtainable according to the method of the present invention, the particles of powder bound to the mycelium cannot be separated from the mycelium without destroying the mycelium culture. Preferably, the separation of the particles of powder not bound to the mycelium is performed by washing. More preferably, the particles of powder bound to the mycelium cannot be separated from the mycelium without destroying the fungal cells. Thus, without being bound by theory, the inventors hypothesize that it is the binding of the particles of powder by the mycelium that is desirable in the composition obtainable according to the method of the present invention.

[0096] Thus, preferably, the binding of particles by mycelium refers to their being substantially permanently attached to one another.

[0097] The inventors have surprisingly found that the binding of powder particles by mycelium, in other words the capture of powder particles by mycelium, preferably occurs during the growth of the mycelium. Thus, in the embodiment of the present invention in which at least one additive that preferably produces the color of the composition comprises a powder, the additive is preferably supplemented to the growth medium during the induction phase in batch fermentation, or the additive is added continuously in the case of a continuous process such that their residence time in the fermenter (and preferably their concentration in the fermenter) allows the growth of mycelium bound to the powder particles. Even more preferably, when batch fermentation is used, the additive is supplemented to the growth medium substantially at the beginning of the cultivation of the fungal biomass for batch fermentation.

[0098] In other words, in the method for producing a composition comprising a fungal biomass, preferably characterized in that the composition has a particular color, step (d) is carried out before the start of step (c) (preferably in the case of batch fermentation).

[0099] According to the inventors, in embodiments of the invention where at least one additive that produces the color of the composition comprises a powder and the additive is replenished during the exponential growth phase (in the case of fed-batch or continuous operation) or during the deceleration or stationary phase of growth in the case of batch operation, the particles can be washed out of the mycelium to a substantially greater extent than if the additive is replenished during the lag phase or before step (c).

[0100] In an embodiment of the invention where the at least one additive that produces the color of the composition comprises a powder, the composition obtained by the method of the invention comprises a fungal biomass and said powder. Preferably, the particles of powder bound to the mycelium constitute up to 25% w / w of the composition comprising the fungal biomass, preferably up to 20% w / w of the composition comprising the fungal biomass, more preferably up to 15% w / w of the composition comprising the fungal biomass, even more preferably up to 10% w / w of the composition comprising the fungal biomass. Alternatively, the particles of powder bound to the mycelium may constitute up to 50% w / w. The amount of particles bound to the mycelium present in the composition relative to the mycelium, when expressed as w / w%, may also be referred to as the loading of powder particles to the mycelium. Preferably, the particles referred to here are substantially permanently attached to the mycelium. It should be noted that this amount depends on the stage at which the powder is added, resulting in different shades depending on the concentration present.

[0101] In a preferred embodiment, the particles of powder bound to mycelium represent 20% w / w to 25% w / w of the composition comprising fungal biomass.

[0102] It is further noted that the inclusion of powder particles in a composition comprising a fungal biomass of the present invention may be further beneficial to the composition, for example by improving the nutritional properties of the flavoring agent, improving baking properties, or improving properties relevant for use in cosmetics, pharmaceuticals and / or dietary supplements.

[0103] In other words, therefore, the inventors have demonstrated that the addition of at least one additive that produces the color of the composition (the additive being a powder) during the lag phase of the fungal culture increases the loading of powder particles onto the mycelium compared to addition at a different time point, e.g. during the logarithmic growth phase or the deceleration phase.

[0104] It should be understood that different degrees of staining of the fungal biomass, i.e. different intensities of the resulting color, can be obtained by controlling the concentration of the staining agent. A second factor known to those skilled in the art to affect color intensity is the contact time between the fungal mycelium and the staining agent. It is submitted by the inventors that the color intensity can be preferably controlled by controlling the concentration of the staining agent and its contact time with the mycelium. Without being bound by theory, the inventors put forward the hypothesis that the accumulation of the staining agent in the fungal mycelium can be considered an equilibrium process, as demonstrated in Example 7 and especially in Table 2, which shows that a similar proportion of the staining agent accumulated in the mycelium regardless of the concentration applied.

[0105] Thus, in one embodiment, during steps (d) and (e) of the method of the invention, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90% of the added colorant is accumulated in the biomass. In a preferred embodiment, the proportion of the provided colorant accumulated in the biomass is preferably 60-95%, more preferably 65-85%, even more preferably 70-80%. These considerations are particularly applicable when the colorant is astaxanthin. In other words, preferably, steps (d) and (e) are carried out such that at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90% of the added colorant is accumulated in the biomass, or that the accumulation in the biomass is preferably 60-95%, more preferably 65-85%, even more preferably 70-80% of the provided colorant is accumulated in the biomass.

[0106] In one embodiment of the present invention, the at least one color producing additive of the composition comprises a colorant that is assimilated by at least one fungal strain.

[0107] As preferably understood herein, a colorant is a compound characterized by a particular color. In other words, a colorant is a compound characterized by the absorption of electromagnetic radiation of a particular wavelength, preferably in the visible light spectrum. Thus, the compound appears to have a color when visually inspected and exposed to white light. Certain preferred colorants are described herein.

[0108] The coloring agent referred to herein is one that is assimilated by at least one fungal strain. Assimilation of the coloring agent preferably refers to its uptake into the fungal cell or fungal mycelium. The mechanism of assimilation is not particularly limited and may include passive diffusion into the cell through the cell membrane, active transport of the coloring agent into the cell by one or more cell surface transporters, mechanisms of endocytosis, etc. Preferably, the absorbed coloring agent is not easily removed from the mycelium, for example by washing the mycelium with water or an aqueous solution. It should be understood that the coloring agent, upon assimilation, changes the color of the fungal mycelium or fungal biomass, such that the fungal biomass or the composition comprising it is characterized by a particular color.

[0109] Thus, the colorant is not particularly limited, so long as it can be assimilated by the fungal mycelium and so long as the fungal biomass obtained upon assimilation or the resulting composition containing same is characterized by a particular color.

[0110] Preferably, the colorant is a terpene compound. The term "terpene compound" can be used interchangeably with the terms "isoprenoid", "isoprenoid compound", "terpene", "terpenoid" and "terpenoid compound" and is known to those skilled in the art. Terpene compounds contain or are composed of, and preferably contain, so-called (C5) isoprene units. The number of C atoms present in an isoprenoid is typically divisible by 5 (e.g., C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, ​​C47, C48, C49, C50, C51, C52, C53, C54, C55, C56, C57, C58, C59, C61, C62, C63, C64, C65, C66, C67, C68, C69, C70, C71, C72, C73, C74, C75, C75, C76, C77, C78, ​​C79, C81, C82, C83, C84, C85, C96, C97, C98, C99, C99, C100, C111, C122, C131, C141, C151, C152, C163, C174, C185, C19 10 , C 15 , C 20 , C 25 , C 30 and C 40 Irregular epoliterpene isoprenoids have been reported and are also included in the definition of "isoprenoid." Terpene compounds include, but are not limited to, monoterpenes, sesquiterpenes, triterpenes, polyterpenes, and diterpenes.

[0111] Terpene compounds include, but are not limited to, carotene compounds (A) and xanthophyll compounds (B), as shown in the following scheme (reproduced from Molecules 2020, 25, 1038; doi:10.3390 / molecules25051038).

[0112] [ka]

[0113] Preferably, the terpene compound is a tetraterpene compound, preferably a carotene compound or a derivative thereof, preferably a carotene compound. As understood herein, a derivative of a terpene compound that is a colorant is a compound in which preferably at least 80% of the atoms, preferably at least 90% of the atoms, remain the same and are bonded in the same manner as the parent colorant, and which itself meets the definition of a colorant. Non-limiting examples of derivatives include -OH, -C 1~4 Alkyl, -COOH, -COO(C 1~4 alkyl), O< and ═O, preferably OH, C 1~4 Alkyl, -COOH, and -COO(C 1~4 The terpene compound is substituted with one or more optional substituents selected from the group consisting of alkyl, aryl ...

[0114] An alkyl, as referred to herein, is a monovalent radical derived from a saturated hydrocarbon by removing one of its hydrogen atoms. An alkyl can be linear or branched. Preferably, the term alkyl refers to a C 1~4 Particularly preferred alkyl groups are methyl or ethyl groups, more preferably methyl groups.

[0115] The heterocycle referred to is preferably a monocyclic ring, either saturated or partially unsaturated, which contains one or more (such as 1, 2, 3 or 4) ring heteroatoms independently selected from O, S and N, preferably the ring contains one O heteroatom, the remaining ring atoms being carbon atoms. Preferably the heterocycle is a 3-6 membered heterocycle.

[0116] Preferably, the colorants are selected from carotene terpenes, xanthophyll terpenes, chlorophylls, phycobilins and anthocyanins. Particularly preferred are carotene terpenes and xanthophyll terpenes.

[0117] In one embodiment, the colorant is selected from flavonoid compounds.As understood herein, flavonoid compounds include a 15 carbon atom skeleton that includes two phenyl rings and a heterocyclic ring fused to one of the phenyl rings.Preferred flavonoid compounds include the following skeleton:

[0118] [ka]

[0119] In one embodiment, the colorant is selected from fungal pigments, such as carotenoids, melanins, flavins, phenazines, quinones, monascin, violacein, indigo, anthraquinones, naphthaquinones, dihydroxynaphthalene melanins, flavins, monascorubamin, lycopene, ankaflavin, chrysophanol, cynodontin, helminthosporin, tritisporin, erythroglaucin, riboflavin, rubropunctatin, or pigments of microbial origin, such as Monascus species, Xanthophyllomyces dendrorhous, Penicillium oxalicum, Ashbya gossypii, Blakeslea trispora, Erwinia uredovora, Rhodotorula mucilaginosa, and Fusarium sporotrichioides.

[0120] The terms dye and colorant are preferably used interchangeably herein.

[0121] More preferably, the colorant is astaxanthin or a derivative thereof, more preferably the colorant is astaxanthin.

[0122] The inventors have surprisingly found that in embodiments of the present invention where the at least one additive that produces the color of the composition comprises a coloring agent that is assimilated by at least one fungal strain, the color that characterizes the composition obtainable according to the method of the present invention depends on the timing of supplementing the growth medium with said at least one additive. Without being bound by theory, terpene compounds tend to degrade upon assimilation into the mycelium. If the addition of said terpene compounds is made during the cultivation stage, i.e. after the logarithmic growth phase or preferably during the deceleration phase, the metabolic activity of the growing mycelium is reduced. The presence of certain reactive functional groups in terpene compounds, such as oxygen-containing functional groups in xanthophylls (i.e. astaxanthin), may increase the tendency of said terpene compounds to be degraded upon assimilation. Furthermore, it is known to those skilled in the art that in the presence of oxygen or reactive oxygen species (e.g. O2, superoxide radical, H2O2 or hydroxyl radical), terpene compounds, such as carotene compounds, may be oxidized in a process called bleaching. Thus, without being bound by theory, the inventors note that the oxygen availability in the fungal culture during the deceleration phase may be lower than during the phase preceding the deceleration phase, depending on the particular fermentation procedure. It is therefore believed that the assimilation of the colorant by the at least one fungal strain depends on the oxygen availability to the at least one fungal strain. In the present specification, assimilation is understood to include the process of assimilation and the process of decomposition of said colorant during assimilation. In the case of a continuous process, the exposure to oxygen or determining the effect of oxygen is regulated by a set residence time in the fermenter. Thus, preferably, according to the present invention, the oxygenation or aeration (or, in other words, air / oxygen flow) is controlled, especially in those embodiments in which the oxygen level may be considered to affect the decomposition of the colorant / pigment. Nevertheless, in all processes encompassed by the present invention, it is preferred that the aeration is controlled (and set to a sufficient value, which can be easily determined by the skilled person). For example, in one preferred embodiment, the oxygenation rate is set to control the desired dissolved oxygen (DO) level in the medium, with reference to the percentage of oxygen saturation. Preferably, this range is maintained at 10-40% v / v.

[0123] Without wishing to be bound by theory, the terpene compounds may also be degraded by enzymes produced by the mycelium. The most important enzymes catalyzing these redox reactions in this context are azoreductase, laccase and peroxidase. These enzymes are capable of degrading the terpene compounds.

[0124] Therefore, as understood herein, the assimilation of coloring agent by at least one fungal strain depends on the oxygen availability to at least one fungal strain. It should be understood that the control of oxygen supply, which is known in principle to those skilled in the art, may be important for the method of the present invention. It has been demonstrated that a large scale using fermenters with well-controlled oxygen supply and / or microenvironment works better than the flasks used to evaluate the process on a small scale, because oxygen availability is more controlled.

[0125] The present invention therefore provides a method for producing a composition comprising a fungal biomass of the present invention, wherein at least one additive that produces the color of the composition comprises a coloring agent that is assimilated by at least one fungal strain, and the coloring agent is added in such a way that its degradation by the mycelium is substantially minimized. As understood herein, by minimizing the degradation of the coloring agent by the mycelium, a more intense color of the composition obtainable according to the method of the present invention is obtained. Thus, the inventors have surprisingly found that supplementation of the growth medium with the coloring agent must occur at a certain stage of mycelial growth in order to allow efficient assimilation and avoid degradation. The inventors have further surprisingly found that when supplementing the growth medium with the coloring agent, the cultivation of the fungal biomass should be carried out for a sufficient time to allow assimilation, but at the same time, the cultivation period of the fungal biomass at the time of addition of the coloring agent should be minimized in order to avoid degradation of the coloring agent by the mycelium.

[0126] Thus, preferably, in the embodiment of the present invention where the at least one additive causing the color of the composition comprises a coloring agent assimilated by at least one fungal strain, step (d) is carried out once the culture in step (c) has reached the deceleration phase (in the case of batch fermentation). Step (e) is therefore carried out thereafter for not more than 72 hours, preferably not more than 48 hours. Furthermore, step (e) is preferably carried out for at least 15 minutes, preferably for at least 30 minutes, even more preferably for at least 1 hour.

[0127] Thus, the present invention provides a preferred embodiment in which the at least one additive that produces the color of the composition comprises a colorant that is assimilated by the at least one fungal strain, and steps (c) to (e) are as follows: (c) culturing at least one fungal strain in a growth medium until a slowdown phase is reached; (d) supplementing the growth medium with at least one additive that produces a color in the composition, preferably the at least one additive that produces a color in the composition comprises a colorant that is assimilated by the at least one fungal strain; (e) further culturing the at least one fungal strain in a growth medium supplemented with at least one additive that produces the color of the composition, preferably for a period of not more than 72 hours, more preferably not more than 48 hours.

[0128] It is envisioned that the methods of the present invention can be generalized by one of skill in the art to a method for producing a composition comprising a fungal biomass and terpene compounds assimilated by the biomass. The terpene compounds can include, but are not limited to, cannabis terpenes, for example.

[0129] In one embodiment of the invention, the at least one additive that produces the color of the composition comprises a colored microorganism, which is understood herein as an organism whose cells are characterized by a particular color, are capable of developing a particular color under particular conditions, or are capable of secreting a colorant (as defined above) into the growth medium.

[0130] It should be understood that pigmented microorganisms, whose cells are characterized by a particular color or that are capable of developing a particular color under certain conditions (e.g., treatment with agents that should not be construed as limiting examples), will be included in the composition comprising the fungal biomass obtainable according to the method of the present invention, and thus their presence in the composition will confer said particular color to said composition.

[0131] Alternatively, in the case of microorganisms capable of secreting a colouring agent which can be assimilated by the fungal mycelium, said colouring agent will be expected to be assimilated by the mycelium during step (e) of the method of the invention.

[0132] Preferably, pigmented microorganisms are understood herein as organisms whose cells are characterized by a particular color.

[0133] Pigmented microorganisms are known to those skilled in the art. It is understood that the microorganism may produce pigments either intracellularly or extracellularly. The pigmented microorganisms added may be fungi, bacteria, archaea, algae, or any microorganism capable of producing pigments. Non-limiting preferred examples of pigmented microorganisms are red algae, green algae, brown algae, and cyanobacteria.

[0134] Preferred red algae are selected from Cyanidioschyzon merolae, Atractophora hypnoides, Gelidiella calcicola, Lemanea, Palmaria palmata, Schmitzia hiscockiana, Chondrus crispus, Mastocarpus stellatus, Acrochaetium efflorescens, Audouinella, Polysiphonia ceramiaeformis, and Vertebrata simulans.

[0135] Preferred green algae are selected from Caulerpa, Codium, Acetabularia, Cladophora, Trentepohlia and Monostroma.

[0136] Preferred cyanobacteria are selected from the families Chroobacteria, Gloeobacteria, and Hormogoneae, Prochloraceae and Prochlorotrichaceae, and the genera Folisarcina, Halospirulina, Phyllonema, Prochlorococcus, Prochloron, Prochlorothrix, Rubidibacter, and Schmidlea.

[0137] Preferred brown algae are selected from the Phaeophyceae family.

[0138] Therefore, preferably, the pigmented microorganism is a microorganism selected from red algae, green algae, brown algae and cyanobacteria. For example, the pigmented microorganism is selected from algae strains such as Haematoccus pluvialis, Chlorella vulgaris and cyanobacteria, preferably from Haematoccus pluvialis and Chlorella vulgaris. It should be noted that Haematoccus pluvialis is known to those skilled in the art to produce astaxanthin.

[0139] The pigmented microorganism according to the present invention may be at least one fungus. Preferably, the pigmented fungus comprises a Rhodotorula fungus, such as Rhodotorula mucilaginosa. The use of a Rhodotorula fungus for such purposes is shown in Example 9 and Figure 20. Alternatively, the pigmented fungus is selected from Monascus species, Xanthophyllomyces dendrorhous, Penicillium oxalicum, Ashbya gossypii, Blakeslea trispora, Erwinia uredovora, Rhodotorula mucilaginosa, and Fusarium sporotrichioides.

[0140] Preferably, the Monascus species added to the fungal biomass during the deceleration phase, to allow the fungal biomass to become colored, is selected from Monascus purpureus (also known as M. anka and M. pilosus), Monascus pilosus, Monascus purpureus, and Monascus ruber. Preferably, the Monascus species is added in the form of a powder, which is commercially available. Alternatively, a Monascus species culture is grown in a separate tank and the culture is then added as described above.

[0141] The use of genetically modified pigmented microorganisms is also contemplated for the present invention. Thus, in one embodiment, the pigmented microorganism is a genetically modified microorganism, such as a genetically modified Saccharomyces cerevisiae.

[0142] The use of CO2 fixing microorganisms as pigmented microorganisms is also encompassed by the present invention.

[0143] The use of colored CO2 fixing microorganisms to give the mycelium its color allows for the improvement of the carbon dioxide output of the process by capturing the carbon dioxide formed during mycelium growth, as well as the adjustment of the nutritional profile of the final mycelium product by being able to include nutritional compounds such as omega-3 or vitamin B12 that are not otherwise produced by these fungi. In other words, the CO2 produced in this case from fungal fermentation is used for the growth of the colored CO2 fixing microorganism (e.g., algae), as discussed in Examples 6 and 10. For example, FIG. 21 shows the trend of the percentage of CO2 fixed (by volume) by the green microalgae Haematococcus pluvialis, calculated by subtracting the percentage of CO2 in the effluent of the microalgae culture from the percentage of CO2 feed provided by the effluent of the Pleurotus pulmonarius mycelium fermentation. The trend of fixed CO2 is shown in this figure to increase over the time frame shown during the microalgae culture. This configuration may be advantageous, for example, to use CO2 produced during fungal fermentation, either in its normal state or after compression, to grow CO2-fixing microorganisms more efficiently in another fermenter, and then add it to the fermenter with at least one fungal strain. Similarly, captured CO2 from other sources can be added to the second fermenter to further promote the growth of the CO2-fixing microorganisms, resulting in a carbon-neutral or carbon-negative process. It is also envisioned to use commercially available CO2 as input for the growth of at least one pigment-producing microorganism. If necessary, the fermenter can also be equipped with various light systems that can operate at various wavelengths (e.g., ultraviolet light, blue-violet light, green light, blue light, red light, etc.) to stimulate the growth of certain microorganisms (e.g., algae) or the production of certain compounds (e.g., vitamin D).

[0144] Preferably, the colored microorganisms referred to herein can be co-cultured with at least one fungal strain of the present invention. This co-culture can be started by having both the at least one fungal strain and the colored microorganism present from the beginning of the co-culture, or by adding the at least one fungal strain once the colored microorganism has grown to a certain extent (i.e., for example, preferably during the deceleration phase of the growth of the colored microorganism). Alternatively, the colored microorganisms can be cultured separately (e.g., in a separate reactor) from the at least one fungal strain described herein and added to the at least one fungal strain provided in the present invention (i.e., during the deceleration phase of the fungal strain).

[0145] As understood herein, optionally in all scenarios, more substrate can be added to the culture if further co-fermentation is required. As will be understood by those skilled in the art, yeast is added during the slowdown phase of the fungus, followed by the addition of new substrate, so that the yeast grows faster and consumes the substrate added before the fungus, while the fungus is still in the slowdown phase. As will be further understood by those skilled in the art, this is the case when pigmented microorganisms are used, including any microorganism that grows significantly faster than the fungus.

[0146] By co-cultivating a colored microorganism, preferably a CO2-fixing microorganism, with at least one fungal strain provided by the present invention, the CO2-fixing organism (which may also be referred to as a microorganism or microbe) can use the CO2 produced by the at least one fungal strain. This may be the case when the CO2-fixing organism is co-cultivated with the at least one fungal strain, or when the CO2-fixing microorganism and the at least one fungal strain are cultivated in separate reactors, but the CO2 produced by the at least one fungal strain is provided to the CO2-fixing organism, for example, by a fluid connection between the two reactors. Thus, the present invention further relates to an embodiment in which the CO2 produced by the at least one fungal strain is fixed by a colored microorganism, which is preferably a CO2-fixing microorganism herein. Preferably, CO2 should be added when sufficient CO2 is produced in the fermenter to allow the growth of the CO2-fixing microorganism.

[0147] It should be understood that the CO2 fixing organism is an organism that can convert CO2 fixation into organic compounds in the presence of light, for example selected from cyanobacteria. Preferably, the CO2 fixing organism is selected from the list of pigmented microorganisms listed herein. In particular, the CO2 fixing organism is selected from cyanobacteria, algae (all types), bacteria, and unicellular organisms (diatoms).

[0148] As understood herein, adding a pigmented microorganism after separate growth can involve either adding a color extracted from the pigmented microorganism, excluding the microorganism itself, or adding a whole culture containing the microorganism. Adding biomass recovered from a culture of a pigmented microorganism can also be envisioned as being encompassed by the present invention. The microorganism can be added inactivated to avoid further co-fermentation, or active, i.e. viable.

[0149] Preferably, the colored microorganism is added to the culture of fungal biomass not before the logarithmic phase of growth. This is especially the case for embodiments where a continuous process is not used. More preferably, it will be understood by those skilled in the art that depending on the timing and supplementation amount (in the case of batch fermentation) of step (d) and the culture conditions, different ratios of colored microorganisms and fungal biomass may be obtained in the final composition of the present invention. It is further noted that the growth of certain colored microorganisms, such as certain algae, requires certain CO2 concentrations. Those skilled in the art will be able to adjust the culture conditions so that the desired amount of fungal mycelium and colored microorganisms are present in the composition. Since certain colored microorganisms can grow faster than fungi, those skilled in the art will know to take care when supplementing a fungal culture with a colored microorganism (or vice versa) to avoid possible overgrowth of the fungal culture by the colored microorganism. It should be noted that when fungal mycelium is co-cultured with pigmented microorganisms, continuous culture is not preferred since it is difficult to maintain a constant composition (i.e., w / w ratio of fungus (i.e., target fungal strain, or at least one fungal strain referred to in step (b) of the method of the present invention) to pigmented microorganism (e.g., pigmented fungal strain / microorganism).

[0150] In embodiments where continuous culture is used, the non-stabilized pigmented microorganisms (which are no longer active) are induced to grow as fast as the target fungal strain (i.e., limiting their growth). Alternatively, for slower growing pigmented microorganisms, the target fungus is induced to grow at the same rate as the pigmented microorganisms (i.e., growth limitation of the target fungus is induced, so that the target fungus grows slower than it is practically capable of).

[0151] In a further embodiment, when continuous culture is used, the supplementation in step (d) is carried out in the logarithmic phase. The pigmented microorganism is continuously added to the fermenter at an optimal rate and concentration that results in the target pigmentation of the biomass after the optimal residence time and pigment concentration have been established in the fermenter. The optimal controllable addition rate that ensures that the pigment is not degraded defines the residence time of the pigment in the fermenter, which is the volume of the fermenter divided by the volumetric flow rate, i.e. the addition rate of the pigmented microorganism.

[0152] A continuous process carried out in steady state is preferably understood to be a continuation of a batch process before reaching the log phase (i.e. a transient state similar to the start of the batch mode). Thus, in the case of a continuous process operating constantly in the log phase at steady state conditions, step (c) is still carried out under batch operation, step (d) is a step of adding coloring agent in the log phase, and step (e) reflects the continuous biomass growth with the presence of coloring agent added continuously at a certain volumetric flow rate, thus determining the residence time, which is a relevant factor to be considered in order to avoid color deterioration and reach the target color. Step (f) will be understood herein as preferably continuously recovering the fungal biomass produced in step (e). Step (d) may also (further) include continuous replenishment of at least one additive in parallel with the cultivation in step (e) so that the concentration of at least one additive is kept constant during step (e).

[0153] If step (d) is performed before the start of step (c), the colorant is added directly in the fugitive phase before reaching the continuous phase (ie, similar to a batch mode operation).

[0154] Also encompassed by the invention are embodiments in which a growth medium containing growing fungal mycelium is added to a culture of a pigmented microorganism.Further encompassed by the invention are embodiments in which the fungal mycelium and the pigmented microorganism are cultivated separately, i.e., in separate reactors / fermentors, and mixed together to obtain the composition of the invention.

[0155] In a further embodiment, where the pigmented microorganisms are added in a sequence according to the above mentioned growth scenarios, the pigmented microorganisms constitute up to 25% w / w of the composition comprising the fungal biomass, preferably up to 20% w / w of the composition comprising the fungal biomass, more preferably up to 15% w / w of the composition comprising the fungal biomass, even more preferably up to 10% w / w of the composition comprising the fungal biomass. It should be understood that the pigmented microorganisms, when used in the method of the present invention, should preferably not overgrow the at least one fungal strain.

[0156] The possibility of combining different additives that produce the color of the composition is envisioned and embraced by the inventors.For example, the growth medium in the method of the present invention can be supplemented with a powder and a soluble colorant.This can occur together (e.g., with a composition that includes both the powder and the soluble colorant, such as a terpene compound), separately (with an additional culture step between the two supplementation steps), or sequentially.Optionally, such growth medium can be further supplemented with at least one colored microorganism, which can be co-cultured with fungal mycelium.

[0157] Further embodiments are encompassed by the present invention, in which the at least one additive that produces the color of the composition supplemented in step (d) of the method of the present invention may comprise an additive selected from vitamin B12, vitamin B6, vitamin B2, vitamin B3 (also called niacin), riboflavin, thiamine, vitamin A, vitamin E, omega-3 fatty acids, vitamin D2, folic acid, iodized salts (NaCl, further comprising iodized salts in an amount up to 5% w / w), minerals (calcium, iron, potassium, etc.), flavorings (salt, pepper, oil, etc.).

[0158] Step (e) of the method for producing a composition comprising a fungal biomass according to the invention, characterized in that the composition has a particular color, comprises further culturing at least one fungal strain in a growth medium supplemented with at least one additive that produces the color of the composition. As understood herein, the growth of the biomass is continued after the supplementation of step (d). Preferably, the culturing in step (e) is continued for at least 24 hours, at least 48 hours or at least 72 hours. However, in the case of co-cultivation with algae, longer periods can be envisaged, for example at least 96 hours or at least 120 hours. Preferably, the culturing in step (e) is continued until the growth of the biomass reaches a stationary phase (in the case of batch fermentation). Preferably, step (e) is carried out according to the same settings as step (c).

[0159] In step (f) of the method for producing a composition comprising a fungal biomass according to the invention, wherein said composition is characterized by having a particular color, the composition comprising a fungal biomass, wherein said composition is characterized by having a particular color, is recovered from the growth medium, i.e. the biomass or the composition comprising the biomass is separated from the liquid components of the growth medium.

[0160] The recovery described herein can be carried out according to any method known to those skilled in the art, and is not meant to be particularly limited herein.

[0161] After fermentation, the biomass-containing composition is separated by filtration, centrifugation, or other state-of-the-art techniques, washed with water, and concentrated to a concentration suitable for food production.

[0162] In one embodiment, the method of the invention further comprises recovering the supernatant in step (f).

[0163] The supernatant produced during fermentation in the method according to the invention is expected to maintain its characteristic color and can therefore also be used to develop specific health drinks containing antioxidants and the specific aromas, tastes and flavors generated during fermentation with edible mushrooms. Mushroom strains are known to produce pleasant volatiles and other compounds with, for example, apple or almond tastes. They also produce compounds known to regulate blood glucose levels.

[0164] It is also envisaged that the supernatant of the present invention may be further processed, for example to extract certain components thereof, such as proteins, particularly enzymes, polysaccharides, peptides, antioxidants, etc., produced by the microorganisms cultured in the medium.

[0165] The present invention further relates to a composition obtainable according to the method for producing a composition comprising a fungal biomass according to the invention, said composition being characterized in that it has a particular color, in particular a color selected from the color characteristic of the food products mentioned above. Depending on the additives used in the method according to the invention, said composition may further comprise particles of a powder, said particles being bound to the mycelium.

[0166] Preferably, the composition of the present invention is characterized in that the composition comprises at least 1 mg of coloring agent per 100 g of fungal biomass, preferably at least 5 mg of coloring agent per 100 g of fungal biomass, more preferably at least 10 mg of coloring agent per 100 g of fungal biomass, even more preferably at least 15 mg of coloring agent per 100 g of fungal biomass, even more preferably at least 30 mg of coloring agent per 100 g of fungal biomass. As shown in Example 8, in the exemplary case where the coloring agent is astaxanthin, it is demonstrated that the composition comprises such an amount of coloring agent. However, it should be understood that in the case of other coloring agents, the composition can also comprise more than the values ​​given above per 100 g of fungal biomass. In embodiments of the present invention in which the coloring agent is added during the slowdown phase of growth, it can be envisaged to use a lower amount / concentration of said coloring agent.

[0167] In a further embodiment the composition of the invention is characterised in that the composition comprises preferably 1-50 mg of colouring agent per 100 g fungal biomass, preferably 30-40 mg, preferably 20-30 mg of colouring agent per 100 g fungal biomass, more preferably 10-20 mg of colouring agent per 100 g fungal biomass, more preferably 1-10 mg of colouring agent per 100 g fungal biomass. Preferably, in this specification the stated numbers are understood to refer to wet biomass, preferably containing 3-30% water.

[0168] In one embodiment, the composition of the present invention comprises at least 0.18% by weight of a colorant (preferably astaxanthin or lycopene), preferably the composition of the present invention comprises at least 0.35% by weight of said colorant. It is further preferred that the composition of the present invention comprises no more than 5% by weight of said colorant, more preferably no more than 1% by weight of said colorant. It should be understood that the weight percentages mentioned herein are preferably based on the dry weight of the composition. In an exemplary embodiment, the water content of the composition of the present invention is 5-20%, preferably about 10%, more preferably 10%.

[0169] It is understood that in this specification weight percent always refers to a dry weight basis unless indicated to the contrary, e.g. unless indicated to refer to a wet weight basis.

[0170] In a further embodiment, the composition of the present invention preferably comprises 0.01-5 wt.-%, preferably 0.1-5 wt.-%, more preferably 0.1-2.5 wt.-%, even more preferably 0.1-1 wt.-%, most preferably 0.1-0.5 wt.-% of a colorant. Preferably, the colorant is a xanthophyll, a carotene or a chlorophyll, preferably a xanthophyll or a carotene. Exemplary xanthophylls and carotenes are astaxanthin and lycopene, respectively.

[0171] In the composition of the present invention, preferably, at least one additive that produces the color of the composition is stably bound to the fungal mycelium. In other words, preferably, at least one additive that produces the color of the composition is irreversibly bound to the fungal mycelium. Example 6 describes a leaching test of the composition of the present invention, demonstrating that no color transfer occurs on the glove upon contact with the composition. Furthermore, it is quantitatively shown that no or very little leaching occurs as detected by UV-vis spectroscopy. Thus, preferably, "irreversibly bound" can be understood as not suitable or substantially not suitable to be washed off or removed otherwise from the fungal mycelium without changing the structure of the fungal mycelium. In the present specification, preferably, the term "substantially not suitable to be washed off or removed otherwise" means that at least 80% of the colorant, preferably at least 90% of the colorant, more preferably at least 95% of the colorant is not suitable to be washed off or removed otherwise from the fungal mycelium without changing the structure of the mycelium. In other words, altering the structure of the mycelium may mean involving the integrity of the mycelium resulting in the release of colorants that are otherwise stably bound thereto.

[0172] In one embodiment of the present invention, the at least one additive causing the color of the composition comprises at least one extract of the lignocellulosic materials listed above. Preferably, the lignocellulosic materials are selected from brewer's grains, cereal bran, wheat bran, cocoa, and coffee, more preferably from brewer's grains, cereal bran, wheat bran, and cocoa, even more preferably from brewer's grains, cereal bran, and wheat bran, even more preferably from brewer's grains and wheat bran. Preferably, the colored liquid extract is added during the fermentation process at the beginning (X1) of the fermentation or in the deceleration phase (X2) (Figure 1), preferably in the deceleration phase (X2) taking into account the sugar content in the extract so that the fermentation remains in the deceleration phase, even more preferably in step (d) provided in the method of the present invention listed herein to obtain a biomass with a uniform color and improved taste. Preferably, the extract has a dark barley color, or a dark brown / black color, or a dark brown / red color, and preferably results in mycelium with a suitable light brown color to be used in the development of meat substitutes (e.g. cooked minced meat). FIG. 22 shows both the mycelium with a light brown color and the lignocellulosic extract from wheat bran with a dark black / brown solution.

[0173] In a further embodiment, the coloured supernatant obtained from the first fermentation, starting at X1, containing the sugar-rich extract, is preferably reused as colouring agent for at least one further fermentation, said supernatant colouring agent being preferably added at the start of the next fermentation (X1) or at the slowdown phase (X2) of the next fermentation, preferably at the slowdown phase (X2) of the next fermentation, even more preferably at step (d) of the next fermentation as provided in the methods of the invention recited herein. Preferably, said coloured supernatant contains up to 20% by weight of sugars and has a similar colour to the extract described herein.

[0174] In a further alternative embodiment, the coloured lignocellulosic extract and / or coloured supernatant is preferably added to the mycelial biomass after harvesting in order to colour the mycelial biomass and improve its taste.

[0175] Preferably, the extract is prepared according to the published methods listed in WO2022136708, which discloses liquid extraction and steam extraction techniques.

[0176] Particularly preferred is an extraction process combining prehydrolysis with steam and an extraction / washing step carried out with liquid water. Preferably, the lignocellulosic material, preferably industrial and / or agricultural by-streams, is contacted with steam at a temperature above 100°C, preferably at a temperature between 150°C and 300°C, more preferably at a temperature between 160°C and 180°C, even more preferably at a temperature of about 170°C, for up to 20 minutes. The solids so treated are then washed with liquid water, preferably at a temperature between 50°C and 100°C, more preferably at a temperature between 50°C and 70°C, even more preferably at a temperature between 50°C and 60°C. Preferably, the washing step as defined herein is carried out for a time period between 5 and 60 minutes. Preferably, the liquid extraction comprises the extraction of the lignocellulosic material, preferably industrial and / or agricultural by-streams, with water at a temperature of 120-220° C., more preferably 130-200° C., and at a pressure of 1.25 bar to 220 bar, preferably 2-220 bar, more preferably 6-35 bar, even more preferably 20-35 bar, for a period of preferably 5-200 minutes, preferably 10-200 minutes, more preferably 10-100 minutes.

[0177] The compositions of the present invention may contain further concentrates (e.g., additives), such as omega-3 fatty acids derived from microalgae. In one embodiment, the compositions contain at least 1, 2.5, 5, 10, 15, 20% by weight of the omega-3 fatty acids.

[0178] The present invention further relates to a food product comprising the composition of the present invention.

[0179] Preferably, the food product of the present invention comprises at least 10% w / w of the composition of the present invention, preferably at least 20% w / w of the composition of the present invention, more preferably at least 30% w / w of the composition of the present invention, even more preferably at least 35% w / w of the composition of the present invention. Preferably, the food product of the present invention comprises from 35% w / w to 100% w / w of the composition of the present invention.

[0180] In one embodiment, the mycelium-based or mycelium-containing food product of the present invention comprises 0.0035 to 5 wt. %, preferably 0.01 to 5 wt. %, more preferably 0.01 to 2.5 wt. %, even more preferably 0.035 to 0.5 wt. %, even more preferably 0.05 to 0.35 wt. % of the colorant composition of the present invention.

[0181] For example, low concentrations of astaxanthin, preferably 0.9 g / L or less, provide color and make the resulting colored biomass suitable for use in developing fish substitutes, i.e. red fish, such as salmon, caviar, or tuna, while high concentrations, preferably greater than 0.9 g / L and less than 1.8 g / L, provide a red color and make the resulting colored biomass suitable for use in developing meat substitutes, i.e. beef-like red meat. It is understood that by varying the concentration of the coloring agent used in the method of the present invention, the color of the resulting composition can be influenced and therefore controlled to obtain the desired color.

[0182] As referred to herein, a mycelium-based food product preferably comprises at least 90% by weight of a mycelium composition (i.e. a composition obtainable according to the method of the present invention).

[0183] As referred to herein, a mycelium-containing food product preferably comprises the mycelium composition (ie the composition obtainable according to the method of the present invention) in an amount of less than 90% by weight.

[0184] The food products understood herein may be dairy products, such as yogurt, milk drinks and ice cream. The food products understood herein may also relate to various embodiments of seafood products, such as crab cakes, fish cakes, tuna, salmon or shrimp, as well as various desserts, including chocolates, brownies, puddings or cookies.

[0185] The present invention also relates to the use of the colored edible fibrous mycelium mass for producing an edible meat substitute product, preferably selected from meatballs, sausages, tartare, minced meat, meat spreads, processed meat, Mett meat, foie gras, steaks, beef jerky, burger patties, fillets, nuggets, salami, whole meat, bacon, hot dogs, prosciutto, dried meat and products replacing extruded products.

[0186] The present invention also relates to the use of colored edible fibrous mycelium mass for producing an edible dairy substitute product, preferably selected from products replacing milk, yogurt, fresh cheese, whey cheese, cream cheese, medium hard cheese, hard cheese, and soft molded cheese.

[0187] The present invention also relates to the use of the colored edible fibrous mycelium mass to produce edible substitute fish or seafood products, such as crab cakes, fish cakes, tuna, salmon, or shrimp.

[0188] In some embodiments, the meat substitute or meat-like food product is understood to have a similar consistency or resemblance or taste to animal meat in all its forms (breasts, fillets, thighs, ribs, wings, chunks, steaks, etc.), preferably selected from meat beef, poultry, fish, chicken, duck, goose, turkey, beef, pheasant, lamb and mutton, white meat, pork, ham, veal, deer or venison, seafood, shrimp, crab, salmon, cod, pangasius, sardines, mussels and oysters.

[0189] In a preferred embodiment, the soft meat substitute is preferably a meatball. In another preferred embodiment, the soft meat substitute is preferably selected from meatballs, sausages, fish fingers, tartare, minced meat, meat spreads, processed meat, Mett meat, luncheon meat, and foie gras.

[0190] In another preferred embodiment, the non-tender meat substitute is preferably selected from steaks, beef jerky, burger patties, fillets, nuggets, salami, whole meat, bacon, hot dogs, prosciutto, dehydrated meats, and extruded products.

[0191] In another embodiment, the concepts of non-soft meat and soft meat may be interchangeable only if the ingredients used to produce the traditional non-soft meat result in a meat substitute that is softer in terms of consistency compared to the traditional definition.

[0192] In a preferred embodiment, the soft dairy substitute is preferably selected from cream cheese.In another preferred embodiment, the soft dairy substitute is preferably understood as cream cheese, cheese spread, processed cheese, whey cheese, pizza cheese, shredded mozzarella cheese, mozzarella cheese, soft cheese, semi-soft cheese, feta cheese, ricotta cheese, cottage cheese, camembert cheese, roquefort cheese, gorgonzola cheese, brie cheese, blue cheese, bouchet cheese, goat cheese, quark, cream, coffee creamer, whipped cream, sour cream, milk chocolate spread, margarine, butter, dessert, custard.In another embodiment, the non-soft or hard dairy substitute is preferably understood as hard cheese, semi-hard cheese, cheddar cheese, parmesan cheese, etc.

[0193] The food may be a textured or textured food. Thus, the food of the present invention comprises all amino acids required for the daily intake of humans that cannot be synthesized de novo. Furthermore, the textured food of the present invention is preferably heat-resistant, boil-resistant and suitable for cooking. For example, the fungus-based food of the present invention may be a meat substitute product, as described herein. It is noted that preferably, the meat substitute product is a textured or textured food. It is further noted that the structure of the textured food improves the acceptance of the textured food by consumers. It is further noted that the unique fibrous texture of the fungal biomass of the present invention may be beneficial for producing textured or textured foods without using traditional texturization methods such as extrusion.

[0194] The food products of the present invention can be further supplemented with, for example, preservatives, antioxidants and acidity regulators, thickeners, stabilizers and emulsifiers, pH regulators and anticaking agents, flavor enhancers, improvers, stabilizers, thickeners, colorants, and / or glazing agents and sweeteners.Accordingly, the present invention further relates to food products further comprising additives selected from preservatives, antioxidants and acidity regulators, thickeners, stabilizers and emulsifiers, pH regulators and anticaking agents, flavor enhancers, improvers, stabilizers, thickeners, colorants, and / or glazing agents and sweeteners.

[0195] Preferably, the preservatives include calcium carbonate, acetic acid, potassium acetate, sodium acetate, calcium acetate, lactic acid, sorbic acid, and malic acid.

[0196] Preferably, the antioxidants and acidity regulators include ascorbic acid, sodium ascorbate, calcium ascorbate, fatty acid esters of ascorbic acid, high tocopherol extracts, α-tocopherol, γ-tocopherol, δ-tocopherol, lecithin, sodium lactate, potassium lactate, calcium lactate, citric acid, sodium citrate, potassium citrate, calcium citrate, tartaric acid (L(+)), sodium tartrate, potassium tartrate, sodium potassium tartrate, sodium malate, potassium malate, calcium malate, calcium tartrate, and triammonium citrate.

[0197] Preferably, the thickener, stabilizer and emulsifier (or hydrocolloid) is selected from the group consisting of alginic acid, sodium alginate, potassium alginate, ammonium alginate, calcium alginate, agar, carrageenan, processed euterpe rhoeas seaweed, locust bean gum, guar gum, tragacanth, gum arabic (acacia gum), xanthan gum, tara gum, gellan gum, sorbitol, mannitol, glycerol, konjac, pectin, cellulose, methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, ethylmethylcellulose, sodium carboxymethylcellulose, and sodium carboxymethylcellulose. Examples of suitable cellulose gums include cellulose gum, cellulose gum, enzymatically hydrolyzed carboxymethylcellulose, sodium-potassium and calcium salts of fatty acids, magnesium salts of fatty acids, mono- and diglycerides of fatty acids, acetate esters of mono- and diglycerides of fatty acids, lactate esters of mono- and diglycerides of fatty acids, citrate esters of mono- and diglycerides of fatty acids, tartrate esters of mono- and diglycerides of fatty acids, microcrystalline cellulose-cellulose gel, mono- and diacetyl tartaric acid esters of mono- and diglycerides of fatty acids, mixed acetate and tartrate esters of mono- and diglycerides of fatty acids, sorbitol and mannitol.

[0198] Preferably, the pH adjuster and anti-caking agent include sodium carbonate, potassium carbonate, ammonium carbonate, magnesium carbonate, hydrochloric acid, potassium chloride, calcium chloride, magnesium chloride, sulfuric acid, sodium sulfate, potassium sulfate, calcium sulfate, sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide, magnesium hydroxide, calcium hydroxide, magnesium hydroxide, fatty acids, gluconic acid, glucono-delta-lactone, sodium gluconate, potassium gluconate, and calcium gluconate.

[0199] Preferably, the flavor enhancers include glutamic acid, monosodium glutamate, monopotassium glutamate, calcium diglutamate, monoammonium glutamate, magnesium diglutamate, guanylic acid, disodium guanylate, dipotassium guanylate, calcium guanylate, inosinic acid, disodium inosinate, dipotassium inosinate, calcium inosinate, calcium 5'-ribonucleotides, disodium 5'-ribonucleotides, and glycine and its sodium salts.

[0200] Preferably, the improver includes L-cysteine.

[0201] Preferably, the stabilizers include invertase and polydextrose.

[0202] Preferably, the thickening agent includes polydextrose, oxidized starch, monostarch phosphate, distarch phosphate, phosphorylated distarch phosphate, acetylated distarch phosphate, acetylated starch. The thickening agent also includes acetylated distarch adipate, hydroxypropyl starch, hydroxypropyl distarch phosphate, sodium starch octenyl succinate, starch-based components, and acetylated oxidized starch. More preferably, the thickening agent includes psyllium husk and / or starch-based components.

[0203] Preferably, the coloring agents include riboflavin, chlorophyll and chlorophyllin, anthocyanin, betanin, lycopene, copper complexes of chlorophyll and chlorophyllin, terpene compounds such as carotene compounds and xanthophyll compounds, plain caramel, caustic sulfite caramel, ammonia caramel, sulfite ammonia caramel, vegetable carbon, calcium carbonate, iron oxide and iron hydroxide, curcumin, tartrazine, cellulose gel, cochineal, carminic acid, carmine, azorubin, carmoisine, lutein, cocoa powder (melanoidin), beet powder, tomato extract, duckweed powder, spirulina powder, paprika powder (capsanthin and / or capsorubin), turmeric powder, blueberry powder, strawberry powder, berry pigment powder, heme powder, lycopene powder, betanin powder, alfalfa powder, saffron powder, mint powder, and annatto extract. More preferably, the colorants include riboflavin, chlorophyll and chlorophyllin, copper complexes of chlorophyll and chlorophyllin, plain caramel, caustic sulfite caramel, ammoniacal caramel, sulfite ammoniacal caramel, vegetable carbon, calcium carbonate, iron oxide and hydroxide, curcumin, tartrazine, cellulose gel, cochineal, carminic acid, carmine, azorubine, carmoisine, and lutein.

[0204] Preferably, the glazing agents and sweeteners include isomalt, maltitol, acesulfame potassium, aspartame, cyclamate, saccharin, sucralose, alitame, steviol glycosides, neotame, lactitol, xylitol, and erythritol.

[0205] The food product of the present invention may be further supplemented with additives selected from vitamin B12, vitamin B6, vitamin B2, vitamin B3 (also called niacin), riboflavin, thiamine, vitamin A, vitamin E, omega-3 fatty acids, vitamin D2, folic acid, iodized salts (NaCl, further including iodized salts in an amount of up to 5% w / w), minerals (e.g., salts containing calcium, iron, and / or potassium, etc.), flavorings (salt, pepper, oils, herbs and spices), and natural aromatic compounds. As defined herein, herbs and spices include natural aromatic compounds such as methyl acetate, linalool, limonene, vanillin, or synthetic compounds such as aprifloren, cinnamyl propionate, cyclohexadecanolide, and ethyl levulinate.

[0206] Preferably, the further additives defined as compositional ingredients are selected from vitamin B12, vitamin B6, vitamin B2, vitamin B3 (also called niacin), riboflavin, thiamine, vitamin A, vitamin E, omega-3 fatty acids, vitamin D2, folic acid, iodized salts (NaCl, further comprising iodized salts in an amount of up to 5% w / w), enzymes (e.g. transglutaminase, amylase), minerals (e.g. salts containing calcium, iron and / or potassium etc.), flavourings or flavouring ingredients (salt, pepper, garlic, onion, mushroom fruiting body pieces, vegetable pieces, ginger, turmeric, curry, sugars (i.e. sucrose, glucose, mono- or disaccharides), oils, lemon juice, orange juice, herbs and spices, yeast flakes), texturizing vegetable proteins and natural aromatic compounds. As defined herein, herbs and spices include natural aromatic compounds such as methyl acetate, linalool, limonene, vanillin, or synthetic compounds such as aprifloren, cinnamyl propionate, cyclohexadecanolide, and ethyl levulinate. Such additional additives may improve optical visibility, flavor, nutrients, and provide additional texture.

[0207] Preferably, the nutrients are selected from high protein ingredients (e.g. pea protein isolate, chickpea protein isolate, wheat gluten, egg white powder, and / or mung bean protein isolate), high carbohydrate / dietary fiber ingredients (e.g. cereal flours, cereal starches, legume starches, fruit fibers, polysaccharides, starch-based ingredients, psyllium husk, inulin, wheat starch, corn starch), high vitamin / mineral ingredients, and / or high lipid ingredients (e.g. all types of edible oils and butter). High fiber ingredients are preferably used to improve freeze-thaw stability and / or juiciness.

[0208] The composition obtainable according to the process of the invention is therefore useful in the manufacture of the food product of the invention. Thus, in another aspect, the invention relates to the use of the composition of the invention in the manufacture of the food product of the invention.

[0209] In a further embodiment, the present invention relates to a supernatant obtainable by recovery of the supernatant in step (f) of the method of the present invention. The supernatant is useful in the manufacture of food products. Thus, in a further embodiment, the present invention relates to the use of the supernatant in the manufacture of food products.

[0210] The supernatant of the present invention can also be used for the production of beverages, such as alcoholic beverages or sweetened health drinks. It can also be used to produce fermented beverages that promote intestinal and digestive health and enhanced immune system. The supernatant can also be dried to obtain a colored powder. This powder can be used in various fields, especially in cosmetic formulations, where there is growing interest in exopolysaccharides and antioxidants for skin care, for example. One possible application in cosmetics would be as a moisturizing agent. The supernatant of the present invention can also be used in dietary supplements and / or pharmaceutical applications.

[0211] Thus, preferably, the supernatant of the present invention is preferably mycelium-free or substantially mycelium-free. This supernatant can serve as a base for soft drinks, health fermented beverages, or alcoholic beverages. The latter can also be supported by choosing the fermentation conditions and production strains such that alcohol production is enhanced during the process and an increase in fermentation broth is achieved over time, as can be observed in typical beer fermentations with brewer's yeast. This liquid solution is then further processed to adjust the taste, either by adding additional ingredients known to those skilled in the art (e.g. sugars, alcohols, acidifiers, etc.), by enzymatically modifying the compounds, or by adjusting the alcohol and sugar levels by methods known to those skilled in the art (e.g. distillation). Alternatively, depending on the end use, the supernatant preferably contains mycelium, preferably a mycelium extract, since mushroom mycelium is known for its ability to produce antioxidants, exopolysaccharides, functional peptides, and other metabolites that may be relevant for addressing health aspects such as regulating blood glucose levels. Thus, the beverage produced can also find application as a health beverage in the future.

[0212] Preferably, the production of functional compounds includes information on compounds produced by the mycelium, such as antioxidants, exopolysaccharides, functional peptides, and other metabolites. Preferably, the functional compounds, also called active compounds, refer to any substrate that preferably has a beneficial (demonstrated or proven) effect on biological functions, and are preferably active compounds from the mycelium selected from ergothioneine, ergosterol, lovastatin, resveratrol, glutathione, eritadenine, lentinan, and concanavalin A that may be produced in the supernatant. However, this list is not meant to be interpreted as being particularly limiting, and may include additional compounds produced in the mycelium, as will be recognized by those skilled in the art. Exemplary compounds from Pleurotus ostreatus have been recently reviewed (Mishra et al., Int J Biol Macromol, 2021, 182, 1628-1637).

[0213] The invention further relates to a method for producing a beverage, such as kombucha, characterized in that a supernatant having at least 0.1% by weight of filamentous fungi is used.

[0214] Preferably, the supernatant used to produce the beverage comprises filamentous fungi in the range of 0.1-20% by weight, more preferably in the range of 1-15% by weight, even more preferably in the range of 1-10% by weight. Alternatively, the supernatant comprises filamentous fungi in the range of 25-80% by weight, more preferably in the range of 25-50% by weight. Preferably, in this specification, the stated numbers are understood to refer to wet biomass, preferably containing 3-30% water. For example, if the supernatant comprises 15% by weight of filamentous fungi, this means that 150 g of wet biomass is mixed with 850 g of supernatant. The method of the present invention preferably comprises a step of transferring functional substances from the mycelium or filamentous fungal cell walls to the supernatant for further enrichment with functional substances. Preferably, the process comprises a heat treatment under stirring, wherein the supernatant is heated at a temperature in the range of 50-150°C, preferably 85-100°C, even more preferably about 85°C, preferably for up to 3 hours, more preferably for up to 2 hours, even more preferably for up to 1 hour, under vigorous stirring, preferably in the range of 500-1500 rpm, more preferably 500-1200 rpm, even more preferably 500-1000 rpm, even more preferably 700-1000 rpm. In another preferred embodiment, the operating temperature is about 120°C. In another embodiment, the heat treatment is preferably performed for up to 30 hours. Alternatively, in case of fungal autolysis, the temperature of the heat treatment is preferably 30-60°C. It is worth noting that the addition of acid / base and / or the use of enzymes such as proteases can achieve the same effect as the heat treatment. The addition of acid / base and / or the use of enzymes such as proteases can also be combined with the heat treatment.

[0215] In a preferred embodiment, the supernatant is further concentrated with functional substances and then the mycelium is thoroughly filtered, the supernatant being a key component for kombucha.

[0216] In another preferred embodiment, the mycelium is preferably filtered after the heat treatment step, so that the concentrated supernatant so obtained for use in beverage preparation contains no more than 5% by weight of mycelium, preferably no more than 1% by weight, preferably no more than 0.1% by weight.

[0217] In an alternative embodiment, up to 10% by weight of mycelium is present throughout the kombucha fermentation process.The mycelium not only enriches the beverage with functional substances, but also contributes to a better taste.Preferably, after filtering the mycelium, the kombucha has a sweet taste that is more suitable for food use.

[0218] In a further alternative embodiment, the functional material is preferably extracted by extraction, evaporation and purification techniques known to those skilled in the art (e.g., ethanol extraction) to produce a powder that can be added either during the kombucha fermentation process or after the kombucha has been strained.

[0219] Thus, preferably, the beverage of the present invention, preferably the kombucha of the present invention, is characterized in that it comprises an extract derived from the mycelium of a filamentous fungus.Preferably, the content of said extract in the final kombucha beverage is at most 10% by weight, even more preferably at most 5% by weight, even more preferably at most 2.5% by weight, even more preferably at most 1% by weight, even more preferably at most 0.5% by weight, even more preferably at most 0.1% by weight.Preferably, said extract comprises ergothioneine, ergosterol, lovastatin, resveratrol, glutathione, eritadenine, lentinan, concanavalin A, or a combination thereof.

[0220] Preferably, the obtained supernatant has an ergothioneine content in the range of 1 to 1000 mg / L, more preferably 1 to 900 mg / L, more preferably 1 to 800 mg / L, more preferably 1 to 700 mg / L, more preferably 1 to 600 mg / L, more preferably 1 to 500 mg / L, more preferably 1 to 400 mg / L, more preferably 1 to 300 mg / L, more preferably 1 to 200 mg / L, more preferably 1 to 100 mg / L, more preferably 1 to 50 mg / L, and more preferably 1 to 25 mg / L.

[0221] In a preferred embodiment, the supernatant has an ergothioneine content in the range of 25-1000 mg / L, preferably 55-800 mg / L, more preferably 80-800 mg / L, even more preferably 350-800 mg / L, even more preferably 400-800 mg / L, even more preferably 400-700 mg / L. As understood herein, ergothioneine value preferably refers to the amount of ergothioneine expressed in mg ergothioneine per liter of supernatant.

[0222] For example, depending on the fermentation conditions used, the ergothioneine content in the supernatant after heat treatment is 1000mg / L, 800mg / L, 650mg / L, 500mg / L, 350mg / L, 150mg / L, 100mg / L, 50mg / L, or 25mg / L using Pleurotus pulmonarius, such values ​​are preferably achieved in about 5-5.5 days starting with X1 (see FIG. 1). Ergothioneine is preferably measured by a high performance liquid chromatography (HPLC) method (injection volume of about 10 μL, mobile phase preferably contains 3% acetonitrile and 0.1% acetic acid in water). External calibration with ergothioneine dissolved in water (5 μg mL-1 to 100 μg mL-1) is preferably used for quantification. This measurement is preferably performed on a HPLC analysis of ergothioneine performed on a Prominence system (Shimadzu) equipped with an LC-20AD high performance liquid chromatography (HPLC) pump, a SIL-20AC HT autosampler, an SPD-M20A diode array detector (DAD), a CBM-20A communication bus module, and a LabSolutions Multi LC Data System Manager.

[0223] Thus, preferably in this scenario, the filamentous fungus is an ergothioneine producing strain and is selected from Basidiomycota, Ascomycota, Hymenochaetaceae, Agaricomycetes, Sordariomycetes, Tremellomycetes, with preferred species selected from Cordyceps spp., Inonotus spp., Grifola spp., Pleurotus spp., Ganoderma spp., Lentinula spp., Tremella spp., Trametes spp., Lepista spp., Tricholoma spp., Aspergillus spp., and / or Panus spp.

[0224] Alternatively, the filamentous fungus comprises at least one of the ergothioneine producing fungal strains above in combination with a non-ergothioneine producing fungal strain, such as any of the other fungal strains listed in the embodiments described herein.

[0225] However, the filamentous fungi may also include only non-ergothioneine fungal strains.

[0226] According to the literature, China Patent Publication No. 103184246(A) discloses a method for producing ergothioneine using liquid culture of wild Pleurotus sapidus, Pleurotus pulmonarius or Lepista sordida to produce ergothioneine with a low yield of 51 mg / L in a 10-day culture time. For example, China Patent Publication No. 110283856(A) discloses a method for producing ergothioneine with a yield of 300 mg / L by fermenting the 3210 fungal strain Pleurotus ostreatus, but the process requires at least 25 days, with mycelium grown on PD for 15 days followed by a 10-day fermentation time. In the patent literature, it has been observed that a higher content of ergothioneine can be obtained by co-fermenting two or more fungal strains (CN 112195215 or CN 114214387). Finally, CN 109939027(A) discloses a method for producing ergothioneine by fermenting Hericium erinaceus with glucose and peptone with a yield of 331 mg / L, but the production cost of the substrate is high and the process requires a long time (about 25 days).

[0227] An exemplary method for producing such a beverage is provided below.

[0228] Methods for producing mycelium-based kombucha include: (1) A supernatant is used having at least 0.1% by weight of filamentous fungi, preferably in the range of 0.1-20% by weight, more preferably in the range of 0.1-15% by weight, even more preferably in the range of 0.1-10% by weight of filamentous fungi. The supernatant is provided from a fermentation based on at least one fungal strain. Preferably, the supernatant is colored. (2) Heat the supernatant at a temperature of 70 to 120°C, preferably 85 to 100°C, and stir vigorously at 500 to 1000 rpm for a period of 1 to 120 minutes, preferably 1 to 60 minutes, and even more preferably 1 to 30 minutes. (3) Add additional sugar, preferably crude cane sugar or a food grade sweetener, in the range of 5-30%. (4) The resulting mixture is mixed with unpasteurized Kombucha SCOBY liquid (Symbiotic Culture of Bacteria and Yeast) in a ratio of SCOBY liquid to supernatant of 1:15, preferably 1:12, more preferably 1:10, followed by the addition of one solid SCOBY piece in the same ratio. (5) Cover it with cloth or cheesecloth and keep it in a room with a temperature of 18°C ​​to 29°C. (6) After one week, the sugar content is measured using a refractometer, and the fermentation is stopped when the Brix score obtained is 5 to 40°Bx, preferably 8 to 35°Bx, and even more preferably 10 to 25°Bx, and the pH is 4 to 6, and preferably 4 to 5. (7) Once the desired characteristics are reached, the SCOBY and mycelium are removed or filtered, followed by straining and bottling the kombucha.

[0229] The mycelium may be filtered prior to step (4).The Brix refractometer is calibrated using the Brix scale, where 1 degree Brix (°Bx) is equal to 1% sucrose by weight.

[0230] Further additives are preferably added along with the sugar added in step (3) to further flavor the beverage or are added in step (7) to further dilute the kombucha with additives. Preferably, such additives are selected from any tea (e.g., green tea, white tea, black tea, fruit tea, herbal tea, or combinations thereof), syrup, aloe vera, coffee, fruit juice, vegetable juice, ginger extract, herbs / spices, dried fruit, and fruit / nut extracts.

[0231] It is understood herein that the functional compounds of interest, i.e. in the scenario of producing health drinks, are those present in the supernatant and not in the mycelium. The presence of mycelium in the supernatant is used to enrich the supernatant with more extract, which is then further fermented into a health drink (e.g., kombucha).

[0232] The present invention further relates to a beverage, preferably a kombucha, obtainable according to the method of the present invention and characterized in that it is derived from a supernatant containing fungal mycelium as described above. Thus, preferably, the beverage of the present invention, preferably the kombucha of the present invention, is characterized in that it comprises an extract derived from the mycelium of a filamentous fungus. Preferably, the mycelium of the filamentous fungus is derived from a fungal species that produces ergothioneine. Thus, preferably, the beverage of the present invention (the kombucha of the present invention) comprises ergothioneine.

[0233] It is understood that the final food product based on the disclosed composition may include the supernatant that can be obtained in the manufacturing process of the composition of the present invention.Thus, the final fungal-derived food product based on the disclosed composition may be the supernatant itself, or the colored biomass or a combination thereof, or any related extracts or a combination thereof from each individual product.Thus, the disclosed composition may be applicable to at least one form of these products, as will be apparent to those skilled in the art.

[0234] In a further embodiment, the present invention relates to the use of the composition of the present invention or the supernatant of the present invention in the manufacture of a cosmetic, pharmaceutical or dietary supplement.

[0235] Thus, in one embodiment, the present invention relates to a cosmetic product comprising the composition of the present invention and / or the supernatant of the present invention. In a further embodiment, the present invention relates to a pharmaceutical product comprising the composition of the present invention and / or the supernatant of the present invention. In yet a further embodiment, the present invention relates to a dietary supplement comprising the composition of the present invention and / or the supernatant of the present invention.

[0236] In one embodiment of the present invention, the composition of the present invention is used in the manufacture of a composite material, the material comprising fungal mycelium. It should be understood that the composite material is characterized by a specific color derived from the composition comprising biomass of the present invention. Preferably, the at least one additive supplemented in step (d) of the method of the present invention is selected from phthalocyanine blue, phthalocyanine green, diarylide pigments, quinacridone, alizarin, pyrrole, and natural gilsonite.

[0237] The present invention is illustrated by the following examples which should not be construed as limiting. In all the following examples, the mycelium stained is based on P. pulmonarius unless otherwise indicated. EXAMPLES

[0238] The average RGB scale was measured with a PCE-RGB colorimeter (DIN 5033). This was a useful way to quantify different shades (light vs. dark) with an averaged measurable value. However, in some cases the color differences were obvious and could be evaluated and recognized by the naked eye.

[0239] Example 1. Astaxanthin as a colorant Astaxanthin powder (5 wt. % concentrated) extracted from the microalga Hematococcus pluvarius was used to color the mycelial biomass. A concentrated stock solution of 10 g / L of powder was prepared and autoclaved at 121 °C for 15 min. On the fifth day of fermentation, i.e. during the slowdown phase, the flasks were removed from the incubator and placed on a clean bench. 5 mL of the autoclaved astaxanthin solution (corresponding to a final concentration of 0.9 g / L or 0.09 wt. % astaxanthin) was added to the fermentation culture and the flasks were returned to the incubator to resume fermentation. On the sixth day, the fermentation was stopped and the flasks were collected and drained through cheesecloth. An orange / red mycelial biomass was obtained.

[0240] The same experiment was carried out in parallel over the same time frame, but after fermentation without the addition of coloring agent to reproduce the coloring procedure described above. On the sixth day of this fermentation (corresponding to the fifth day for the first scenario), the biomass was harvested and drained through cheesecloth. It was then immersed for one day at ambient temperature in a solution of 0.9 g / L astaxanthin prepared in a 0.9% by weight NaCl solution, exposing the biomass to the coloring agent for one day in both scenarios. In this case, the biomass was slightly colored.

[0241] The panels of Figure 2 show a comparison of the pigmented biomass in both scenarios. The mycelial biomass obtained from pigmentation during fermentation (Figure 2A) is more pronounced and intense (better matched to the RGB values ​​of red meat) compared to that obtained from pigmentation after fermentation using the same concentration of astaxanthin solution (Figure 2B) (better matched to the RGB values ​​of white meat). Moreover, the addition of astaxanthin at the beginning of fermentation, i.e. during the induction phase, leads to the decomposition of this pigment, showing the importance of the addition time, which is determined by the chemical nature of the colorant.

[0242] Example 2. Cocoa as a colorant Cocoa powder was used to colour the mycelium biomass. A concentrated stock suspension of 100 g / L was prepared and autoclaved at 121° C. for 15 min. 10 mL of autoclaved cocoa suspension (concentration of cocoa suspension 20 g / L, i.e. 2% by weight) was added at t=0 min in one flask, while a control flask without any added cocoa was run in parallel. Both flasks were inoculated from an agar plate and placed in an Innova 44 incubator for 6 days. On the sixth day, both flasks with and without cocoa were harvested by two centrifugation steps. The fermentation broth of both flasks was transferred to a 50 mL falcon and centrifuged at a speed of 5000 rpm for 20 min at 4° C. After the first centrifugation, the supernatant was discarded and the biomass was resuspended in tap water for washing. Both falcons were again placed in the centrifuge under the same conditions. The washing water was then discarded and the washed biomass was isolated. The biomass recovered from the flasks containing cocoa showed a dark brown / black mycelial biomass, while the other biomass was naturally colored (yellowish white). This naturally colored biomass was soaked in a 20 g / L cocoa suspension (same concentration as above) for 6 days to replicate the fermentation period carried out above. The results show that the colored biomass after fermentation has a lighter color compared to the dark colored biomass obtained when this type of colorant is present throughout the entire fermentation process (Figures 3A and 3B). When the cocoa encounters "active mycelium", i.e. actively growing mycelium, the resulting color is darker because the particles diffuse into the mycelium structure and are more actively trapped, as opposed to "resting mycelium", i.e. mycelium that is dormant in the stationary phase, which results in a lighter color due to the particles simply diffusing into the mycelium structure. Furthermore, it has been shown that adding cocoa powder during the deceleration phase results in a better and more uniform coloration compared to adding the colorant on day 0. FIG. 3D shows mycelium biomass with 3 g / L of cocoa powder added on day 0 (left, color: light brown-non-uniform) versus during the deceleration phase (right, color: dark brown-uniform).

[0243] Example 3. Paprika as a coloring agent Paprika powder (without preservatives) was used for coloring the mycelial biomass. A concentrated suspension of 100 g / L was prepared and autoclaved at 121 °C for 15 min. In four different flasks, the paprika suspension was added at two different time frames (start of fermentation (day 0) and day 5 of fermentation) and at two concentrations (5 g / L and 10 g / L). The same fermentation and harvesting procedures were performed as mentioned in the previous examples. In the flasks where the suspended paprika was added on day 0, the color disappeared within 24-48 hours and the mycelial biomass became natural in color. In the flasks where the colorant was added on day 5, the mycelial biomass was found to have a light or dark orange hue depending on the concentration used, i.e., 5 g / L and 10 g / L, respectively. The experimental results are shown in Figure 4.

[0244] Example 4. Comparison of browning performance of pigmented biomass during and after fermentation using astaxanthin as a colorant The coloring experiment was designed to compare coloring performance by allowing the same contact time between the biomass and the colorant astaxanthin as described in Example 1. The biomass was molded into four patties of 50 g each and treated according to the scheme shown in Figure 5A for both scenarios (coloring during or after fermentation). The patties kept in the refrigerator were kept at 4°C. All other samples were kept under vacuum conditions in bags.

[0245] The cooking performance of different biomasses was tested. Figure 5BC shows a comparison of fried and non-fried patties made from biomasses colored after fermentation (B) and during fermentation (C, darker). Before frying, the patties were cooked (heat treatment) at 65°C for 15 minutes (A, left) and at 80°C for 15 minutes (A, right). The bottom photo shows patties with the same treatment but for biomasses colored during fermentation. The coloring performance and browning effect of the patties made from biomasses colored during fermentation was significantly better compared to the patties made from biomasses colored after fermentation. In other words, the browning effect was more uniform and with a color that was attractive to eat.

[0246] All samples were stored at 4°C for 21 days and non-fried patties were fried under the same conditions. The colouring performance and browning effect obtained are shown in Figure 6. The upper figure shows half of a patty made from biomass coloured after fermentation, fried for 5 minutes at the lowest level of the heating stove (level 1) on day 0 and day 21 (after the storage period). The lower figure shows half of a patty made from biomass coloured during fermentation, stored and fried in the same way.

[0247] The difference in coloring performance and browning effect between the samples without resting period and the samples resting for 21 days was significant. The browning performance was significantly improved in both scenarios (fermentation and post-fermentation) after 21 days of storage period. Water was lost during storage period and better crusting effect was observed in both halves of the patties made from both scenarios. The color did not change during storage period, indicating that the color was stable throughout this period.

[0248] Example 6. Bioreactor configuration FIG. 8 shows an exemplary bioreactor configuration for carrying out the methods of the invention.

[0249] [1] Preparation of dye solution In the first step, a pigment solution is prepared. This step aims to provide natural pigments to be used in the subsequent fermentation. The easiest way to achieve this goal is to purchase commercial powders or liquid products containing natural pigments, such as carotenoids, chlorophyll, phycobilins, anthocyanins, or others. Alternatively, other materials can be ground before being added to the medium, if necessary. Ideally, these compounds provide color to the mycelium, but may also be advantageous in terms of nutrition or shelf-life stability of the product (e.g., antioxidants, omega-3 fatty acids). Powders can also be freeze-dried or liquid suspensions of microorganisms such as algae.

[0250] Another way to prepare pigments for fermentation would be to use industrial by-streams that contain these pigments. If the by-stream used is a liquid that already contains the pigments, it can be added directly to the fermentation medium. The color can possibly be fine-tuned by varying the temperature and time of sterilization, or it can remain the original color if the by-stream is sterilized using a bactericide (e.g. HClO). A second possibility is that the by-stream is a powder, which can be resuspended in a liquid and used later as a liquid by-stream. Finally, other solid by-streams, such as plant waste from farms, etc., can also be obtained, and the pigments must be extracted using a solvent and then ground.

[0251] [2] Fungal fermentation in colored culture medium Mixing different pigments is also an option to obtain a finely tuned color. The same by-stream may have different shades. For example, cocoa powders may have different browning grades, possibly related to different browning processes in the manufacture of cocoa. The pigments are preferably derived from fruits, vegetables and plants, but may also be "inorganic pigments" that contain trace elements of interest for fermentation.

[0252] After the dye solution is prepared, the solution is sterilized or pasteurized and added to the fermentation medium, or it may be added to the medium before sterilization or pasteurization depending on the most efficient method to achieve the desired color. The fungus is then grown on the color medium and, depending on the nature of the pigment, either assimilates the pigment or grows on it during fermentation.

[0253] [3]~[5] Co-fermentation of edible fungi and coloring microorganisms Another option for coloring the fungal biomass is to produce color by co-fermenting at least one fungal strain with at least one other microorganism producing a pigment either intracellularly or extracellularly (i.e., a pigmented microorganism). In that case, the at least one pigmented microorganism is added to the fermenter at a time that allows efficient coloring of the fungal biomass but does not result in overgrowth of said microorganism in the fermenter, meaning that the at least one added microorganism does not exhaust the nutrients of the medium before the at least one fungal strain has had enough time to grow to the desired extent. The pigmented microorganism added can be a fungus, a bacterium, an archaea, an algae, or any microorganism capable of producing a pigment. In certain embodiments, even colorants from animal origin can be used to color the fungal biomass. Preferably, the at least one pigmented microorganism added to the fermenter is not a GMO organism, although GMO organisms are not excluded. Alternatively, the medium used to perform the co-culture also contains at least one other pigment, which can be added as a powder or solution as described in step [2], allowing fine-tuning of the final color of the fungal mycelium. Depending on the time required to have a significant amount of biomass of at least one pigment-producing microorganism, it is also possible to start the fermentation with at least one pigment-producing microorganism and later add at least one fungal strain of interest to the fermenter.

[0254] If the growth conditions (medium, pH, optimum temperature, shear sensitivity, etc.) required by the at least one pigment-producing microorganism are too different from the growth conditions of the at least one selected fungal strain, or if the growth time from the at least one pigment-producing microorganism is too different from the growth time of the at least one fungal strain, the fermentation can be carried out in two separate fermenters, with the at least one pigment-producing microorganism being added at a given time to the fermenter containing the at least one fungal strain [4][5]. This configuration can be advantageous, for example, to use CO2 produced during fungal fermentation in its normal state or after compression to grow CO2-fixing microorganisms, such as algae or cyanobacteria, more efficiently in another fermenter, and then add it to the fermenter with the at least one fungal strain [5]. Similarly, CO2 captured from other sources can be added to the second fermenter to further promote the growth of the CO2-fixing microorganisms, thereby resulting in a carbon-neutral or carbon-negative process. It is also envisaged to use commercially available CO2 as an input for the growth of the at least one pigment-producing microorganism [5]. If desired, the fermentor can also be equipped with various light systems that can operate at different wavelengths to stimulate the growth of certain microorganisms (e.g., algae) or the production of certain compounds (e.g., vitamin D).

[0255] [6] Biomass separation and processing After fermentation, the biomass is separated by filtration, centrifugation, or other state-of-the-art techniques, washed with water, and concentrated to a concentration suitable for food production. The biomass can be pure mycelium incorporating soluble pigments, or mycelium grown on solid (pigment) particles, or a mixture of mycelium and other colored microorganisms. Depending on the composition, it can also be enriched with vitamins, especially B12, antioxidants, and omega-3 or other fatty acids.

[0256] [7] Recovery and use of the supernatant as a beverage, e.g., an alcoholic beverage or a sweetened health drink. After separation of the fermentation broth, the supernatant has a residual color and an attractive taste and aroma (e.g., almond, maple, raspberry, kiwi, etc.) due to the volatile compounds that the mushroom mycelium can produce during fermentation. This supernatant can serve as the base for a soft drink or an alcoholic beverage. The latter can also be supported by selecting the fermentation conditions and the producing strains so that the alcohol production is enhanced during the process and increases over time in the fermentation broth, as can be observed in a typical beer fermentation with brewer's yeast. This liquid solution is then further processed to adjust the taste, either by adding additional ingredients (e.g., sugars, alcohols, acidifiers, etc.), enzymatically modifying the compounds, or adjusting the level of alcohol by methods known to those skilled in the art (e.g., distillation). Mushroom mycelium is also known for its ability to produce antioxidants, exopolysaccharides, functional peptides, and other metabolites that may be relevant for addressing health aspects such as regulating blood glucose levels. Thus, the beverage produced can also find application as a health drink in the future.

[0257] [8] Drying of the supernatant for other uses Alternatively, the functional compounds found in the supernatant obtained after fermentation can be purified by methods known to those skilled in the art (e.g., preparative chromatography, crystallization, solvent extraction, etc.) and dried to deliver a high-purity solution for the nutraceutical and pharmaceutical industries. Another interesting aspect can be to dry the colored supernatant and incorporate it as a powder into a functional cosmetic composition, especially for skin care applications. In fact, it is known that exopolysaccharides, antioxidants, functional peptides, and other fungal-based metabolites have been of increasing interest in the cosmetic industry in the last few years, and delivering coloring materials with functionality would be of interest to this industry. Similarly, the functional compounds found in the colored extract generated from the by-stream in step [1] can also be recovered in the same way and have found applications in the nutraceutical, pharmaceutical and pharmaceutical industries.

[0258] Example 6. Leaching test The color stability is investigated by UV-vis absorption method using a photolab 7600 UV-Vis. The colored biomass containing astaxanthin is immersed in water. Samples are taken every hour up to 12 hours and another sample is taken after 24 hours and placed in the spectrophotometer to generate the UV-vis spectrum. The UV-vis spectrum of the control solution (water without immersed biomass) shows a high match with the spectrum of the collected solution at least up to 24 hours, indicating that the color is not leaching into the solution.

[0259] Example 7. Astaxanthin as a colorant: stability, content analysis, addition time, and scale-up [1] Addition of different concentrations of astaxanthin during the deceleration phase As an example, 0.9 g / L and 1.8 g / L of astaxanthin powder (5 wt% astaxanthin) were added during the slowdown phase of fermentation. The fermentation was carried out aerobically in 100 mL shake flasks. The color difference shows that the intensity of the red color can be controlled accordingly based on the concentration added (Figure 9).

[0260] [2] Cleaning protocol The harvested biomass shown in Figure 9 was washed with tap water to mimic downstream processes of large scale operations and to confirm color stability when stored in solution. Therefore, the biomass was immersed in tap water and spectrophotometer readings were recorded on the wash water after 2, 4 and 6 hours. Table 1 below shows the amount of astaxanthin that leached into solution and was also shown to be negligible to the naked eye in Figure 11. The same observation was observed after 24 hours (see Figure 10).

[0261] [Table 1]

[0262] [3] Content analysis

[0263] [Table 2]

[0264] Table 2 shows that for both concentrations of astaxanthin added during the deceleration phase (0.9 g / L and 1.8 g / L), the amount of astaxanthin accumulated in the biomass was comparable, 73.4% on average, which ultimately helps to estimate and quantify the amount of colorant required to reach the desired product properties and composition.

[0265] [4] Different addition times Figure 12 shows the mycelium biomass obtained by adding astaxanthin during the induction phase (X1), logarithmic phase (X3), and deceleration phase (X2). It can be observed that the later the colorant is added, the more color is retained. This confirms that adding astaxanthin during the deceleration phase is preferable over the induction phase or logarithmic phase. Furthermore, adding the colorant later is more cost-effective since less pigment is needed to reach the target product composition.

[0266] [5] Heating test

[0267] [Table 3]

[0268] The purpose of this test was to mimic the effect of cooking on the color of biomass. Biomass was immersed in tap water and heated to 85°C. As can be observed from the table above, no color loss is detectable up to 60 minutes, indicating that color does not leach out during cooking. This is also shown in Figure 13, which shows that there is almost no coloring of the water after the 80 minutes of heating mentioned above.

[0269] [6] Coloration after fermentation, i.e. after biomass recovery To mimic the addition of astaxanthin during the slowdown phase, 100 mL flasks (50 mL medium) were inoculated. At X2, the biomass was harvested and soaked in 50 mL of 0.9 g / L astaxanthin. One was placed in a 4°C refrigerator and the other in a 25°C incubator for 2.5 days, resulting in the same total incubation time of 6.5 days.

[0270] The pigmented biomass was then harvested and washed three times after 2, 4 and 24 hours to understand the stability of the pigment compared to the pigmentation during the slowdown phase of the fermentation.

[0271] [Table 4]

[0272] According to the figures, the samples cannot be distinguished visually between the biomass soaked at 4°C and the biomass soaked at 25°C. However, according to the above table, it is clear that the color stability is low when using this coloring method (i.e., after fermentation). In addition, it was clear in the laboratory that the colored biomass after fermentation, whether soaked at 4°C or 25°C, did not retain the color well because the astaxanthin leached onto the gloves while working with the colored biomass. Furthermore, in the first photographs of Figures 14 and 15, it is clear that the biomass was not colored uniformly, since white clumps of biomass are still visible.

[0273] [7] Scaling up fungal growth with astaxanthin Mycelium added with astaxanthin at time 0 and fermented in a 1 L fermenter with controlled dissolved oxygen (DO), agitation and aeration also showed degradation of astaxanthin compared to addition during the deceleration phase. Figures 16 and 17 show that the process is scalable, and in the first figures it is clear that the dye degrades over time similarly to that observed at flask scale, but with a slightly higher intensity. Taking into account the fact that in the fermenter additional parameters such as pH and dissolved oxygen (which can have a significant effect on cell behavior) are controlled compared to the flask. The results are relatively comparable, and as can be seen in Figures 17 and 18, adding the dye during the deceleration phase still proves to result in a more uniformly colored biomass and less astaxanthin degradation.

[0274] Example 8. Lycopene Lycopene powder (10% lycopene by weight) was added either on day 0 or during the retardation phase, as shown in Figure 19. Adding lycopene during the retardation phase resulted in a more colored biomass, similar to the case of astaxanthin, and the later (i.e., toward the retardation phase) the colorant was added, the greater the color retention (i.e., higher color intensity, less leaching).

[0275] Example 9. Fungal co-fermentation Pleurotus pulmonarius and species including at least the genus Rhodotorula were fermented aerobically together. Rhodotorula is a genus of pigmented yeasts that are part of the phylum Basidiomycota. Rhodotorula was added from the beginning because it has a slower growth rate than Pleurotus pulmonarius. In Figure 20 above, a salmon-like pigmented mycelium biomass can be observed, which is suitable for a fish substitute. The same co-fermentation concept can be carried out with algae strains such as Haematoccus pluvialis, Chlorella vulgaris, and / or cyanobacteria.

[0276] Example 10. Microalgae as a coloring agent and its CO2 fixation Pleurotus pulmonarius was cultivated aerobically in a 1 L fermenter. The fermentation was fed with air with a 0.05% CO2 composition. The exhaust air of the fermenter was split in two, one of which was fed to another fermenter in which microalgae were cultivated, and one of which was connected to a gas analyzer to quantify the CO2 in the exhaust air of the fermenter. A second fermenter containing the microalgae Haematococcus pluvialis species (undisrupted) was cultivated in BG11 commercial medium supplemented with a trace element mixture and fed with the effluent of the Pleurotus pulmonarius fermentation (as described above). It was also equipped with an LED light (3000K warm color). The exhaust air of this second fermenter was connected to a gas analyzer to quantify the CO2 in the exhaust air and to calculate the CO2 fixed by the microalgae accordingly. Figure 21 shows the trend in the percentage of CO2 fixed by microalgae (by volume), calculated by subtracting the percentage of CO2 in the effluent of the microalgae culture from the percentage of CO2 supply provided by the effluent of the Pleurotus pulmonarius mycelium fermentation. The trend in fixed CO2 is shown in this figure to increase over the time frame shown during the microalgae culture.

[0277] Scenario 1: After pigmentation of Haematococcus pluvialis, microalgae broth containing microalgae is added during the deceleration phase of an additional fermenter of Pleurotus pulmonarius running in parallel. The resulting biomass is green in color and enriched in omega-3 fatty acids.

[0278] Scenario 2: After Haematococcus pluvialis starts to pigment, stress is induced by nitrogen starvation or salt addition to produce astaxanthin in situ, which is then added during the slowdown phase of Pleurotus pulmonarius fermentation, resulting in a reddish biomass.

[0279] Example 11. Kombucha beverage preparation 850 g of the supernatant was mixed with 150 g of mycelium (wet mass) and heated at 95°C for 60 min with vigorous stirring at 650 rpm. The mycelium was then filtered to 99% after cooling the mixture, followed by the addition of 17% by weight of raw cane sugar. Then 100 g of unpasteurized kombucha SCOBY liquid (Symbiotic Culture of Bacteria and Yeast) was added. All the liquid was placed in a jar or fermentation vessel, one piece of kombucha SCOBY (125 g) was placed in the liquid, the jar was covered with a cloth and kept at 22°C. After 7 days, the SCOBY was removed, the liquid was strained and diluted with black tea to reach a sugar content of 8°Bx, before being bottled.

[0280] Further examples and / or embodiments of the present invention are disclosed in the following numbered paragraphs. 1. A method for producing a composition comprising a fungal biomass, the composition being characterized in that it has a particular color, the method comprising: (a) providing a growth medium; (b) providing at least one fungal strain; (c) culturing at least one fungal strain in a growth medium; (d) supplementing the growth medium with at least one additive that produces a color in the composition; (e) further culturing the at least one fungal strain in a growth medium supplemented with at least one additive that produces the color of the composition; (f) recovering a composition comprising a fungal biomass from the growth medium, the composition being characterized as having a particular color; The method wherein the particular color is due to at least one additive replenished in step (d). 2. The method according to item 1, wherein the at least one fungal strain is an edible fungal strain. 3. The method according to item 1 or 2, wherein the at least one fungal strain is selected from Basidiomycota and Ascomycota. 4. The method according to any one of items 1 to 3, wherein the at least one fungal strain is selected from Pezizomycotina and Agaricomycotina. 5. The method according to any one of items 1 to 4, wherein the at least one fungal strain is selected from Peziomycetes, Agaricomycetes and Sordariomycetes. 6. The method according to any one of items 1 to 5, wherein the at least one fungal strain is selected from Pezizales, Boletales, Cantharellales, Agaricales, Polyporales, Russulales, Auriculariales, Sordoriales and Hypocreales. 7. The method according to any one of items 1 to 6, wherein the at least one fungal strain is selected from Morchellaceae, Tuberaceae, Pleurotaceae, Agariaceae, Marasmiaceae, Cantharellaceae, Hydnaceae, Boletaceae, Meripillaceae, Polyporaceae, Strophariaceae, Lyophyllaceae, Tricholomataceae, Omphalotaceae, Physalacriaceae, Schizophyllaceae, Sclerodermataceae, Ganodermataceae, Sparassidaceae, Hericiaceae, Bondarzewiaceae, Cordycipitaceae, Auriculariaceae, Sordoriaceae, Nectriaceae and Fistulinaceae. 8. The method according to any one of items 1 to 7, wherein the at least one fungal strain is P. pulmonarius, P. ostreatus, P. citrinopileatus or P. salmoneostramineus, or the at least one fungal strain is M. esculenta, M. angusteps, M. deliciosa or M. rufobrunnea. 9. The method according to any one of items 1 to 8, wherein the at least one fungal strain is P. pulmonarius. 10. The method according to any one of items 1 to 9, wherein the growth medium further comprises vitamin B12 and / or omega-3 fatty acids. 11. The method according to any one of items 1 to 10, wherein the at least one additive that produces the color of the composition comprises a powder, preferably, the powder forms a dispersed phase when supplemented with the growth medium in step (d). 12. The method according to item 11, wherein the powder is cocoa powder, duckweed powder, spirulina powder, paprika powder, turmeric powder, heme powder or beet powder, or a combination thereof. 13. The method according to item 11 or 12, wherein the composition comprising a fungal biomass comprises mycelium bound to particles of powder dispersed in the growth medium, preferably the growing mycelium captures particles of powder dispersed in the growth medium. 14. The method according to item 13, wherein the particles of powder bound to the mycelium cannot be separated from the mycelium, preferably by washing, without disrupting the mycelium structure and preferably without disrupting the fungal cells. 15. The method according to item 13 or 14, wherein the particles of powder bound to mycelium constitute up to 25% w / w of the composition comprising fungal biomass. 16. The method according to any one of items 11 to 15, wherein step (d) is carried out before the start of step (c). 17. The method according to any one of items 1 to 10, wherein the at least one additive that produces a color in the composition comprises a colorant that is assimilated by the at least one fungal strain. 18. The method according to item 17, wherein assimilation of the coloring agent by the at least one fungal strain depends on oxygen availability to the at least one fungal strain. 19. The method according to item 17 or 18, wherein step (e) is carried out for 72 hours or less, preferably for 48 hours or less. 20. The method according to any one of items 17 to 19, wherein the colorant is a terpene compound. 21. The method according to item 20, wherein the colorant is astaxanthin or a derivative thereof. 22. The method according to any one of items 1 to 10, wherein the at least one additive that produces a color in the composition comprises a colored microorganism. 23. The method according to item 22, wherein the pigmented microorganism is a microorganism selected from red algae, green algae, brown algae and cyanobacteria. 24. The method according to item 22 or 23, wherein the composition comprising a fungal biomass further comprises a biomass of a pigmented microorganism, preferably selected from red algae, green algae, brown algae and cyanobacteria. 25. The method according to any one of items 1 to 24, wherein the supplementation in step (d) is carried out during the lag phase, acceleration phase, logarithmic phase, deceleration phase or stationary phase of biomass growth, preferably, the supplementation in step (d) is carried out during the deceleration phase or logarithmic phase of biomass growth, more preferably, the supplementation in step (d) is carried out during the deceleration phase of biomass growth. 26. The method according to any one of items 1 to 25, wherein the supplementation in step (d) is carried out in the logarithmic phase, preferably as submerged fermentation, in which the cultivation is carried out as a continuous process. 27. The method according to any one of items 1 to 65, further comprising recovering the supernatant in step (f). 28. A composition obtainable according to the method according to any one of items 1 to 27. 29. A food product comprising the composition of claim 28. 30. The food according to item 29, further comprising additives selected from preservatives, antioxidants and acidity regulators, thickeners, stabilizers and emulsifiers, pH regulators and anticaking agents, flavor enhancers, improvers, stabilizers, thickeners, colorants, and / or glazing agents and sweeteners. 31. Use of the composition according to item 28 in the production of food products. 32. A supernatant obtainable according to item 27. 33. Use of the supernatant according to item 32 in the production of a food product.

[0281] Further examples and embodiments of the present invention are disclosed in the following numbered paragraphs. 1. A method for producing a composition comprising a fungal biomass, the composition being characterized in that it has a particular color, the method comprising: (a) providing a growth medium; (b) providing at least one fungal strain; (c) culturing at least one fungal strain in a growth medium; (d) supplementing the growth medium with at least one additive that produces a color in the composition; (e) further culturing the at least one fungal strain in a growth medium supplemented with at least one additive that produces the color of the composition; (f) recovering a composition comprising a fungal biomass from the growth medium, the composition being characterized as having a particular color; The method wherein the particular color is due to at least one additive replenished in step (d). 2. At least one fungal strain is an edible fungal strain; Preferably, the at least one fungal strain is selected from Basidiomycota and Ascomycota, more preferably, the at least one fungal strain is selected from Pezizomycotina and Agaricomycotina; Even more preferably, the at least one fungal strain is selected from Peziomycetes, Agaricomycetes, and Sordariomycetes; Even more preferably, the at least one fungal strain is selected from the orders Pezizales, Boletales, Cantharellales, Agaricales, Polyporales, Russulales, Auriculariales, Sordoriales, and Hypocreales, and even more preferably, the at least one fungal strain is selected from the orders Morchellaceae, Tuberaceae, Pleurotaceae, Agariaceae, Marasmiaceae, Cantharellaceae, Hydnaceae, Boletaceae, Meripilis, and the like. laceae, Polyporaceae, Strophariaceae, Lyophyllaceae, Tricholomataceae, Omphalotaceae, Physalacriaceae, Schizophyllaceae, Sclerodermataceae, Gan selected from odermataceae, Sparassidaceae, Hericiaceae, Bondarzewiaceae, Cordycipitaceae, Auriculariaceae, Sordoriaceae, Nectriaceae and Fistulinaceae, Even more preferably, the at least one fungal strain is P. pulmonarius, P. ostreatus, P. citrinopileatus or P. salmoneostramineus, or the at least one fungal strain is M. esculenta, M. angusteps, M. deliciosa or M. rufobrunnea; Even more preferably, the method of paragraph 1, wherein the at least one fungal strain is P. pulmonarius. 3. The method of paragraph 1 or 2, wherein the growth medium further comprises vitamin B12 and / or omega-3 fatty acids. 4. The method of any one of paragraphs 1-3, wherein the at least one additive that produces the color of the composition comprises a powder, preferably, the powder forms a dispersed phase when supplemented with the growth medium in step (d). 5. The powder is cocoa powder, duckweed powder, spirulina powder, paprika powder, turmeric powder, heme powder, or beet powder, or a combination thereof; Preferably, the composition comprising a fungal biomass comprises mycelium bound to particles of powder dispersed in the growth medium, preferably the growing mycelium captures particles of powder dispersed in the growth medium; Preferably, the method according to paragraph 4, wherein the particles of the powder bound to the mycelium cannot be separated from the mycelium, preferably by washing, without disrupting the mycelium structure, preferably without disrupting the fungal cells. 6. The particles of powder bound to mycelium constitute up to 25% w / w of the composition containing fungal biomass; and / or 6. The method of paragraph 4 or 5, wherein step (d) is performed before the start of step (c). 7. At least one additive that produces a color in the composition comprises a colorant that is assimilated by at least one fungal strain, and preferably, assimilation of the colorant by the at least one fungal strain is dependent on oxygen availability to the at least one fungal strain; Preferably, the method according to any one of paragraphs 1 to 3, wherein step (e) is carried out for not more than 72 hours, preferably not more than 48 hours. 8. The method of any one of paragraphs 6 or 7, wherein the colorant is a terpene compound, preferably the colorant is astaxanthin or a derivative thereof. 9. At least one additive that produces color in the composition comprises a colored microorganism, preferably selected from red algae, green algae, brown algae and cyanobacteria; 4. The method according to any one of paragraphs 1 to 3, wherein the composition comprising a fungal biomass further comprises a biomass of a pigmented microorganism, preferably selected from red algae, green algae, brown algae and cyanobacteria. 10. The supplementation in step (d) is carried out during the lag phase, acceleration phase, logarithmic phase, deceleration phase, or stationary phase of biomass growth; Preferably, the supplementation in step (d) is carried out during the deceleration or exponential phase of biomass growth, more preferably during the deceleration phase of biomass growth. Or preferably, the method according to any one of paragraphs 1 to 9, wherein the supplementation in step (d) is carried out during the induction phase. 11. The method of any one of paragraphs 1 to 10, wherein the replenishment in step (d) is performed during a deceleration phase. 12. The method according to any one of paragraphs 1 to 10, wherein the supplementation in step (d) is carried out in the logarithmic phase, preferably as submerged fermentation, in which the cultivation is carried out as a continuous process. 13. The method of any one of paragraphs 1 to 12, further comprising recovering the supernatant in step (f). 14. A composition obtainable according to the method according to any one of paragraphs 1 to 13. 15. A food product comprising the composition according to paragraph 14. 16. Use of the composition according to paragraph 14 in the manufacture of a food product. 17. A supernatant obtainable according to paragraph 13. 18. Use of the supernatant described in paragraph 17 in the manufacture of a food product.

Claims

1. 1. A method for producing a composition comprising a fungal biomass, the composition being characterized by having a particular color, the method comprising: (a) providing a growth medium; (b) providing at least one fungal strain; (c) culturing said at least one fungal strain in said growth medium; (d) supplementing the growth medium with at least one additive that produces the color of the composition; (e) further culturing said at least one fungal strain in said growth medium supplemented with said at least one additive that produces the color of said composition; (f) recovering a composition comprising the fungal biomass from the growth medium, wherein the composition is characterized by having a particular color; The method wherein the particular color is due to the at least one additive replenished in step (d).

2. The method of claim 1 , wherein the replenishment in step (d) is performed during a deceleration phase.

3. 3. The method of claim 1 or 2, wherein the at least one fungal strain is an edible fungal strain.

4. 2. The method of claim 1, wherein the at least one fungal strain is selected from Basidiomycota and Ascomycota.

5. 2. The method of claim 1, wherein the at least one fungal strain is selected from Pezizomycotina and Agaricomycotina.

6. 2. The method of claim 1, wherein the at least one fungal strain is selected from Peziomycetes, Agariomycetes, and Sordariomycetes.

7. 2. The method of claim 1, wherein the at least one fungal strain is selected from Pezizales, Boletales, Cantharellales, Agaricales, Polypolares, Russulares, Auriculares, Sordriales, and Hypocreales.

8. The at least one fungal strain is selected from the group consisting of Morchellaceae, Tuberaceae, Pleurotaceae, Agaricaceae, Marasmiaceae, Cantharellaceae, Hydnaceae, Boletaceae, Meripilaceae, Polyporaceae, Strophariaceae, Lyophyllaceae, Tricholomataceae, Omphalotaceae , Physalacriaceae, Schizophyllaceae, Sclerodermataceae, Ganodermataceae, Sparassidaceae, Hericiaceae, Bondarzewiaceae, Cordycipitaceae, Auriculariae, Sordoriae, Nectriaceae and Fistulinaceae.

9. 2. The method of claim 1, wherein the at least one fungal strain is selected from Pleurotus pulmonarius, Pleurotus florida, Pleurotus citrinopileatus, Pleurotus salmoneostramineus, Morchella esculenta, Morchella angusticeps, Morchella deliciosa, and Morchella rufobrunnea.

10. 10. The method of claim 1, wherein the at least one fungal strain is P. pulmonarius or Morchella rufobrunnea.

11. 10. The method of claim 1, wherein the growth medium further comprises vitamin B12 and / or omega-3 fatty acids.

12. 10. The method of claim 1, wherein the at least one additive that produces the color of the composition comprises a powder, preferably the powder forms a dispersed phase when supplemented with the growth medium in step (d).

13. 13. The method of claim 12, wherein the powder is cocoa powder, duckweed powder, spirulina powder, paprika powder, turmeric powder, heme powder, or beet powder, or a combination thereof.

14. 14. The method of claim 12 or 13, wherein uniform coloring of the composition is achieved when supplementing the growth medium with the powder in step (d).

15. 13. The method of claim 12, wherein the composition comprising the fungal biomass comprises mycelium bound to particles of the powder dispersed in the growth medium, preferably the growing mycelium captures the particles of the powder dispersed in the growth medium, more preferably the growing mycelium uniformly captures the particles of the powder dispersed in the growth medium.

16. 16. The method of claim 15, wherein the particles of the powder bound to the mycelium cannot be separated from the mycelium, preferably by washing, without disrupting the mycelium structure and preferably without disrupting the fungal cells.

17. 16. The method of claim 15, wherein the particles of the powder bound to the mycelium constitute up to 25% w / w of the composition comprising the fungal biomass.

18. 13. The method of claim 12, wherein step (d) occurs before the start of step (c).

19. 10. The method of claim 1, wherein the at least one additive that produces color in the composition comprises a colorant that is assimilated by the at least one fungal strain.

20. 20. The method of claim 19, wherein assimilation of said colorant by said at least one fungal strain is dependent on oxygen availability to said at least one fungal strain.

21. 21. The method according to claim 19 or 20, wherein step (e) is carried out for not more than 72 hours, preferably not more than 48 hours.

22. 20. The method of claim 19, wherein the colorant is a terpene compound, preferably a carotenoid.

23. 20. The method of claim 19, wherein the colorant is selected from xanthophylls, carotenes and chlorophylls, preferably the colorant is selected from xanthophylls and carotenes, more preferably the colorant is xanthophylls.

24. 23. The method of claim 22, wherein the colorant is astaxanthin or a derivative thereof.

25. The coloring agent may be selected from the group consisting of carotenoids, melanins, flavins, phenazines, quinones, monascin, violacein, indigo, anthraquinones, naphthaquinones, dihydroxynaphthalene melanins, flavins, monascorbamine, lycopene, ankaflavin, chrysophanol, cynodontin, helminthosporin, trithysporin, erythroglaucin, riboflavin, rubropunctatin, and the like, selected from the group consisting of Monascus species, Xanthophyllomyces dendrorhous, Penicillium oxalicum, Ashbya gossypii, Blakeslea trispora, Erwinia uredovora, Rhodotorula mucilaginosa, and Fusarium 20. The method of claim 19, wherein the pigment is selected from pigments of microbial origin, such as sporotrichioides.

26. The method of claim 1 , wherein the at least one additive that produces color in the composition comprises a colored microorganism.

27. 27. The method of claim 26, wherein the pigmented microorganism comprises a fungus, preferably a Rhodotorula fungus.

28. 27. The method of claim 26, wherein the pigmented microorganism is a microorganism selected from red algae, green algae, brown algae, and cyanobacteria.

29. 27. The method of claim 26, wherein the pigmented microorganism is a CO2-fixing microorganism.

30. The CO2-fixing microorganisms may be selected from the group consisting of CO2-fixing microorganisms produced by culturing the at least one fungal strain in step c) or e) for their growth. ２ 30. The method of claim 29, wherein

31. The method of claim 1 , wherein the at least one additive that produces color in the composition comprises a lignocellulosic liquid extract.

32. 32. The method of claim 31 , wherein the lignocellulosic liquid extract is selected from extracts of brewer's grains, cereal bran, wheat bran, cocoa, and coffee, preferably the lignocellulosic liquid extract is selected from extracts of brewer's grains, cereal bran, and wheat bran, more preferably the lignocellulosic liquid extract is selected from extracts of brewer's grains and wheat bran.

33. 32. The method of claim 31 , wherein the lignocellulosic liquid extract is selected from an extract of wheat bran.

34. 2. The method of claim 1, wherein the supplementation in step (d) is carried out during the lag phase, acceleration phase, logarithmic phase, deceleration phase, or stationary phase of the biomass growth, and preferably, the supplementation in step (d) is carried out during the logarithmic phase of the biomass growth.

35. 2. The method of claim 1, wherein the supplementation in step (d) is carried out during the logarithmic phase, and preferably the culturing is carried out as a submerged fermentation carried out as a continuous process.

36. The method of claim 1, further comprising recovering the supernatant in step (f).

37. A composition obtainable according to the method of claim 1.

38. 38. The composition of claim 37, wherein the composition comprises at least 15 mg of colorant per 100 g of the fungal biomass, preferably at least 30 mg of colorant per 100 g of the fungal biomass.

39. 38. The composition of claim 37, wherein the composition comprises 0.01 to 5 wt.-%, preferably 0.1 to 5 wt.-%, more preferably 0.1 to 2.5 wt.-%, even more preferably 0.1 to 1 wt.-%, and most preferably 0.1 to 0.5 wt.-% of said colorant.

40. 40. The composition of any one of claims 37 to 39, wherein the at least one additive that produces color in the composition is irreversibly bound to the fungal mycelium.

41. A food product comprising the composition of any one of claims 37 to 39.

42. 42. The food product of claim 41, further comprising additives selected from preservatives, antioxidants and acidity regulators, thickeners, stabilizers and emulsifiers, pH adjusters and anti-caking agents, flavor enhancers, improvers, stabilizers, thickeners, colorants, and / or glazing agents and sweeteners.

43. 42. The food product of claim 41, wherein the food product comprises 0.0035 to 5 wt.%, preferably 0.01 to 5 wt.%, more preferably 0.01 to 2.5 wt.%, even more preferably 0.035 wt.% to 0.5 wt.%, even more preferably 0.05 to 0.35 wt.% of the colorant composition of the present invention.

44. Use of a composition according to any one of claims 37 to 39 in the manufacture of a food product.

45. 37. A supernatant obtainable according to claim 36.

46. 46. ​​The supernatant of claim 45, characterized in that the supernatant contains at least 0.1% by weight of filamentous fungi.

47. 47. Use of the supernatant of claim 45 or 46 in the manufacture of a food product.

48. 48. The use according to claim 47, wherein the food product is a beverage, preferably kombucha.

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