Production of a fungal fermentation medium from beer brewing lees

Abstract

The present invention relates to a method for producing a fungal fermentation medium from beer brewing spent grain (BSG) and a fermentation medium obtainable thereby, a method for producing fungal biomass by submerged fermentation of at least one fungal strain and fungal biomass obtainable thereby, and a food obtainable by using the instant fungal biomass of the present invention.

Description

Technical Field

[0001] The present invention relates to a method for producing a fungal fermentation medium from brewer's spent grain (BSG) and a fermentation medium obtainable thereby, a method for producing fungal biomass by submerged fermentation of at least one fungal strain and a fungal biomass obtainable thereby, and a food obtainable by using the instant fungal biomass of the present invention.

Background Art

[0002] In recent years, the production of food from animals has attracted attention due to its unsustainability and growing concerns about animal welfare. In relation to climate change, many plant-based meat alternatives have emerged with the aim of reducing CO2 emissions and alleviating animal suffering. However, these products are currently produced from three major monocrops (soybeans, peas, and rice), the cultivation of which requires a lot of land and water, is highly dependent on chemical agents (insecticides and fertilizers), and generates a lot of waste because only the proteins isolated from these crops are used in the production of meat alternatives. In addition, these isolates have a strong bitter taste and no inherent texture, and thus their use in food requires additional processing steps and the addition of additional ingredients including, but not limited to, flavoring agents, texturizing agents, and / or coloring agents. Therefore, plant-based alternatives are not necessarily healthy, and their production causes other environmental problems such as deforestation, a significant decline in biodiversity, soil pollution, and / or water pollution. It should be noted that the present inventors can use the above waste by-products in the processes described herein, and thus contribute to the improvement of the sustainability of these plant-based processes.

[0003] The production of food using fermentation processes seems to address some of these drawbacks. It allows for better use of land as fermenters can be expanded vertically and enables local food production in urban or rural areas. Furthermore, they require less water per kilogram of product than plant proteins, and with the continuous development and improvement of filtration and treatment technologies, this water can be reused in the process. The production of fungal mycelium as a new food using novel fermentation processes is disclosed herein, and the growth medium as well as the final product are at least partially produced using lignocellulosic materials such as industrial by - products and / or agricultural by - products as raw materials. In that sense, the processes described herein contribute to the effort to build a circular economy where industrial, food, and agricultural waste is minimized and resources are maximized. Another advantage of fungal fermentation over the production of conventional plant isolates is the resulting raw material - the fungal biomass, which naturally already has the desired fibrous texture and provides a balanced nutritional profile with not only complete proteins but also dietary fiber, vitamins, and micronutrients that offer healthy products to consumers. In particular, the use of mycelium isolated from the fruiting bodies of known edible mushrooms further provides a typical mushroom flavor characteristic of this group, which varies slightly between species (e.g., morel, truffle, or button mushroom), enabling the production of clean and delicious products with a very short list of ingredients.

[0004] The increasing demand for sustainable food supply chains and the management of GHG emissions by seeking food alternatives is a major global issue. The use of renewable waste lignocellulosic biomass as a raw material for food production creates a more environmentally conscious roadmap for achieving environmentally and economically sustainable processes.

[0005] Brewers' spent grain (BSG) is a lignocellulosic waste generated as a by-product by the brewing industry. BSG is obtained as a solid residue after wort production in the brewing process. The product is initially wet and has a short shelf life, but can be dried and processed in various ways. BSG, being a lignocellulosic material, is rich in carbohydrates (hexoses, pentoses, and lignin), and thus has value in utilizing the sugars locked within its structure. The use of BSG as a by-product reduces the problems associated with its disposal in the environment and contributes to a circular economy.

[0006] As is apparent to those skilled in the art, the carbohydrates available in BSG exist in the form of dietary fiber, cellulose, and hemicellulose, and thus it is recognized that it is difficult to obtain such carbohydrates from BSG.

[0007] German Patent No. 10201410884 describes a process for deodorizing lignin, which includes a step of extracting a lignocellulosic substrate with a supercritical fluid or a supercritical fluid mixture.

[0008] German Patent No. 102016110653 relates to a food / fermented product containing fungal mycelia.

[0009] Chinese Patent No. 101838673(A) discloses the fermentation of fungi of the family Basidiomycota in a liquid fermentation medium supplemented with rice distillers' grains.

[0010] Chinese Patent No. 1078872(A) discloses a method for preparing a beverage comprising culturing fungi in a fermentation medium containing, among other components, a distillation residue.

[0011] International Publication No. 2017 / 208255(A1) relates to a method for preparing an edible fungus (of the phylum Ascomycota) by culturing in a medium containing a distillation residue.

[0012] International Publication No. WO 2002 / 090527 (A1) relates to a method for preparing edible fungi (e.g., Fusarium species).

[0013] International Publication No. WO 2017 / 181085 (A1) discloses a specific method for producing fungal mycelia.

[0014] International Publication No. WO 2019 / 046480 (A1) relates to the preparation of edible filamentous fungi by growing filamentous fungal biomass.

[0015] Russian Patent Application Publication No. 2006 / 126554 relates to a method for producing food and feed biomass on a nutrient medium based on waste from a distillery's distillation waste liquid, which includes continuous cultivation of baker's yeast Saccharomyces cerevisiae and an edible basidiomycete selected from the group including, for example, especially Pleurotus ostreatus and Pleurotus pulmonarius.

[0016] U.S. Patent No. 5,846,787 discloses a process for treating cellulosic materials.

[0017] Papadaki (doi:10.3390 / microorganisms7070207) discloses the cultivation of Pleurotus species (P. pulmonarius and P. ostreatus) by solid-state fermentation and semi-liquid fermentation using grape pomace as a by-product.

[0018] Kim Min-Keun et al. (Korean Journal of Mycology, DOI:10.4489 / KJM.2012.40.1.049) disclose the development of an optimal medium for the mycelial culture of Pleurotus eryngii using a hot water extract of the raw material. The described process is solid-state fermentation for the production of fruiting bodies, and steam is not used.

[0019] Platt M.W. et al. (Eur. J. Appl. Microbiol. Biotechnol vol. 17, pages 140 - 142, 1983) disclose an increase in the decomposition of straw by Pleurotus ostreatus sp. ‘florida’. The disclosed process is solid-state fermentation.

[0020] Beltran-Garcia M.J. et al. (Revista de la Socidad Quimica de Mexico, vol 45, pages 77 - 81, 2001) disclose that lignin degradation products from corn stalks significantly enhance the radial growth of basidiomycete mushroom mycelium, which is carried out as solid-state fermentation.

[0021] Chinese Patent No. 108 203 693(A) discloses a specific Rhizopus oryzae species culture medium.

[0022] Korean Patent No. 2013 / 0057507 discloses a specific culture method for Cordyceps militaris.

[0023] Spanish Patent No. 2’370’215 discloses a specific means for fungal culture.

[0024] Sidana Arushdeep et al. (Chinese Journal of Biology, vol 2014, pages 1 to 5) disclose sugarcane bagasse as a potential medium for fungal culture.

[0025] U.S. Patent No. 9,206,446 discloses a specific extraction method from plant biomass.

[0026] Kemppainen et al. (Appl. Biochem. Biotechnol, 16 April 2016, doi:10.1007 / s12010 - 016 - 2085 - 9) disclose a specific aspect of steam explosion of spent beer grains.

[0027] Parchami et al. (Bioresource Technology 337 (2021) 125409 - doi:10.1016 / j.biortech.2021.125409) disclose certain aspects of the recovery of starch and protein from beer brewing spent grains using hydrothermal pretreatment and their conversion to edible fungi.

Summary of the Invention

[0028] Means and methods for utilizing lignocellulosic materials, preferably agricultural and / or industrial waste, herein industrial by - products and / or agricultural by - products, particularly beer brewing spent grains, are particularly desirable as they are cost - effective and more sustainable. A method leading to the production of fungal biomass having an amino acid composition reflecting that of a complete protein according to the FAO definition, and thus to food, is even more particularly desirable (www.fao.org, https: / / en.wikipedia.org / wiki / Complete_protein).

[0029] A method for the production of fungal biomass that is resistant to contamination by other microorganisms, such as bacteria, is even more particularly desirable. Thus, a fungal fermentation medium resistant to bacterial contamination that can be obtained by using lignocellulosic materials, preferably industrial by - products and / or agricultural by - products (i.e., waste), particularly beer brewing spent grains, is particularly desirable.

[0030] The object of the present invention was to provide improved means and methods for the production of fungal - based foods, a method for the production of a fungal fermentation medium from lignocellulosic materials, preferably agricultural by - products and / or industrial by - products, and means and methods for the production of fungal biomass for use in the production of fungal - based foods.

[0031] The inventors have surprisingly found that beer brewing spent grain can be extracted according to the method disclosed herein, and (optionally, supplementing the extract with at least one non-carbohydrate nutrient for fungal culture) a fungal fermentation medium with a high sugar content where the sugar content is mostly C5-complex sugars can be obtained. Thus, since C5-complex sugars cannot support the growth of most bacteria and are therefore resistant to contamination, the medium is fungal-specific. Notably, although the carbohydrates available in BSG are present in the form of dietary fiber, cellulose, and hemicellulose which make the extraction process more difficult, they could be extracted according to the method of the present invention.

[0032] Thus, the problems described herein are solved by the embodiments described below and characterized in the claims.

[0033] The present invention is summarized in the following embodiments.

[0034] In a first embodiment, the present invention is a method for producing a fungal fermentation medium from beer brewing spent grain (BSG), the method comprising: (a1) extracting C5-sugars from the lignocellulosic material contained in BSG by steam pretreatment, followed by a washing step with liquid water at a temperature of 50 °C or lower; and (b) combining the extract thus obtained with at least one non-carbohydrate nutrient for fungal culture.

[0035] In a second embodiment, the present invention is a method for producing a fungal fermentation medium from beer brewing spent grain (BSG), the method comprising: (a2) extracting C5-sugars from the lignocellulosic material contained in BSG via a liquid extraction treatment with water at a temperature of 145 °C to 155 °C and / or for a time up to a maximum of 70 minutes, preferably up to a maximum of 50 minutes, preferably at a pressure of 30 - 50 bar; and (b) combining the extract thus obtained with at least one non-carbohydrate nutrient for fungal culture.

[0036] In the third embodiment, the present invention relates to a fungal fermentation medium that can be obtained according to a method for producing a fungal fermentation medium from the spent beer grains of the present invention.

[0037] In the fourth embodiment, the present invention relates to the use of the medium of the present invention in fungal culture.

[0038] In the fifth embodiment, the present invention relates to a method for producing fungal biomass, the method comprising a step of submerged fermentation of at least one fungal strain using the fungal fermentation medium of the present invention.

[0039] In the sixth embodiment, the present invention relates to fungal biomass produced according to a method for producing the fungal biomass of the present invention.

[0040] In the seventh embodiment, the present invention relates to the use of the fungal biomass of the present invention in the production of fungal-based foods.

[0041] In the eighth embodiment, the present invention relates to fungal-based foods prepared using the fungal biomass of the present invention.

Brief Description of the Drawings

[0042]

Figure 1

Figure 2

Figure 3

Figure 4

Best Mode for Carrying Out the Invention

[0043] The present invention will be described in detail below. It should be understood that all disclosed features can be combined with each other unless otherwise specified.

[0044] In one embodiment, the present invention relates to a method for producing a fungal fermentation medium from beer brewing spent grain (BSG).

[0045] Beer brewing spent grain is preferably understood as the residue or by - product of the brewing industry. Preferably, the spent grain is the material remaining after the grinding process and preferably has a dry matter content of 10% - 30%. However, the dry matter content described herein is not meant to be limiting, and those skilled in the art recognize that the dry matter content can be increased in the pretreatment, for example, by compression, by drying, or by other methods known to those skilled in the art. Further, spent grain from other industries (for example, spent grain obtained as a by - product of food production) can also be used within the scope of the present invention.

[0046] Preferably, the beer brewing dregs suitable for use in the method of the present invention are characterized by a particle size distribution having a maximum value of 0.3 to 1 mm, preferably a particle size called the coarse particle size by those skilled in the art, preferably a maximum value of 0.4 to 0.8 mm. Preferably, the particle size of the beer brewing dregs is determined in the sieving process that results in the production of the beer brewing dregs. Preferably, the particle size distribution includes a maximum value of 2.0 to 4.0 mm and a second maximum value of 1.0 to 2.0 mm without pretreatment. When mechanical treatment is performed, a particle size distribution including a single maximum value of 1.0 mm or less is used. Thus, as discussed in the literature (Ozturk et al., J. Inst. Brew. 108(1):23-27, 2002), the beer brewing dregs may be sieved through a set of sieves having apertures of 850, 425, and 212 μm during grinding. Depending on the fraction, the beer brewing dregs preparation may be regarded as coarse grains (425 - 850 μm), medium grains (212 - 425 μm), and fine grains (less than 212 μm). As demonstrated in the Examples section, those skilled in the art may also be in a position to determine the particle size distribution by using a set of sieves different from those disclosed above. It is preferable to use a set of sieves conforming to ASTM standards. It should be further understood that those skilled in the art are also in a position to implement the method of the present invention for other particle size distributions, for example, a particle size distribution including a maximum value of 2.0 to 4.0 mm. Those skilled in the art further recognize that the brewing dregs have a particle size distribution including a maximum value of 2.0 to 4.0 mm and a second maximum value of 1.0 to 2.0 mm without any further mechanical (pre)treatment. When mechanical treatment is performed, generally, the resulting particle size distribution will include a single maximum value of 1.0 mm or less.

[0047] Preferably, BSG contains 20% - 25% w / w cellulose (preferably 22%), 23 - 28% w / w hemicellulose (preferably 25.8%), and / or 20% - 30% w / w protein (preferably 25% w / w protein). These values are understood to refer to the content of BSG with respect to its dry mass.

[0048] The method of the present invention is disclosed and described herein in relation to beer brewing lees. However, it is contemplated by those skilled in the art that the method may also be applicable to any other lignocellulosic material. The lignocellulosic material is preferably defined herein as a material containing dry plant matter. Preferably, the lignocellulosic material contains cellulose, hemicellulose and lignin. Preferably, at least one lignocellulosic material is at least one industrial by-product and / or agricultural by-product as defined herein. More preferably, the lignocellulosic material is preferably solid.

[0049] Examples of lignocellulosic materials include brewing lees, cereal bran, cotton, cottonseed hulls, bagasse, cocoa husks, cocoa, cocoa sheaths, sunflower, peanuts, hazelnuts, palm oil, cotton and press cakes from olives, husks and shells from nuts, grass and leaf waste, wood chips, coffee powder, coffee husks, coffee silver skin, rapeseed, and by-products from the soybean industry such as soybean pulp ("okara").

[0050] Preferred lignocellulosic materials include brewing lees, cereal bran (also called wheat bran) and press cakes. In a particular embodiment, a particularly preferred lignocellulosic material is cereal bran. The water used for pre-hydrolysis of cereal bran with steam preferably contains a dilute base, for example, the base at 1% w / w or less. A concentration up to 0.2% w / w, particularly a concentration of 0.2% w / w of NaOH is particularly suitable. Alternatively, a concentration of 0.2% w / w to 0.8% w / w, more preferably a concentration of 0.6% w / w to 0.8% w / w, particularly a concentration of 0.7% w / w to 0.8% w / w of NaOH is particularly suitable. 0.7% w / w of NaOH is particularly suitable, and more preferably, 0.8% w / w of NaOH is particularly suitable. In an alternative embodiment, 0.2% w / w of NaOH is particularly suitable.

[0051] Therefore, in the method of the present invention, the beer brewing by-products can be replaced with by-products selected from beer lees, brewing by-products (from industries other than beer brewing), cereal bran, bagasse, cotton and hydraulic press cakes from sunflowers, husks and shells from hazelnuts and nuts, grass and leaf waste, wood chips, coffee powder, coffee husks, coffee silver skin, rapeseed, and by-products from the soybean industry such as soybean pulp ("okara"), banana leaves, banana peels, chicory roots, cassava peels, citrus pulp, cocoa, cocoa bean husks, cocoa mucilage, cocoa sheath husks, coconut fiber, coconut husks, coconut endocarp, coffee pulp, corn cobs, corn stover, cotton, cottonseed oil cake, cottonseed, hemp, hop lees, pea by-products, peanut husks, peanut oil cake, peanuts, raw potato peels, potato tubers, eucalyptus bark, lantana weeds, switchgrass, rice bran, rice husks, rice straw, sugar beet lees, sugar beet pulp, sawdust, sugarcane bagasse wet, walnut endocarp, wheat bran, wheat distillers' grains, wheat germ, wheat straw, lupinus seeds, chickpea bran, chickpea sheath husks, chickpea straw, olive waste, grape pomace, pear pulp, sorghum bran, sorghum germ, sorghum stalks, sorghum straw, sunflower waste, and tea waste. Further, those skilled in the art would also consider that in the method of the present invention, the beer brewing by-products can be replaced with the following by-products: timothy grass, pine, coconut palm, apple, apricot, barley lees, broccoli, cabbage, carrot, turnip, eggplant, kiwi, melon, alfalfa, pineapple, pomegranate, plum, watermelon, zucchini, asparagus, beet, cauliflower, garlic, onion, pumpkin, squash and / or the skin or waste or pulp or pomace of tomatoes.

[0052] In one embodiment, a method for producing a fungal fermentation medium from beer brewing by-products (BSG) comprises: (a1) extracting C5-sugars from the lignocellulosic material contained in BSG by steam pretreatment, followed by a washing step with liquid water at a temperature of 50 °C or lower; and (b) combining the extract thus obtained with at least one non-carbohydrate nutrient for fungal culture.

[0053] The C5-sugars defined herein preferably refer to a fraction in which at least 80% w / w of the total sugar content consists of pentoses (sugars including polysaccharides composed of sugar subunits with 5 carbon atoms). It should be noted that the C5-sugars defined as a fraction containing sugars herein may contain other sugars, particularly C6-sugars (sugars having 6 carbon atoms, also called hexoses), as monomers, and / or may be contained within polysaccharides and / or oligosaccharides. Therefore, other sugars besides pentoses may also be extracted in step (a1) or step (a2) of the method of the present invention.

[0054] According to the present invention, the extraction conditions, particularly the extraction temperature, pressure, and time, are set such that preferably at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of the saccharides contained in the lignocellulosic material in the beer brewing residue are recovered by aqueous extraction. As will be understood by those skilled in the art, the extraction time may depend on further conditions, particularly the reactor applied (especially in the context of available agitation as discussed herein), and the specific type of material used in the extraction.

[0055] In step (a1), preferably, the steam pretreatment is carried out with a severity factor of 2.9 to 3.3.

[0056] The severity factor is also called log 10 (Ro), and Ro is defined herein as follows.

[0057]

Equation

[0058] The steam pretreatment in step (a1) may be carried out with a severity coefficient of 2.9 to 3.0, 3.0 to 3.1, 3.1 to 3.2, and / or 3.2 to 3.3. Preferably, the steam pretreatment in step (a1) may be carried out with a severity coefficient of 3.1 to 3.3, and more preferably, the steam pretreatment in step (a1) may be carried out with a severity coefficient of 3.1 to 3.2.

[0059] In one embodiment, the steam pretreatment in step (a1) may be carried out with a severity coefficient of 2.0 to 4.0, more preferably 2.5 to 3.3, and even more preferably 2.9 to 3.3.

[0060] In one embodiment, the steam pretreatment in step (a1) may be carried out with a severity coefficient of 2.6 to 3.0 or 3.1 to 3.3.

[0061] During the steam pretreatment in step (a1), the lignocellulosic material contained in BSG may be brought into contact with steam at a temperature of 130°C to 180°C. The steam pretreatment in step (a1) is preferably carried out at a temperature of 160 to 180°C, more preferably at a temperature of 165 to 175°C. Therefore, the steam pretreatment in step (a1) of the present invention may be carried out at 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179 or 180°C. Preferably, during the steam pretreatment in step (a1), the lignocellulosic material contained in BSG is brought into contact with steam for a time up to a maximum of 30 minutes, preferably up to a maximum of 15 minutes. The steam pretreatment in step (a1) is preferably carried out for at least 5 minutes. Preferably, during the steam pretreatment in step (a1), the lignocellulosic material contained in BSG is at a temperature of 165°C to 175°C and / or for a time up to a maximum of 15 minutes, preferably up to a maximum of 12.5 minutes, more preferably up to a maximum of 10 minutes, even more preferably up to a maximum of 7.5 minutes, even more preferably up to a maximum of 5 minutes, even more preferably up to a maximum of 2.5 minutes, even more preferably up to a maximum of 1 minute, in contact with steam. Therefore, within the scope of the present invention, the extraction in step (a1) may preferably be carried out for about 7.5 minutes, about 5 minutes, about 2.5 minutes or about 1 minute. More preferably, during the steam pretreatment in step (a1), the lignocellulosic material contained in BSG is brought into contact with steam at a temperature of 165°C to 175°C for a time up to a maximum of 15 minutes. More preferably, during the steam pretreatment in step (a1), the lignocellulosic material contained in BSG is brought into contact with steam at a temperature of 165°C to 175°C for a time up to a maximum of 10 minutes.Even more preferably, during the steam pretreatment in step (a1), the lignocellulosic material contained in the BSG is contacted with steam at a temperature of 165°C to 175°C for a time of up to 7.5 minutes, even more preferably up to 5 minutes, even more preferably up to 2.5 minutes, even more preferably up to 1 minute. Thus, as described herein, the extraction in step (a1) may thus preferably be carried out for about 7.5 minutes, about 5 minutes, about 2.5 minutes, or about 1 minute. Preferably, the temperature of 165°C to 175°C refers to a temperature of about 170°C, and more preferably, the temperature of 165°C to 175°C refers to a temperature of 170°C. Preferably, step (a1) is carried out at a pressure not exceeding 10 bar.

[0062] In one embodiment of the present invention, the water used for pre-hydrolysis with steam may contain a dilute acid, for example, such an acid of 1% w / w or less, preferably within the range of 0.8% w / w to 1% w / w, more preferably within the range of 0.8% w / w to 0.9% w / w. Preparations of H2SO4 of 0.2% w / w, or 0.4% w / w, or 0.8% w / w or 0.9% w / w are particularly suitable. However, embodiments in which the water contains up to 2% w / w of such an acid, for example, H2SO4, are also encompassed by the present invention. Preparations of H2SO4 in the range of 0.4% w / w to 1.6% w / w, preferably in the range of 0.6% w / w to 1.6% w / w, more preferably in the range of 0.7% w / w to 1.5% w / w are particularly suitable. In a specific embodiment, the concentration of the acid (preferably H2SO4) is 0.4 to 1% w / w, more preferably 0.4 to 0.9% w / w, still more preferably 0.5 to 0.9% w / w. In a specific embodiment, the concentration of the acid (preferably H2SO4) is 1.1 to 1.6% w / w. This is particularly applicable to embodiments in which the lignocellulosic material is beer brewing spent grains.

[0063] In one embodiment of the present invention, the water used for pre-hydrolysis with steam may contain a dilute base, for example, the base at 1% w / w or less. NaOH at a concentration in the range of 0.2% w / w to 0.8% w / w, more preferably in the range of 0.6% w / w to 0.8% w / w, particularly preferably in the range of 0.7% w / w to 0.8% w / w is particularly suitable. 0.7% w / w of NaOH is particularly suitable, and more preferably, 0.8% w / w of NaOH is particularly suitable. In an alternative embodiment, 0.2% w / w of NaOH is particularly suitable.

[0064] In an alternative embodiment of the present invention, the water used for pre-hydrolysis with steam is replaced with a phosphate buffer having a pH in the range of 4 to 6, more preferably in the range of 4.5 to 5.5.

[0065] In step (a1), following the steam pretreatment, a washing step with liquid water at a temperature of 50°C or lower is performed. The washing step with liquid water is preferably performed using water at a temperature of 40°C or lower, more preferably 30°C or lower, still more preferably 25°C or lower. Preferably, the liquid water used in the washing step is at room temperature, i.e., 20°C to 25°C, preferably at a temperature of about 22°C, more preferably 22°C. In an alternative preferred embodiment, the liquid water used in the washing step is in the range of 30°C to 50°C (especially 30°C to 49°C, preferably 30°C to 48°C), still more preferably 40°C to 50°C (especially 40°C to 49°C, preferably 40°C to 48°C). Therefore, the washing step may be performed using liquid water at a temperature of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50°C. The washing time is preferably at least 15 minutes, more preferably at least 20 minutes, still more preferably at least 25 minutes, and even more preferably at least 30 minutes. Preferably, the time or duration of the washing step is in the range of 15 to 30 minutes, still more preferably 20 to 30 minutes. Therefore, the duration of the washing step may be about 20 minutes, about 25 minutes or about 30 minutes. Depending on the desired result, a time of less than 15 minutes is also possible and is also considered to be included in the present invention.

[0066] Preferably, as described above, the washing step with liquid water at a temperature of 50°C or lower may be performed simultaneously or sequentially with ultrasonic treatment.

[0067] Preferably, the washing step according to the present invention is performed using water with a pH of 1 to 8, preferably 1 to 5, more preferably 1 to 4, still more preferably 1 to 3, and even more preferably 1 to 2. Alternatively, the washing step can be performed using water with a pH of 10 to 14, more preferably 11 to 13.5, still more preferably 12 to 13.5, and even more preferably 12.5 to 13.5. H3O+ or OH - The method of calculating the required corresponding amount of a specific acid or base to be added to reach a specific concentration, i.e., a specific pH value, is well known to those skilled in the art.

[0068] Preferably, in a method for producing a fungal fermentation medium from brewer's spent grain (BSG), the BSG is characterized by a final moisture content of 50% to 75% by weight. Accordingly, the final moisture content may be achieved by dehydrating the BSG by pressing the material to a final moisture content of 50 to 75% by weight using methods known to those skilled in the art. However, this does not mean that it is to be construed as limiting, as it is further apparent to those skilled in the art that other means and methods for dehydrating the BSG may also be applicable here.

[0069] Accordingly, in a preferred embodiment, a method for producing a fungal fermentation medium from brewer's spent grain (BSG) further comprises a step of dehydrating the BSG by pressing the material to a final moisture content of 50 to 75% by weight, and the step of dehydrating precedes step (a1). This step is conventional and known to those skilled in the art and can be carried out, for example, by pressing the by - product using a screw press (or any other suitable press). Alternatively, the lignocellulosic material, preferably the lignocellulosic system, may be dried, for example, by using a fluidized bed dryer. A convection oven may also be used for this purpose. Preferably, in the method of the present invention, the water thus obtained is discarded and not further used in the method of the present invention.

[0070] The method for producing a fungal fermentation medium may further comprise a further step of pretreating the brewer's spent grain. Preferably, the pretreatment of the brewer's spent grain includes changing the particle size or other mechanical properties of the spent grain. For example, the pretreatment of the spent grain may include grinding the spent grain. This step is conventional and known to those skilled in the art.

[0071] The residue of the beer brewing lees obtained after the process (a1) can be further subjected to a pretreatment, for example, by grinding, crushing, micronizing, etc., for example, by decomposing the particles, for example, to increase its available surface area, which is further obvious to those skilled in the art. Such a process disclosed herein as an optional process is known to those skilled in the art. The said process of pretreatment can also be called mechanical pretreatment. In particular, for example, a pretreatment process including the destruction of particles by grinding, crushing, micronizing can also be called mechanical treatment or mechanical pretreatment. The pretreatment process is described herein after the process (a1), but can also be carried out at other stages of the method. For example, the beer brewing lees can be subjected to a mechanical pretreatment process as contemplated herein before being subjected to the process (a1). Preferably, each of these mechanical pretreatment procedures can be carried out using a crusher or a disk refiner. It will be recognized by those skilled in the art that such a crusher or disk refiner is preferably arranged at the extractor outlet (the extractor is understood herein as a reactor in which the extraction process described herein is carried out, and the extractor outlet is defined as an opening in the cavity used to remove the solid residue after extraction). The said crusher or the said disk refiner is configured to treat the beer brewing lees so that the BSG surface area is further increased (i.e., the particle size is further reduced) for higher sugar extraction in the washing process.

[0072] In step (b) of the method for producing the fungal fermentation medium of the present invention, the extract obtained in step (a1) is combined with a non-carbohydrate nutrient for at least one fungal culture.

[0073] Accordingly, the aqueous extract of step (a1) obtained according to the present invention can be further supplemented with a nitrogen source, a carbon source, trace elements, vitamins and / or a protein composition. The nitrogen source as defined herein is preferably selected from ammonia, urea, yeast extract, malt extract, corn steep liquor and peptone. More preferably, the nitrogen source is ammonia and / or urea. The carbon source is preferably selected from glucose, fructose, sucrose, lactose, maltose, xylose, galactose, dextrose, glycerol, and molasses, and more preferably the carbon source is glucose. Preferably, carbon sources other than those derived from BSG are not added to the medium of the present invention. Examples of the trace elements as defined herein may include iron(III) salts, copper(II) salts, zinc salts, manganese(II) salts, molybdenum salts and / or cobalt(II) salts. The vitamins as defined herein preferably include vitamins beneficial for the growth of fungi on the medium that can be obtained according to the method of the present invention, such as folic acid, riboflavin, pantothenic acid or biotin. The protein composition may be further used to supplement the aqueous extract of (a1) of the present invention. Preferably, the protein composition obtained from the protein separated preferably by aggregation or precipitation with CO2 from the aqueous extract of (a1) of the method of the present invention is preferably used in the method of the present invention.

[0074] Preferably, according to step (b), the extract of step (a1) is preferably supplemented with at least one nitrogen source as described above.

[0075] In step (b), it is more preferable that the extract of step (a1) is not substantially diluted. As used herein, the term "not substantially diluted" preferably means a dilution of 15% or less (which is preferably understood to mean that the final concentration of the extract at time point (a1) in the medium of step (b) does not decrease by more than 15% in step (b), more preferably a dilution of 10% or less). Thus, preferably, the said extract of step (a1) constitutes at least 85% v / v of the total medium obtained in step (b), preferably, the said extract of step (a1) constitutes at least 90% of the total medium obtained in step (b), more preferably, the said extract of step (a1) constitutes 90 - 99% v / v of the total medium obtained in step (b), even more preferably, the said extract of step (a1) constitutes 94 - 98% v / v of the total medium obtained in step (b).

[0076] Preferably, in the method for producing the fungal fermentation medium of the present invention, step (b) includes the addition of a solid substance, preferably the addition of a solid nitrogen source, as known to those skilled in the art.

[0077] The inventors have preferably found that the medium obtainable by the method of the present invention, which is a method including step (a1), i.e., a method including steam pretreatment, is substantially free of fungal growth inhibitors such as hydroxymethylfurfural or furfural. Thus, the medium obtainable as described herein can preferably support fungal growth without a further dilution step to reduce the concentration of fungal growth inhibitors.

[0078] Those skilled in the art will recognize that when the medium obtainable according to the present invention is subjected to a post-treatment by removal of fungal growth inhibitors, the use of a dilution step, which means reducing their concentration to an acceptable level, can be avoided.

[0079] Further examples of fungal growth inhibitors that can be formed in the extraction process are weak acids (e.g., acetic acid, formic acid, levulinic acid), furans (furfural, hydroxymethylfurfural, 2-furoic acid) and / or phenols (vanillin, syringaldehyde, ferulic acid, coumaric acid, coniferyl alcohol, eugenol, acetovanillin, ferulic acid amide, coumaric acid amide).

[0080] The undesirable presence of furfural and / or hydroxymethylfurfural and / or other undesirable compounds can be avoided as described above. Further, undesirable furfural and / or hydroxymethylfurfural and / or other undesirable compounds can be removed, for example, by gas stripping, by heteroazeotropic distillation, or by liquid-liquid extraction. For example, in one embodiment, furfural and / or other fungal growth inhibitors may be removed by using a suitable membrane. Optionally, furfural can be recovered and used for other industrial applications. During the extraction described herein, the concentration of furfural does not exceed 0.6 g / L, preferably does not exceed 0.2 g / L, preferably does not exceed 0.15 g / L, more preferably does not exceed 0.1 g / L, still more preferably does not exceed 0.05 g / L, and most preferably does not exceed 0.01 g / L. Preferably, when the extraction is carried out according to the method of the present invention, the concentration of furfural in the final extract is as described herein.

[0081] Accordingly, the present invention further relates to embodiments in which, in the medium obtained before step (b), the concentration of furfural does not exceed 0.6 g / L, preferably does not exceed 0.2 g / L, preferably does not exceed 0.15 g / L, more preferably does not exceed 0.1 g / L, still more preferably does not exceed 0.05 g / L, and most preferably does not exceed 0.01 g / L.

[0082] In one embodiment of the method for producing a fungal fermentation medium from beer brewing spent grain (BSG) of the present invention, the method includes step (a2) which involves a liquid extraction treatment of BSG with water.

[0083] Therefore, in step (a2) of the method of the present invention, C5 sugars from the lignocellulosic material contained in BSG are extracted through the liquid extraction treatment with water.

[0084] Within the scope of the method of the present invention, the extraction in step (a2) for obtaining C5 may be carried out at a temperature of 140°C to 180°C. The extraction in step (a2) is preferably carried out at a temperature of 145°C to 175°C, more preferably at a temperature of 145°C to 170°C, even more preferably at a temperature of 145°C to 160°C, and even more preferably at a temperature of 145°C to 155°C. In a preferred embodiment of the present invention, the extraction may be carried out at a temperature of about 150°C. Therefore, within the scope of the present invention, the extraction in step (a2) may be carried out at 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179 or 180°C.

[0085] As those skilled in the art recognize, temperature and pressure are in a linear relationship. Within the scope of the present invention, the extraction in step (a2) for obtaining C5 sugars may be carried out at a pressure of 30 to 50 bar. Therefore, the extraction in step (a2) may be carried out at 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 bar. The extraction in step (a2) is preferably carried out at 50 bar.

[0086] Within the scope of the method of the present invention including step (a2), extraction is carried out to obtain C5 sugars from the lignocellulosic material contained in the BSG. Therefore, the extraction is carried out over a minimum time sufficient to obtain C5 sugars. Within the scope of the present invention, the extraction is carried out up to a maximum of 70 minutes. The extraction in step (a2) is preferably carried out up to a maximum of 50 minutes, more preferably up to a maximum of 30 minutes, more preferably up to a maximum of 25 minutes, more preferably up to a maximum of 15 minutes, more preferably up to a maximum of 10 minutes. Most preferably, the extraction in step (a2) is carried out up to a maximum of 5 minutes to obtain C5 sugars. Therefore, within the scope of the present invention, the extraction in step (a2) may preferably be carried out for a time of 5 to 15 minutes, more preferably for about 15 minutes, about 10 minutes or about 5 minutes. The extraction in step (a2) is preferably carried out for at least 5 minutes. Further, the extraction may be carried out at a pressure of 5 to 50 bar. The extraction is preferably carried out at a pressure of 10 to 50 bar, more preferably at a pressure of 30 to 50 bar. Preferably, the pressure and temperature are selected such that water remains in the liquid state under the conditions of the extraction in step (a2).

[0087] Preferably, step (a2) of the method of the present invention is carried out with a severity coefficient of 2.9 to 3.3. Therefore, step (a2) may be carried out with a severity coefficient of 2.9 to 3.0, 3.0 to 3.1, 3.1 to 3.2, and / or 3.2 to 3.3. Preferably, the steam pretreatment in step (a2) may be carried out with a severity coefficient of 3.1 to 3.3, more preferably, the steam pretreatment in step (a2) may be carried out with a severity coefficient of 3.1 to 3.2.

[0088] In one embodiment, step (a2) of the method of the present invention may be carried out with a severity coefficient of 2.0 to 4.0, more preferably with a severity coefficient of 2.5 to 3.3, and even more preferably with a severity coefficient of 2.9 to 3.3. An embodiment in which step (a2) is carried out at a severity coefficient of 2.9 to 3.3 and a temperature of 145°C to 175°C, preferably at a temperature of 145°C to 170°C, more preferably at a temperature of 145°C to 160°C, even more preferably at a temperature of 145°C to 155°C, and even more preferably at a temperature of about 150°C is particularly preferred. Thus, in this particularly preferred embodiment, step (a2) of the present invention may be carried out at a temperature of 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, or 155°C. In an alternative embodiment, step (a2) of the method of the present invention may be carried out at 130°C to 140°C. In this particular embodiment, the pH of the water used in step (a2) is preferably 1.0 to 3.0.

[0089] Alternatively, step (a2) of the method of the present invention is preferably carried out with a severity coefficient of 2.0 to 3.0.

[0090] In one embodiment of the present invention, the water used for liquid extraction with water may contain a dilute acid, for example, the acid at 1% w / w or less, preferably in the range of 0.8% w / w to 1% w / w, more preferably in the range of 0.8% w / w to 0.9% w / w. Preparations of H2SO4 at 0.2% w / w, or 0.4% w / w, or 0.8% w / w or 0.9% w / w are particularly preferred. However, embodiments in which the water contains the acid, for example, H2SO4, up to 2% w / w are also encompassed by the present invention. This is particularly applicable to embodiments in which the lignocellulosic material is spent beer grains. Preparations of an acid, preferably H2SO4, in the range of 0.4% w / w to 1.6% w / w, preferably in the range of 0.6% w / w to 1.6% w / w, more preferably in the range of 0.7% w / w to 1.5% w / w are particularly preferred. In a specific embodiment, the concentration of the acid (preferably H2SO4) is 0.4 to 1% w / w, more preferably 0.4 to 0.9% w / w, still more preferably 0.5 to 0.9% w / w. In a specific embodiment, the concentration of the acid (preferably H2SO4) is 1.1 to 1.6% w / w. This is particularly applicable to embodiments in which the lignocellulosic material is spent beer grains. It should be understood that this embodiment can be combined with the embodiments of the three preceding paragraphs that describe the preferred temperature and severity coefficient for the (a2) step.

[0091] In one embodiment of the present invention, the water used for liquid extraction with water may contain a dilute base, for example, the base at 1% w / w or less. NaOH at a concentration in the range of 0.2% w / w to 0.8% w / w, more preferably in the range of 0.6% w / w to 0.8% w / w, particularly in the range of 0.7% w / w to 0.8% w / w is particularly preferred. 0.7% w / w of NaOH is particularly preferred, and more preferably, 0.8% w / w of NaOH is particularly preferred. Alternatively, 0.2% w / w of NaOH is particularly preferred.

[0092] In an alternative embodiment of the present invention, the water used for the liquid extraction treatment is replaced with a phosphate buffer having a pH in the range of 4 to 6, more preferably in the range of 4.5 to 5.5.

[0093] Preferably, in the method of the present invention, the lignocellulosic material contained in the BSG extracted in step (a2) of the method is untreated, and preferably, the material is not dehydrated.

[0094] Preferably, the step (a2) of aqueous extraction of the lignocellulosic material according to the present invention, preferably industrial by-products and / or agricultural by-products, is carried out using water having a pH of 2.0 to 12.0, preferably 3.0 to 10.0, more preferably 4.0 to 8.0, and even more preferably 5.0 to 8.0. However, it is also preferred that the step (a2) of aqueous extraction of the lignocellulosic material according to the present invention, preferably industrial by-products and / or agricultural by-products, is carried out using water having a pH of 1.0 to 12.0, preferably 1.0 to 10.0, more preferably 1.0 to 8.0, and even more preferably 1.0 to 8.0. The pH value as understood herein is measured at a pressure of 1.0 bar and a temperature of 25 °C, even if the extraction itself is carried out under different conditions, as disclosed herein. Preferably, the pH is adjusted before the water is placed in contact with at least one lignocellulosic material, preferably industrial by-products and / or agricultural by-products. It is further understood herein that, as described herein, the addition of an acid or base to water to a final concentration exceeding 1% w / w should preferably be avoided.

[0095] More preferably, the aqueous extraction step (a2) of the lignocellulosic material that requires acid treatment, preferably the industrial by-products and / or agricultural by-products according to the present invention, is carried out using water with a pH of 1 to 5, more preferably a pH of 1 to 4, even more preferably a pH of 1 to 3, and still more preferably a pH of 1 to 2. This is particularly applicable to the embodiment where the lignocellulosic material is brewery spent grains. Alternatively, when using base treatment, in the aqueous extraction step (a2) of the lignocellulosic material that requires base treatment, preferably the industrial and / or agricultural by-products according to the present invention, it is preferably carried out using water with a pH of 10 to 14, more preferably a pH of 11 to 13.5, even more preferably a pH of 12 to 13.5, and still more preferably a pH of 12.5 to 13.5. This is particularly applicable to the embodiment where the lignocellulosic material is wheat bran. H3O + or OH - The method for calculating the specific concentration of, that is, the corresponding amount of the specific acid or base to be added required to reach a specific pH value is well known to those skilled in the art.

[0096] The aqueous extraction step (a2) can be carried out by any technical method known to those skilled in the art. For example, the aqueous extraction step (or each of the steps) can be carried out as a batch process. However, preferably, the aqueous extraction step (a2) of the method of the present invention is carried out as a continuous extraction process, as known to those skilled in the art.

[0097] Preferably, the batch extraction is carried out in a closed system, and the spent grains and water are charged only once at first. The reactor is preferably pressurized and remains closed until the mixture is cooled to at least 40°C. The continuous extraction is preferably carried out at the same rate of continuous supply and recovery of the raw materials, resulting in only a pressure drop when the material produced by steam explosion is recovered. In a semi-continuous process, the feed and the reaction mixture continuously enter and leave the system. The supply and recovery of the feedstock are not at the same rate, and the raw materials are supplied periodically.

[0098] As understood herein, as described in step (a1), the step of washing with water can also be carried out as a batch process or as a semi - continuous process. Thus, in one embodiment, as described herein, the step of washing with water in step (a1) is carried out as a batch process as described herein. Preferably, in the batch process, at least 50% of the C5 - sugars are recovered, more preferably at least 60% of the C5 - sugars are recovered. In one embodiment where the step of washing with water in step (a1) is carried out as a batch process as described herein but in the presence of dilute acid (up to 2% w / w), at least 70%, preferably at least 80%, more preferably at least 90% of the C5 - sugars are recovered. In one embodiment, as described herein, the step of washing with water in step (a1) is carried out as a semi - continuous process as described herein. Preferably, in the semi - continuous process, at least 80% of the C5 - sugars are recovered, more preferably at least 90% of the C5 - sugars are recovered, and even more preferably at least 96% of the C5 - sugars are recovered.

[0099] The method of the present invention for producing a fungal fermentation medium from beer - brewing spent grain (BSG) comprising step (a2) further comprises step (b), namely, combining the extract thus obtained with at least one non - carbohydrate nutrient for fungal culture. Step (b) in the embodiments described herein of the method is as described above, that is, as described with respect to the method of the present invention for the production of a fungal fermentation medium from beer - brewing spent grain comprising step (a1).

[0100] Preferably, in the method of the present invention, if applicable, sugars other than those present in the extract of step (a1) or the extract of step (a2) are not substantially added to the medium. "Substantially" means, in this specification, if applicable, preferably, no more sugar is added than an amount corresponding to 5% w / w of the sugar present in the extract of step (a1) or the extract of step (a2), more preferably, no more sugar is added than an amount corresponding to 1% w / w of the sugar present in the extract of step (a1) or the extract of step (a2), and even more preferably, no additional sugar is added to the sugar present in the extract of step (a1) or the extract of step (a2). Thus, preferably, in the method of the present invention, if applicable, sugars other than those present in the extract of step (a1) or the extract of step (a2) are not added to the medium.

[0101] Optionally, in the method of the present invention comprising step (a1) or in the method of the present invention comprising step (a2), the lignocellulosic material, preferably brewery spent grain, may be subjected to a further pretreatment step before being subjected to the method for producing the fungal fermentation medium of the present invention. Thus, the lignocellulosic material as used herein (e.g., as contained in brewery spent grain as described herein) can be pretreated according to a method selected from washing, solvent extraction, solvent swelling, grinding, milling, dilute acid pretreatment, hot water pretreatment, alkali pretreatment, lime pretreatment, wet oxidation, hydrothermal explosion, ammonia fiber explosion, organic solvent pretreatment, biological pretreatment, ammonia leaching, ultrasonic, electroporation, microwave, supercritical CO2, supercritical H2O, ozone, and gamma ray irradiation. Preferably, further pretreatment steps other than those described in step (a) are not included in the method of the present invention for producing the fungal fermentation medium.

[0102] As understood herein, in the method of the present invention including step (a1) or in the method of the present invention including step (a2), the extraction step (a1) or (a2) of extracting the lignocellulosic material contained in the beer brewing residue is carried out, where applicable, in a suitable reactor known to those skilled in the art. At least one lignocellulosic material, preferably an industrial by - product and / or an agricultural by - product, is preferably fed into the reactor with a solid input of 5 - 70% weight / volume (w / w), preferably 10 - 55% (w / w), and treated with water or steam. The solid input as understood herein is defined as the weight ratio of the dry solid by - product (the material fed in) to the total reaction volume (including the water used for extraction and at least one solid by - product), and is preferably expressed as a percentage. The weight of the material fed in is understood herein as the dry weight. The solid input required in the present invention depends on the material fed in and the reactor characteristics. As will be understood by those skilled in the art, the amount of the material fed in should preferably not affect the stirring and heat transfer inside the reactor. It also depends on the amount of the liquid extract that has to be recovered and its composition. As understood herein, the reactor can preferably be filled with dry lignocellulosic biomass (also called dry solid by - product) up to 55% w / w. It should be noted that in certain embodiments, alternatively, the reactor can preferably be filled with wet lignocellulosic biomass. As is known to those skilled in the art, the preferred input depends on the material, i.e., the lignocellulosic material, preferably an industrial by - product and / or an agricultural by - product.

[0103] Optionally, in the method of the present invention including step (a1) or in the method of the present invention including step (a2), the aqueous extract obtained in step (a1) or step (a2) may be subjected to further treatment. Treating the aqueous extract obtained in (a1) / (a2) before step (b) may preferably include separation of proteins from the aqueous extract of (a1) / (a2). Any separation method known to those skilled in the art and suitable for the purpose can be used in the method of the present invention. Preferably, the protein is separated from the aqueous extract preferably by aggregation or by precipitation with CO2, and preferably then mechanical separation is carried out, for example, by a decanter centrifuge. Optionally, the protein thus obtained can be further prepared as a solution containing 50% w / w or more of polypeptides and / or amino acids. Further optionally, the protein thus obtained is preferably hydrolyzed by using a proteolytic enzyme, particularly one selected from alkaline protease, papain, proteinase K, and trypsin. The proteolytic enzyme can be used at a concentration of 0.01% to 5% (w / w) (understood herein as the total concentration of all enzymes used herein) and / or at a temperature of 15 to 100 °C and / or for a time of 0.5 to 96 hours. The solution thus obtained containing the hydrolyzed protein (i.e., polypeptides and amino acids) can optionally be further used in step (b) of the method of the present invention. It should be further noted that further treatment of the aqueous extract obtained in step (a1) or step (a2) can remove any contaminants and / or fungal growth inhibitors.

[0104] As used herein, the term "polypeptide" includes proteins, peptides, and polypeptides, and the protein, peptide, or polypeptide may or may not be post-translationally modified. Post-translational modifications can be, for example, phosphorylation, methylation, and / or glycosylation. However, other post-translational modifications recognizable to those skilled in the art are also included.

[0105] Preferably, in the method for producing a fungal fermentation medium according to the present invention, the protein is not separated from the extract, i.e., from the product of the method. The inventors have found that it is beneficial to retain the protein as defined herein in the medium as a nitrogen source. As understood herein, the protein may comprise amino acids, peptides and / or proteins.

[0106] Preferably, step (a1) or step (a2) in the method for producing a fungal fermentation medium is characterized by a reactor solids input of 1 to 25%, preferably 1 to 15%, more preferably 4 to 14% w / w, even more preferably 8 to 14% w / w, even more preferably 10 to 14% w / w, even more preferably 12 to 14% w / w. In a particular embodiment, the reactor solids input is 5 to 15%.

[0107] As understood herein, the product of step (b) of the method of the present invention may be further processed. Optionally, the water contained in the product of step (b) can be removed, for example, by spray drying, drum drying, belt drying, or freeze drying to obtain a dried fungal fermentation medium, which can have an improved storage time as is known to those skilled in the art. Optionally, the fungal fermentation medium of the present invention may be further sterilized or pasteurized within the scope of the method of the present invention. Optionally, the fungal fermentation medium obtainable according to the method of the present invention may be further supplemented, for example, with salts (preferably sodium chloride, sodium nitrate, magnesium sulfate, calcium chloride, calcium carbonate, ammonium chloride, diammonium phosphate, ammonium sulfate, potassium phosphate, disodium phosphate, and / or monosodium phosphate), antibiotics, or water. The water used herein is preferably used to optimize the concentration of the components in the medium.

[0108] More preferably, in a method for producing a fungal fermentation medium from at least one lignocellulosic material of the present invention, preferably industrial by-products and / or agricultural by-products, the pH of the fungal fermentation medium obtainable can preferably be set to a desired value by adding a buffer. In the present specification, a citrate buffer system or a phosphate buffer system is particularly useful. Further, in a fermenter, the pH can be adjusted by adding an appropriate amount of urea, NaOH, ammonia, sulfuric acid, phosphoric acid, or hydrochloric acid.

[0109] Preferably, the fungal fermentation medium of the present invention contains a complex C5-sugar (also referred to as a complex C5-polysaccharide). As understood herein, the complex C5-sugar is preferably a C5-sugar in which at least 60%, at least 70%, at least 80%, or at least 90% is composed of molecules containing two or more sugar units.

[0110] Accordingly, preferably, the complex C5-polysaccharide constitutes at least 50% of the total sugars in the medium, preferably, the complex C5-polysaccharide constitutes at least 65% of the total sugars in the medium, and more preferably, the complex C5-polysaccharide constitutes at least 80% of the total sugars in the medium. It should be understood that the medium obtainable in the method of the present invention including step (a') does not necessarily meet this condition because the product of step (a') contains C6 sugars. Accordingly, preferably, the complex C5-polysaccharide constitutes at least 50% of the total sugars in the extract produced in step (a1) or step (a2), preferably, the complex C5-polysaccharide constitutes at least 65% of the total sugars in the extract, and more preferably, the complex C5-polysaccharide constitutes at least 80% of the total sugars in the extract.

[0111] Preferably, the medium obtainable according to the method of the present invention is suitable for culturing at least one fungal strain selected from Pezizales, Boletales, Cantharellales, Agaricales, Polyporales, Russulales, Auriculariales, Sordoriales and Hypocreales, preferably at least one fungal strain selected from P. pulmonarius, P. ostreatus, P. citrinopileatus, P. salmonostramineus, M. rufobrunnea, M. esculenta, M. angusticeps and M. deliciosa, preferably at least one fungal strain selected from P. pulmonarius and M. rufobrunnea.

[0112] Also included in the present invention are embodiments in which, after the steam treatment followed by washing with liquid water in step (a1), treatment with liquid water as in step (a2) of the obtained solid residue follows, and then step (b) follows only thereafter. Alternatively, after step (a2), step (a1) performed on the solid residue obtained after step (a2) may follow, and then step (b) may follow. Further included in the present invention is a method comprising step (a1) as defined herein and / or step (a2) as defined herein, followed by step (b) as defined herein. According to the inventors, performing two or more steps on the BSG (or the residue obtained in the respective preceding step) selected from step (a1) and step (a2) as described herein makes it possible to recover more sugars and avoid the mass production of fungal growth inhibitors.

[0113] In one embodiment, after the steam treatment step (a1) followed by washing with liquid water, another steam treatment step (a1) followed by washing with liquid water, which is performed on the solid residue obtained in the first step (a1), follows, and then step (b) follows only thereafter. Optionally, step (a') may be performed before step (b).

[0114] In one embodiment, after the step (a2) of liquid water treatment, another step (a2) of liquid water treatment is performed on the solid residue obtained in the first step (a2), and then only the step (b) follows. Optionally, the step (a') may be performed before the step (b).

[0115] In one embodiment, after the step (a1) of steam treatment followed by cleaning by liquid water treatment, another step (a2) of liquid water treatment is performed on the solid residue obtained in the first step (a1), and then only the step (b) follows. Optionally, the step (a') may be performed before the step (b).

[0116] In one embodiment, after the step (a2) of liquid water, another step (a1) of steam treatment followed by cleaning with liquid water is performed on the solid residue obtained in the first step (a2), and then only the step (b) follows. Optionally, the step (a') may be performed before the step (b).

[0117] In the method of the present invention, in the step (a1) or the step (a2), a solid lignocellulosic residue is generated. The residue may be further used in the method of the present invention. Therefore, preferably, the method for producing a fungal fermentation medium from beer brewing residue further includes a step (a') of enzymatically hydrolyzing the solid lignocellulosic residue obtained in the step (a1) or the step (a2) with cellulase and separating the liquid product of hydrolysis from the solid residue. Therefore, the lignocellulosic residue is charged into a second reactor (preferably at an input amount of 5 to 50% w / w), where it is hydrolyzed with cellulase. Preferably, the step (a') is performed at a temperature of 15 to 100 °C, more preferably at a temperature of 40 to 80 °C. Preferably, the step (a') is performed at a pH of 3.0 to 8.0, more preferably at a pH of 4.0 to 7.0. Preferably, the step (a') is performed for a time of 10 to 200 hours.

[0118] Preferably, the solid lignocellulosic residue generated in the step (a1) or the step (a2) does not undergo further treatment before being subjected to the step (a').

[0119] Preferably, the extract obtained in step (a') contains a C6 sugar. The C6 sugar is preferably defined as a hexose (i.e., a sugar containing a sugar unit of 6 carbon atoms) or an oligosaccharide and a polysaccharide composed of hexose units.

[0120] Preferably, the extract obtained in step (a') is mixed with the extract obtained in step (a1) or the extract obtained in step (a2). Preferably, the ratio of the extract of step (a2) to the mixed extract in step (a1) is not particularly limited. The ratio may depend on the requirements of specific medium production and is the result of the solid input of the reactor applied to each of these steps.

[0121] In a further embodiment, the present invention relates to a fungal fermentation medium obtainable in a method for producing a fungal fermentation medium of the present invention. The fungal fermentation medium of the present invention obtainable in a method for producing a fungal fermentation medium from at least one lignocellulosic material of the present invention, preferably industrial by-products and / or agricultural by-products, may further optionally contain a nitrogen source, a carbon source, trace elements, vitamins and / or a protein composition. The nitrogen source as defined herein is preferably selected from ammonia, urea, yeast extract, malt extract, corn steep liquor and peptone. More preferably, the nitrogen source is ammonia and / or urea. The carbon source is preferably selected from glucose, fructose, sucrose, lactose, maltose, xylose, galactose, dextrose, glycerol, and molasses, and more preferably the carbon source is glucose or xylose. However, it is preferred that carbon sources other than those obtained through the extraction of BSG are not added herein. Thus, the present invention preferably does not include the addition of further sugars other than those obtained in any one of steps (a1), (a2) and / or (a') of the method of the present invention. Examples of the trace elements as defined herein may include iron(III) salts, copper(II) salts, zinc salts, manganese(II) salts, molybdenum salts and / or cobalt(II) salts. The vitamins as defined herein preferably include vitamins beneficial for the growth of fungi on the medium obtainable according to the method of the present invention as defined herein.

[0122] Preferably, in the fungal fermentation medium of the present invention, substantially all sugars are derived from BSG. "Substantially" means, in the context of this specification, where applicable, preferably no more sugar is added than an amount corresponding to 5% w / w of the sugars present in the extract of step (a1) and / or the extract of step (a2) and / or the extract of step (a'), more preferably no more sugar is added than an amount corresponding to 1% w / w of the sugars present in the extract of step (a1) and / or the extract of step (a2) and / or the extract of step (a'), and even more preferably no additional sugar is added to the sugars present in the extract of step (a1) and / or the extract of step (a2) and / or the extract of step (a').

[0123] In the fungal fermentation medium of the present invention, preferably, the complex C5-polysaccharide constitutes at least 50% of the total sugars in the medium, preferably, the complex C5-polysaccharide constitutes at least 65% of the total sugars in the medium, more preferably, the complex C5-polysaccharide constitutes at least 80% of the total sugars in the medium. It should be understood that the medium obtainable in the method of the present invention including step (a') does not necessarily meet this condition since the product of step (a') contains C6 sugars. Thus, preferably, the complex C5-polysaccharide constitutes at least 50% of the total sugars in the extract produced in step (a1) or step (a2), preferably, the complex C5-polysaccharide constitutes at least 65% of the total sugars in the extract, more preferably, the complex C5-polysaccharide constitutes at least 80% of the total sugars in the extract.

[0124] In an embodiment of the present invention where the extract of step (a1) or the extract of step (a2) is mixed with the extract of step (a'), the complex polysaccharide may constitute less than 50% of the total sugars in the medium.

[0125] The fungal fermentation medium of the present invention may optionally be further processed as defined herein. In particular, within the scope of the present invention, the fungal fermentation medium described herein may be further processed into a dry form. For this purpose, the water contained in the fungal fermentation medium obtainable according to the present invention can be removed, for example, by spray drying, belt drying, drum drying or freeze drying to obtain a dry fungal fermentation medium, which, as is known to those skilled in the art, can have an improved storage period. Optionally, the fungal fermentation medium of the present invention may be further sterilized within the scope of the method of the present invention.

[0126] In a further embodiment, the present invention relates to the use of the fungal fermentation medium of the present invention in the production of fungal biomass. Thus, the fungal fermentation medium of the present invention, i.e., the medium described above and obtainable according to the method disclosed above, can be used in the method of the present invention for producing fungal biomass.

[0127] Thus, in a further embodiment, the present invention relates to a method for producing fungal biomass, the method comprising the step of submerged fermentation of at least one fungal strain using the fungal fermentation medium of the present invention.

[0128] According to the present invention, the submerged fermentation 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 of substances from the fermentation vessel and the inflow of substances into the fermentation vessel.

[0129] As understood herein, submerged fermentation or deep fungal fermentation is defined as the cultivation of fungi in a liquid medium. As is known to those skilled in the art, an alternative to deep fungal fermentation is surface fungal fermentation, also known as solid-state fungal fermentation. As understood herein, a liquid fungal fermentation medium such as the fungal fermentation medium of the present invention in solution or suspension (optionally pH-adjusted) is placed in a sealed container, herein preferably a fermenter, which is typically sterilized according to methods known to those skilled in the art to kill organisms that can interfere with fungal growth. An inoculum of at least one fungal strain as defined herein is introduced into the container (herein preferably a fermenter), and in the case of at least aerobic fungi, air is blown into the container. The contents of the container (herein a fermenter) are preferably agitated according to methods known to those skilled in the art, and preferably such agitation can be incorporated into the fermenter design. Agitation continuously brings the nutrients and oxygen present in the medium into contact with the substance being fermented (herein at least one fungal strain), and preferably, the temperature and pH are controlled at levels suitable for the fungi. After a specific time, typically 1 to 12 days, depending inter alia on the type of fermentation, the fungus, and the exact fermentation conditions, the fungal biomass can be recovered. (As mentioned by those skilled in the art, the timings given herein need not necessarily apply in the case of continuous fermentation). However, as is known to those skilled in the art, mixing may also be achieved by other methods than agitation, and mixing may also affect the morphology of the fungal cells and result in subjecting the fungal cells to shear stress. As understood herein, the method of mixing is not meant to be limiting, and any applicable method known to those skilled in the art is included within the scope of the present invention.

[0130] The batch process is characterized by the absence of the inflow of materials into the fermentation vessel. In the batch process, all nutrients are provided at the start of the culture without further addition during the subsequent bioprocess. No additional nutrients are added during the entire bioprocess, except for gases, acids, and bases. The bioprocess then continues until the nutrients are consumed. This strategy is suitable for rapid experiments such as strain characterization or optimization of the nutrient medium. The drawback of this convenient method is that the biomass and product yields are limited. Usually, due to carbon source and / or oxygen transfer being limiting factors, the microorganisms are not in the logarithmic growth phase for a long time. After the completion of the bioprocess executed in batch mode, only the biomass or the medium is recovered and appropriately processed to obtain the desired product. From the perspective of the bioreactor, this process is repeatedly interrupted by a washing process and a sterilization process, and the biomass is simply produced step by step.

[0131] In the fed-batch process, substrates, nutrients, and other substances can be added to the fermentation vessel to, among other things, extend the possible culture time or increase the yield. The advantage of the supply during culture is that it enables achieving a higher overall production volume. Under specific growth conditions, the microorganisms and / or cells multiply continuously and thus follow the logarithmic growth curve. Therefore, in certain embodiments, the supply rate can also be increased logarithmically. Generally, the substrate is pumped from the supply bottle into the culture vessel through, for example, a silicone tube. The user can manually set the supply (linearly, logarithmically, pulsingly) at any time, or add nutrients when specific conditions are met, such as when a specific biomass concentration is reached or when the nutrients are depleted. The fed-batch process provides a wide range of control strategies and is also suitable for highly specialized applications. However, the fed-batch process can increase the processing time and potentially cause inhibition due to the accumulation of toxic by-products.

[0132] Preferably, in the method of the present invention, the submerged fermentation is operated as a continuous process. After the batch growth phase, an equilibrium is established (also called a steady state) with respect to certain components. Under these conditions, the same amount of fresh culture medium as that removed is added (chemostat). These bioprocesses are called continuous cultures and are particularly suitable when excess nutrients would result in inhibition due to, for example, the accumulation of acids or ethanol or excessive heating. Other advantages of this method include reduced product inhibition and improved space-time yields. When the medium is removed, the cells are recovered, which is why the inflow rate and the outflow rate must be less than the doubling time of the microorganism. Alternatively, the cells can be retained in a variety of ways called perfusion (e.g., in a spin filter). In a continuous process, the space-time yield of the bioreactor can be further improved compared to that of a fed-batch process.

[0133] Thus, the fungal fermentation medium obtainable according to the method of the present invention is provided in a fermenter suitable for growing fungal mycelia. Suitable fermenters are known to those skilled in the art. For example, a suitable stirred tank equipped with a specific agitator useful for reducing shear stress, or an air-lift fermenter, is useful within the scope of the present invention. The fungal fermentation medium can be obtained according to the method of the present invention and is understood as the medium disclosed herein. As understood herein, the fungal fermentation medium can be further sterilized in certain embodiments of the present invention. As known to those skilled in the art, sterilization may be performed by exposing the medium to a high temperature for a certain period of time. Typically, sterilization is performed at a temperature of 150 to 200 °C for a time of 30 seconds to 10 minutes. However, the conditions described herein are not meant to limit the scope of the present invention. For example, the process can be carried out at a temperature of 120 to 200 °C, preferably 150 to 200 °C, and / or for a time of 30 seconds to 20 minutes, preferably 30 seconds to 20 minutes.

[0134] Preferably, the fungal growth is carried out at a temperature of 15 to 40 °C. Typically, a constant temperature is maintained throughout the process, which can be selected for the optimal growth of a particular fungal strain, as is known to those skilled in the art. For example, in the case of P. ostreatus, the growth is preferably carried out at a temperature of 25 to 30 °C. More preferably, the growth is carried out at a pH of 3.0 to 8.5. The pH of the medium can be adjusted within the scope of the method for producing the fungal fermentation medium of the present invention. As will be understood by those skilled in the art, the choice of pH may depend on the fungal strain being cultured or potential contaminating strains excluded from the growth. More preferably, the growth is carried out for a time of 12 to 240 hours. However, as will be understood by those skilled in the art, when the growth is carried out as a continuous process, the time is preferably not limited to 240 hours.

[0135] As understood herein, for example, the choice of growth conditions including pH, fungal fermentation medium and / or temperature can affect the growth of fungal mycelium, the metabolism of fungal cells, and / or whether the fungus grows as pellets or as mycelium.

[0136] Within the scope of the method for producing fungal biomass by submerged fermentation of at least one fungal strain of the present invention, it is understood that the at least one fungal strain is an edible fungus. Edible fungi are understood herein as fungi that can be consumed by mammals, preferably humans, as food without causing any harmful reactions. Harmful reactions are defined herein as food poisoning or undesirable taste characteristics that would prevent consumption. Edible fungi are not limited herein to their fruiting bodies (mushrooms), and other parts of the fungus, such as mycelium, can also be considered edible mushrooms.

[0137] In the method for producing fungal biomass by submerged fermentation of at least one fungal strain of the present invention, the at least one fungal strain is selected from Basidiomycota and Ascomycota.

[0138] According to the present invention, at least one fungal strain can be selected from Basidiomycota. Preferably, at least one fungal strain that can be selected from Basidiomycota can be a fungal strain selected from Agaromycotina. As defined herein, a fungal strain selected from Agaromycotina can be a fungal strain selected from Agaricomycetes. Preferably, the fungal strain selected from Agaricomycetes can be a fungal strain selected from Boletales, Cantharellales, Agaricales, Polyporales, Russulales, and Auriculariales.

[0139] As defined herein, the fungal strain selected from Boletaceae is preferably B. edulis. In certain embodiments, the fungal strain selected from Agaricomycetes can be a fungal strain selected from Polyporales. Preferably, as defined herein, the fungal strain selected from Polyporales can be a fungal strain selected from Meripilaceae, Polyporaceae, Ganodermataceae, Sparassidaceae.

[0140] As defined herein, the fungal strain selected from Meripilaceae is preferably G. frondosa. As defined herein, the fungal strain selected from Polyporaceae is preferably selected from P. umbellatus and L. sulphureus. As defined herein, the fungal strain selected from Sparassidaceae is preferably S. crispa.

[0141] Preferably, the fungal strain selected from Agaricomycetes can be a fungal strain selected from Cantharellales. More preferably, the fungal strain selected from Cantharellales can be a strain selected from Cantharellaceae and Hydnaceae. As defined herein, the strain selected from Cantharellaceae can be C. cornucopioides or C. cibarius, preferably C. cibarius. As further defined herein, the strain selected from Hydnaceae can be H. repandum.

[0142] Alternatively, the fungal strain selected from Agaricomycetes can be a fungal strain selected from Boletales. Preferably, as defined herein, the fungal strain selected from Boletales can be a fungal strain selected from Boletaceae and Sclerodermataceae.

[0143] Alternatively, the fungal strain selected from Agaricomycetes can be a fungal strain selected from Russulales. More preferably, as defined herein, the fungal strain selected from Russulales can be a fungal strain selected from Hericaceae and Bondarzewiaceae. Preferably, the fungal strain selected from Russulales is a fungal strain selected from Hericaceae, preferably a fungal strain selected from H. erinaceus and H. coralloides. More preferably, the fungal strain selected from Bondarzewiaceae is B. berkeleyi.

[0144] Alternatively, the fungal strain selected from Agaricomycetes can be a fungal strain selected from Auriculiales, more preferably a fungal strain selected from Auriculariaceae. Preferably, the fungal strain selected from Auriculariaceae is A. auricula - judae.

[0145] Preferably, in the method of the present invention, at least one fungal strain is selected from Agaricales. Thus, at least one fungal strain selected from Agaricales can be selected from Tuberaceae, Pleurotaceae, Agaricaceae, Marasmiaceae, Stropharaceae, Lyophyllaceae, Tricholmataceae, Omphalotaceae, Physalaciaceae, Schizophyllaceae, and Fistulinaceae.

[0146] As defined herein, a fungal strain selected from Marasmiaceae is preferably L. edodes. As further defined herein, a fungal strain selected from Strophariaceae is preferably a fungal strain selected from A. aegerita and H. capnoides. As further defined herein, a fungal strain selected from Lyophyllaceae is preferably C. Indica. As further defined herein, a fungal strain selected from Tricholomataceae is preferably a fungal strain selected from H. tesselatus and C. nuda. As further defined herein, a fungal strain selected from Omphalotaceae is preferably C. gigantean. As further defined herein, a fungal strain selected from Physalaciaceae is preferably F. velutipes. As further defined herein, a fungal strain selected from Schizophyllaceae is preferably S. Commune. As further defined herein, a fungal strain selected from Fistulinaceae is preferably F. hepatica.

[0147] At least one fungal strain according to the present invention selected from Agaricales can be selected from Tuberaceae. Preferably, the fungal strain according to the present invention selected from Tuberaceae is T. magnatum, T. aestivum, T. uncinatum, T. indicum, T. rufum or T. melanosporum, more preferably T. melanosporum and T. magnatum.

[0148] More preferably, at least one fungal strain selected from Agaricales can be a fungal strain selected from Pleurotaceae. Even more preferably, at least one fungal strain of the present invention is a fungal strain selected from P. pulmonarius, P. ostreatus, P. citrinopileatus and P. salmoneostramineus, even more preferably a fungal strain selected from P. pulmonarius or P. ostreatus, and most preferably a fungal strain selected from P. pulmonarius.

[0149] At least one fungal strain according to the present invention selected from Agaricles can be selected from Agaricaceae. Preferably, the fungal strain selected from Agaricaceae as defined herein is A. bisporus or A. blazei, more preferably A. bisporus.

[0150] According to the present invention, at least one fungal strain can be selected from Ascomycota. Preferably, at least one fungal strain can be selected from Ascomycota which can be a fungal strain selected from Pezizomycotina. As defined herein, the fungal strain selected from Pezizomycotina can be selected from Pezizomycetes. Preferably, within the scope of the present invention, the fungal strain selected from Pezizomycetes can be a fungal strain selected from Pezizales.

[0151] More preferably, at least one fungal strain defined in the method for producing fungal biomass of the present invention can be selected from Pezizales. Preferably, the fungal strain selected from Pezizales can be selected from Morchellaceae. Preferably, the fungal strain selected from Morchellaceae is M. esculenta, M. angusticeps or M. deliciosa.

[0152] Alternatively, at least one fungal strain selected from Ascomycota can be a fungal strain selected from Sordariomycetes. Preferably, at least one strain as defined herein selected from Sordariomycetes can be a fungal strain selected from Hypocreales. More preferably, the fungal strain selected from Hypocreales can be a fungal strain selected from Cordycipitaceae. Even more preferably, the fungal strain selected from Cordycipitaceae is a fungal strain selected from C. militaris and C. sinensis. Alternatively, the fungal strain selected from Hypocreales can preferably be a fungal strain selected from Nectriaceae. More preferably, the fungal strain selected from Nectriaceae can be a Fusarium strain. In another embodiment, the fungal strain selected from Sordariomycetes can be a fungal strain selected from Sordariaceae. More preferably, the fungal strain selected from Sordariaceae can be a Neurospora strain.

[0153] As disclosed herein, in a method for producing fungal biomass by submerged fermentation of at least one fungal strain of the present invention, the at least one fungal strain can be selected from Pezizomycotina and Agaromycotina.

[0154] As further disclosed herein, in the method of the present invention for producing fungal biomass by submerged fermentation of at least one fungal strain, the at least one fungal strain is preferably selected from Peziomycetes, Agaricomycetes and Sordariomycetes.

[0155] As further disclosed herein, in the method of the present invention for producing fungal biomass by submerged fermentation of at least one fungal strain, the at least one fungal strain is preferably selected from Pezizales, Boletales, Cantharellales, Agaricales, Polyporales, Russulales, Auriculariales, Sordoriales and Hypocreales.

[0156] As further disclosed herein, in the method of the present invention for producing fungal biomass by submerged fermentation of at least one fungal strain, the at least one fungal strain is preferably selected from Morchellaceae, Tuberaceae, Pleurotaceae, Agaricaceae, Marasmiaceae, Cantharellaceae, Hydnaceae, Boletaceae, Meripilaceae, Polyporaceae, Strophariaceae, Lyophyllaceae, Tricholomataceae, Omphalotaceae, Physalacriaceae, Schizophyllaceae, Sclerodermataceae, Ganodermataceae, Sparassidaceae, Hericiaceae, Bondarzewiaceae, Cordycipitaceae, Auriculariaceae, and Fistulinaceae.

[0157] As further disclosed herein, in the method of the present invention for producing fungal biomass by submerged fermentation of at least one fungal strain, the at least one fungal strain is preferably P. pulmonarius, P. ostreatus, P. citrinopileatus or P. salmonostramineus, and even more preferably P. pulmonarius or P. ostreatus.

[0158] As further disclosed herein, in the method of the present invention for producing fungal biomass by submerged fermentation of at least one fungal strain, the at least one fungal strain is preferably M. esculenta, M. angusticeps or M. deliciosa.

[0159] As described herein, the co-fermentation of two or more fungal strains is also encompassed by the present invention. The selection of strains for co-fermentation depends on their compatibility and can be performed by those skilled in the art.

[0160] According to the method of the present invention, it should be understood that the yield is up to 100%, preferably up to 99%, and more preferably 85 - 95% (based on the conversion of the carbon source available in the medium). It is understood that the biomass yield is calculated according to the ratio of the amount of biomass produced to the amount of substrate consumed (e.g., C5 sugar).

[0161] In a further embodiment, the present invention relates to a fungal biomass produced according to a method for producing fungal biomass by submerged fermentation of at least one fungal strain of the present invention. Preferably, the fungal biomass comprises fungal cells of a fungal strain selected from Pleurotaceae, in particular, the fungal strain is P. pulmonarius, P. ostreatus, P. citrinopileatus or P. salmonostramineus, more preferably P. pulmonarius or P. ostreatus is selected. In another embodiment, preferably, the fungal biomass comprises fungal cells of a fungal strain selected from Morchellaceae, and the fungal strain is M. esculenta, M. angusticeps or M. deliciosa. However, the fungal biomass of the present invention is not limited to a single fungal strain. As described herein, co-fermentation of two or more fungal strains to obtain the fungal biomass of the present invention is also encompassed by the present invention. The selection of strains for co-fermentation depends on their compatibility and can be performed by those skilled in the art. Furthermore, the selection of strains for inclusion together in the fungal biomass of the present invention depends on their properties and intended uses, as well as their growth rates, as disclosed herein.

[0162] The fungal biomass of the present invention preferably has a protein content of 10-60% (w / w). As further disclosed herein, the fungal biomass of the present invention preferably has a dietary fiber content of 20-60% (w / w).

[0163] As shown in Example 10, the inventors have demonstrated that the fungal biomass of the present invention is characterized by a high Asp / Asn content. Accordingly, the present invention relates to a fungal biomass of the present invention characterized by an Asp / Asn content of at least 20% of the total amino acid content, preferably at least 30% of the total amino acid content. Preferably, herein, % refers to % w / w. The amino acid composition is preferably determined as described in the Examples section.

[0164] In a further embodiment, the invention relates to the use of the fungal biomass of the invention in the production of fungal-based foods. Accordingly, the invention also relates to fungal-based foods obtainable as described herein. The fungal-based foods of the invention can be prepared in any form known to those skilled in the art. For example, the fungal-based foods of the invention can take the form of balls (i.e., meatball substitutes), dumplings, vegetarian sausages, meat substitute steaks, meat substitute minced meat products, meat substitute products for preparing sandwiches, etc.

[0165] The food according to the invention may be, for example, a nutritional supplement. The nutritional supplement can be in liquid form or in solid forms such as pills, lozenges or tablets. For example, the nutritional supplement of the invention may be a protein supplement and / or a carbohydrate supplement.

[0166] The food as understood herein may be dairy products, such as yogurt, milk beverages and ice cream. The food as understood herein may also relate to different embodiments of seafood products, such as club cakes, fish cakes, tuna, salmon, or shrimp.

[0167] The food may be a texturized food or a texturized food. Accordingly, the food of the invention contains all the amino acids necessary for human daily intake that cannot be newly synthesized. Furthermore, the texturized food of the invention is preferably heat-resistant and boil-resistant and is suitable for cooking. For example, the fungal-based food of the invention may be a meat substitute product as described herein. It should be noted that preferably, the meat substitute product is a texturized food or a texturized food. Attention should be paid to the structure of the texturized food to improve the acceptability of the texturized food by consumers. It should be further noted that the unique fibrous texture of the fungal biomass of the invention can be beneficial for producing texturized foods or texturized foods without using conventional texturization methods such as extrusion.

[0168] According to the inventors of the present invention, the food of the present invention is characterized by an improved taste. The taste characteristics of the food can be determined, for example, as shown in the Examples section.

[0169] As understood herein, the term "about", when referring to temperature, preferably means ±3°C, more preferably ±1°C. When the term "about" refers to other numerical values, it preferably means ±10% of the value, more preferably ±5% of the value, and even more preferably ±1% of the value.

[0170] Further examples and / or embodiments are disclosed in the following numbered items. 1. A method for producing a fungal fermentation medium from beer brewing spent grain (BSG), the method comprising: (a1) extracting C5-sugars from the lignocellulosic material contained in BSG by steam pretreatment, followed by a washing step with liquid water at a temperature of 50°C or lower; and (b) combining the extract thus obtained with at least one non-carbohydrate nutrient for fungal culture. 2. The method according to item 1, wherein the steam pretreatment is carried out with a severity factor of 2.9 to 3.3. 3. The method according to item 1 or 2, wherein during the steam pretreatment, the lignocellulosic material contained in BSG is brought into contact with steam at a temperature of 130°C to 180°C, preferably 160°C to 180°C, more preferably 165°C to 175°C, and / or for a time up to 30 minutes, preferably up to 15 minutes. 4. The method according to any one of items 1 to 3, wherein in step (a1), the washing step with liquid water is carried out at a temperature of 40°C or lower, preferably 30°C or lower, more preferably 25°C or lower. 5. BSG is characterized by a final moisture content of 50% to 75% by weight. Preferably, the method further comprises a step of dehydrating BSG by pressing the material to a final moisture content of 50% to 75% by weight, and the dehydrating step precedes step (a1). The method according to any one of items 1 to 4. 6. In step (b), the extract of step (a1) is not substantially diluted, preferably the extract of step (a1) constitutes 90 to 99% v / v of the whole medium obtained in step (b), more preferably the extract of step (a1) constitutes 94 to 98% v / v of the whole medium obtained in step (b), the method according to any one of items 1 to 5. 7. The method according to any one of items 1 to 6, wherein the medium can support fungal growth without a further dilution step to reduce the concentration of the fungal growth inhibitor. 8. A method for producing a fungal fermentation medium from brewer's spent grain (BSG), the method comprising: (a2) extracting C5 sugars from the lignocellulosic material contained in BSG through a liquid extraction treatment with water at a temperature of 145 °C to 155 °C and / or for a time up to 70 minutes, preferably up to 50 minutes, preferably at a pressure of 30 to 50 bar; and (b) combining the extract thus obtained with at least one non-carbohydrate nutrient for fungal culture. 9. The method according to item 8, wherein step (a2) is carried out with a severity coefficient of 2.9 to 3.3. 10. The lignocellulosic material contained in BSG is untreated, preferably the material is not dehydrated, the method according to item 8 or 9. 11. The method according to any one of items 1 to 10, further comprising step (a') of enzymatically hydrolyzing the solid lignocellulosic residue obtained in step (a1) or step (a2) with cellulase, separating the liquid product of the hydrolysis from the solid residue, and mixing the liquid product with the extract of step (a1) or the extract of step (a2). 12. The method according to any one of items 1 to 11, wherein sugars other than those present in the extract of step (a1) or step (a2) or in the liquid product of the hydrolysis in step (a') are not substantially added. 13. The method according to any one of items 1 to 12, wherein step (a1) or step (a2) is characterized by a solid input to the reactor of 4 to 14% w / w. 14. The composite C5-polysaccharide constitutes at least 50% of the total sugars in the extract of step (a1) or step (a2), preferably, the composite C5-polysaccharide constitutes at least 65% of the total sugars in the extract of step (a1) or step (a2), more preferably, the composite C5-polysaccharide constitutes at least 80% of the total sugars in the extract of step (a1) or step (a2), the method according to any one of items 1 to 13. 15. The medium is suitable for culturing at least one fungal strain selected from P. pulmonarius and M. rufobrunnea, the method according to any one of items 1 to 14. 16. A fungal fermentation medium obtainable according to the method according to any one of items 1 to 15. 17. Substantially all sugars are derived from BSG, the fungal fermentation medium according to item 16. 18. The composite C5-polysaccharide constitutes at least 50% of the total sugars in the medium, preferably, the composite C5-polysaccharide constitutes at least 65% of the total sugars in the medium, more preferably, the composite C5-polysaccharide constitutes at least 80% of the total sugars in the medium, the fungal fermentation medium according to item 16 or 17. 19. Use of the medium according to any one of items 16 to 18 in fungal culture. 20. A method for producing fungal biomass, the method comprising the step of submerged fermentation of at least one fungal strain using the fungal fermentation medium according to any one of items 16 to 18. 21. At least one fungal strain is an edible fungus, the method according to item 20. 22. At least one fungal strain is P. pulmonarius, P. ostreatus, P. citrinopileatus or P. salmonostramineus, or at least one fungal strain is M. rufobrunnea, M. esculenta, M. angusticeps or M. deliciosa, the method according to item 20 or 21. 23. Fungal biomass produced according to the method according to any one of items 20 to 22. 24. The fungal biomass according to item 23, characterized by an Asp / Asn content of at least 20% of the total amino acid content. 25. Use of the fungal biomass according to item 23 or 24 in the production of a food, preferably a fungus-based food. 26. A food, preferably a fungus-based food, prepared using the fungal biomass according to item 23 or 24. 27. The food according to claim 26, characterized by an improved taste.

[0171] Various modifications and variations of the present invention will be apparent to those skilled in the art without departing from the scope of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant art are intended to be encompassed by the present invention.

[0172] The following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the present invention in any way defined by the appended claims.

Examples

[0173] Example 1: Steam treatment (extraction with hot water at 50°C or water at 22°C) Beer brewing spent grain (BSG) is first hydrothermally treated under pressure to release pentose sugars (C5 sugars), namely mainly xylose and arabinose. Subsequently, the pretreated biomass is subjected to enzymatic hydrolysis (EH) to release glucose (C6 sugar). Hydrothermal treatment refers to a thermochemical process for decomposing biomass with water under high temperature and high pressure conditions. Enzymatic hydrolysis (EH) or saccharification is a process of converting complex carbohydrates into simpler sugars. EH was carried out using cellulolytic enzymes. Cellulolytic enzymes mainly decompose cellulose and a certain degree of hemicellulose into hexose (mainly glucose) sugars and pentose (mainly xylose) sugars. The research was carried out at different scales for screening efficient treatment conditions to achieve the maximum amount of sugar in the extract, with little or no use of toxic compounds (mycelial growth inhibitors), providing conditions for maximum mycelial growth. In the following examples, P. pulmonarius was grown on sugars extracted from BSG.

[0174] Determining the composition of the BSG used, it was found that (based on dry mass) cellulose was 22%, hemicellulose was 25.8%, protein was 25%, and the moisture content was about 80 wt%.

[0175] To reduce the moisture content of BSG and improve heat transfer during steam treatment, fresh BSG with a moisture content of about 80% was treated with a hydraulic press at a pressure of 3 bar to dehydrate it to a final moisture content of about 70%.

[0176] To determine the most promising set of temperature and holding time, several experiments were carried out under different process conditions. For the recovery of C5 sugars, experiments on one-step treatment with steam were carried out. For the recovery of C6 sugars, to enhance the enzymatic digestibility of BSG, a two-step steam treatment cascade was carried out before saccharification.

[0177] The process conditions were tested in the range of 150 - 180 °C and short holding times (5 - 20 minutes) to limit energy consumption. Different solid inputs were evaluated in the range of 4 - 14% w / w.

[0178] After steam treatment, extraction was carried out in different process units by washing the material with water at different temperatures. Depending on the solid content of the material (i.e., the dry matter percentage of the pretreated brewing lees), warm water (i.e., 50 °C) or water near room temperature (i.e., 22 °C) was added in a given ratio to reach the desired solid input during extraction. Constant stirring was carried out for at least 20 minutes. A hydraulic press was used to separate the liquid fraction and the solid fraction. The liquid fraction and the solid fraction were recovered for further processing, either fermentation or further enzymatic hydrolysis, respectively.

[0179] After steam treatment, the material was recovered for subsequent extraction or further steam treatment. At the end of the treatment, the material presented a homogeneous dark color, suggesting that the steam had effectively diffused through the bulk material.

[0180] Analysis of the samples showed that C5 sugars (xylose and arabinose) were effectively solubilized from BSG after steam treatment. Furthermore, a small amount of glucose (1 - 3.5 g / L) was detected in the samples.

[0181] Following this value, the highest concentration of total sugars was observed at treatment intensities of LogRo = 3.23 and LogRo = 2.94. Therefore, the best candidates for scale-up correspond to those process conditions with a severity coefficient in the range of 2.9 - 3.2. In addition, the results show that at the above treatment intensity (LogRo = 3.5), a decrease in sugar concentration is detected due to the thermal decomposition of sugars to furfural. Since furfural is a known potential inhibitor of mycelial growth, it is an undesirable compound.

[0182] Several fermentation experiments were carried out to determine the optimal conditions for mycelial growth. Before fermentation, the liquid hydrolyzate was centrifuged to reduce the amount of fine powder that would cause interference in the process. The screening part was carried out by fermenting 50 mL of the liquid hydrolyzate in an Erlenmeyer flask.

[0183] The results show that the growth of biomass during fermentation can be limited by some inhibitors present in the extract. Therefore, dilution of the extract for the fermentation medium is considered an option to reduce the concentration of the inhibitor and confirm its effect on biomass growth. When washed in water at 22 °C, the observed mycelial growth is similar whether the extract is diluted or not, i.e., proportional to the amount of extract added. Therefore, the inventors have surprisingly found that by controlling the washing temperature, the production of unwanted growth inhibitors, such as furfural, can be reduced. Furthermore, as will be apparent to those skilled in the art, the inhibitor can be separated from the obtained extract using methods known to those skilled in the art. Some of the highest mycelial concentrations achieved are shown in the following table.

[0184] The experimental setup is shown in Figure 1. The results are summarized in Tables 1 and 2 below.

[0185] Table 1.1. Experimental results of extraction with water at 22 °C and 50 °C in a BSG steam treatment followed by a batch setup. The values shown below relate to the growth of biomass using media containing (a1) process extracts diluted (50%) and undiluted (100%). The C5 recovery range is based on three tests.

[0186]

Table 1

[0187] The C5 recovery rate defined in this specification refers to the ratio of C5 sugars to the total amount of hemicellulose, which is determined based on the solid input of the reactor and the composition of beer brewing spent grains. The maximum achievable recovery rate of C5 sugars is 53% (i.e., up to a maximum of 60%) obtained for the conditions carried out with an extraction at 170 °C, a holding time of 7.5 minutes, a pressure of 8 bar, a solid input percentage of 14% (w / w), and water at 50 °C. The total sugar concentration is 19.2 g / L, and the biomass concentration was 0.73 and 0.95 [g / L per input%] in the 50% dilution of the extract used for fermentation and the 100% extract, respectively. The effect of the washing temperature at 22 °C is found to be valid for the conditions where the solid input percentage is 8 and 14, but not as much as when using a washing temperature of 50 °C. Therefore, this example clearly shows that a higher washing temperature is more efficient for the preparations disclosed in the present invention, which was also surprising regarding the mycelial growth using sugars extracted at a higher temperature.

[0188] Furthermore, after investigating the effects of solid input and washing temperature on the extract and mycelial growth, the extractor settings were changed from batch to semi - continuous. Condition 4 in Table 1.2 shows a C5 sugar recovery rate of up to 96%, while the same condition in Table 1.1 shows a C5 sugar recovery rate of up to 60%. This shows the influence of the extractor configuration on the extraction yield. In a semi - continuous configuration, the feed and reaction mixture continuously enter and exit the reaction system, and the supply and recovery of the raw material are not at the same rate, i.e., the raw material is fed periodically and does not remain stationary in the extractor as in the case of a batch system, thereby increasing the surface area of the BSG exposed to the steam treatment. Additionally, a crusher or a disk refiner may be present or arranged at the extractor outlet, and the crusher or the disk refiner is configured to process the beer brewing spent grains such that the surface area of the BSG is further increased (i.e., the particle size is further reduced) for higher sugar extraction in the washing step.

[0189] Table 1. Experimental results of BSG steam treatment using 1.2.14% (w / w) solid input and subsequent extraction with hot water at 50 °C in a semi - continuous setting. The values shown below in this specification relate to (a1) the process of biomass growth using a medium containing 50% diluted extract.

[0190] [Table 2]

[0191] To reduce the energy requirement and cost of the process, the holding time was shortened from 7.5 minutes to 5 minutes. The BSG solid input was 14% (w / w) for the conditions implemented, as shown in Table 2 below. The results for C5 sugar recovery, biomass growth, and furfural concentration are listed in Table 2. The percentage of C5 sugar recovered was found to be higher at a holding time of 7.5 minutes. The biomass concentration was found to be higher in the extract treated at a holding time of 7.5 minutes compared to 5 minutes. No effect of the change in holding time on the furfural concentration was observed.

[0192] Table 2. Experimental results of BSG steam treatment with 14% (w / w) solid input, subsequent batch setting, and extraction with hot water at 50 °C with holding times of 5 minutes and 7.5 minutes. The values shown below in this specification relate to (a1) the process of biomass growth using a medium containing 50% diluted extract.

[0193] [Table 3]

[0194] In a further step, in order to improve the energy consumption of the process and the overall economy, sugar extraction was carried out using water at three different temperatures, namely 22 °C, 40 °C, and 50 °C, and the BSG batch was processed at a batch setting of 170 °C for 7.5 minutes with a 14% (w / w) solid input. The concentration (g / L) of C5 sugar in the extracts obtained at three different washing temperatures is shown in Figure 4. The sugar concentration decreased as the temperature of the water used for extraction decreased. The maximum concentration of extracted C5 sugar was found for the extraction conditions with water at 50 °C (19.2 g / L), followed by 40 °C (14.5 g / L), and 22 °C (10.82 g / L). Thus, this example also clearly shows that higher washing temperatures are more efficient for the preparations disclosed in the present invention. Depending on the desired composition of the extract, a washing temperature range of 40 °C to 50 °C is preferred, and even more preferably a washing temperature range of 40 °C to 48 °C is preferred.

[0195] Example 2: Formation of a fungal growth inhibitor in a method comprising steam pretreatment and washing with liquid water. Furfural has been proven to inhibit the growth of mycelia at a low concentration of 0.2 g / L. This compound is a product of the thermal decomposition of hemicellulose. Even at higher furfural concentrations, biomass growth can still occur due to possible effects caused by other inhibitors present (described above).

[0196] The formation of furfural can be partially avoided by careful control of the process conditions. Generally speaking, when the treatment conditions are severe, sugars tend to decompose more strongly into furfural. However, even under mild treatment conditions, the generation of furfural cannot be completely avoided. Furfural formation depends strongly not only on the process conditions but also on the material composition. The more free sugars the material has, the easier it is for those sugars to decompose into furfural.

[0197] BSG is a material with a similar composition every time it leaves the wort filtration process during beer production. However, BSG is not a stable material, and its composition changes rapidly after leaving the wort filtration process. In this regard, the delivered material may contain varying amounts of free sugars from one batch to another. As a result, it is difficult to control the formation of furfural, but when BSG is treated under fairly mild conditions, the appearance of furfural is significantly reduced. The results are summarized in Table 3 below. The furfural concentration was measured by HPLC. Furfural can also be isolated or separated according to methods known in the art.

[0198] The results are summarized in Table 3 below.

[0199] Table 3. Concentration ranges of furfural observed under different steam pretreatment conditions and washing conditions.

[0200]

Table 4

[0201] Example 3: Liquid hydrothermal treatment The experimental setup for this process is shown in Figure 2. The pressure is set constant at 50 bar. Extraction (solubilization of C5 sugars) is carried out in the same unit, and the separation of the liquid and solid fractions is carried out by pressure filtration. In this particular example, to find the optimal conditions for maximum sugar extraction and maximum mycelial growth, the temperature was tested in the range of 110 - 210 °C and the holding time was tested in 10 - minute increments up to 120 minutes. The results are excerpted and summarized in Table 4. Table 5 shows another set of larger-scale conditions that emphasize the increase in the efficiency of liquid extraction by the use of acid by comparing the extraction results in the presence / absence of acid.

[0202] Table 4. Experimental results of BSG liquid hydrothermal treatment on a small scale (30 mL extraction volume) showing the obtained cell dry weight (g) of P. pulmonarius.

[0203]

Table 5

[0204] Table 5. Excerpts of some experimental results of large-scale BSG liquid hydrothermal treatment. The values shown below relate to the growth of biomass using a medium containing undiluted (a2) process extract. In conditions 5 and 7, 0.92% w / w H2SO4 was added, and in condition 6, 0.5% w / w H2SO4 was added. When condition 6 was changed to have the same acid concentration as conditions 5 and 7, the resulting sugar recovery rate was 82%.

[0205] [Table 6]

[0206] Example 4: Contamination test All chemicals required for medium preparation were purchased from Carl Roth (Karlsruhe, Germany), VWR (Darmstadt, Germany), Merck KgaA (Darmstadt, Germany) or Sigma-Aldrich (Steinheim, Germany).

[0207] The medium for the preparation of agar plates was prepared by weighing different compounds according to Table 6 and then dissolving them in water. The medium according to the invention described as C5 extract was prepared as in condition 2 of Example 6. C5 complete means 5.9 g / L of K2HPO4, 9.0 g / L of KH2PO4, 12.0·10 -2 g / L of MgSO4, 8.10·10 -3 of FeCl3, and 10.10·10 -3Refers to the C5 extract described herein, further supplemented with CaCl2. Subsequently, the mixture was autoclaved at 121 °C for 20 minutes, followed by aseptically pouring under a sterile clean bench and allowing to stand until completely solidified. Subsequently, plates prepared with LB medium, malt extract, M9 classical medium, C5 extract, and C5 complete were inoculated with 100 μL of a 1:1000 diluted Escherichia coli suspension obtained from a culture in LB medium incubated overnight at 37 °C. Then, the plates were incubated overnight at 37 °C and photographed the next day. In another experiment, the lids of plates prepared using malt extract, M9 classical medium, C5 extract, and C5 complete were removed and exposed to air contamination to compare growth on different media. In that case, the plates were photographed after 10 days (see Figure 3 for a summary).

[0208] Table 6. Composition of media used in the contamination test.

[0209]

Table 7

[0210] Example 5: Production of a fungal fermentation medium from brewing lees using a liquid extract with added acid Extraction experiments using liquid water were performed on brewing lees according to the above or the general protocol in Table 5, and H2SO4 was added to the water used for extraction at different concentrations. The experiments were conducted according to the conditions described in Table 7.

[0211] Table 7. Summary of extraction conditions for brewing lees with dilute acid.

[0212]

Table 8

[0213] Similar to the previous example, the protocol "Analysis of Sugar and Furfural Content" was used to quantify the recovery rate of C5 sugar and the furfural concentration. Subsequently, fermentation was performed on the obtained extract using the protocol "Cultivation of Fungal Biomass". The results of both extraction and fermentation are summarized in Table 8.

[0214] Table 8. Summary of extraction experiments using brewers' spent grain with dilute acid.

[0215]

Table 9

[0216] Example 6. Hydrolysis of Cellulose from Pretreated Brewers' Spent Grain The extraction experiments were carried out on brewers' spent grain using 10 different conditions defined in Table 9 below, according to the general protocol outlined in the "Thermal Extraction of Lignocellulosic Materials" section. The biomass was then produced using the protocol "Cultivation of Fungal Biomass".

[0217] Thermal Extraction of Lignocellulosic Materials For thermal extraction, the dry matter of the lignocellulosic material was measured using a moisture analyzer (until a constant weight at 160 °C) with 3 g of the material. Based on the obtained dry matter, the required amount of the material was weighed and then introduced into the reactor to adjust the reactor charge. Then, water was added to fill the reactor to its final working volume, and a rubber sealing ring was placed on top of the reactor housing. Next, a lid equipped with a manometer and a temperature probe was sealed to avoid leakage, and then the reactor was pressurized with nitrogen until the desired operating pressure was reached. Subsequently, the desired temperature, pressure, and holding time were set using software, and the extraction was started. During the experiment, the temperature and pressure were constantly monitored and adjusted.

[0218] Once the extraction was completed, the reactor was cooled by immersing it in cold water or, for larger reactors, by using a cooling jacket. When the temperature dropped below 30 °C, the reactor was depressurized by slowly opening the exhaust valve and the lid was opened as soon as the pressure reached atmospheric pressure. In the case of a continuous extraction process, after the mixture was extracted from the reactor, it was cooled using a heat exchanger. Subsequently, the solid and liquid phases (extract) were separated either by pressure filtration or by a decanter centrifuge. The liquid extract was then stored briefly at 4 °C or frozen at -20 °C for long-term storage. The remaining treated solid lignocellulosic material was further subjected to enzymatic hydrolysis to recover glucose from cellulose.

[0219] Cultivation of fungal biomass The liquid extract obtained after hot extraction was used directly as a growth medium or was either mixed and / or supplemented with additional compounds (5.9 g / L of K2HPO4, 9.0 g / L of KH2PO4, 12.0·10 -2 g / L of MgSO4, 8.10·10 -3 of FeCl3, 10.10·10 -3 of CaCl2 and other trace elements according to a typical M9 medium (Miller, 1972, Experiments in Molecular Genetics. Cold Spring Harbor, NY: New York Cold Spring Harbor Laboratory). The resulting mixture was then placed in a suitable container and sterilized (121 °C, 20 min). After sterilization, the medium was cooled to room temperature and inoculated with spores or mycelia from a suspension prepared from a well-grown mycelial agar plate. The broth was then incubated at a temperature of 30 °C for 5 days. During fermentation, the pH was adjusted using acids and bases to maintain an optimal pH of 6.5 for the P. pulmonarius culture strain. Subsequently, the biomass was harvested by centrifugation, washed with water, and finally the dry matter was measured using a moisture analyzer (until a constant weight was achieved at 160 °C). The dry matter was converted to biomass concentration using the broth volume.

[0220] The reference biomass used for comparison with the biomass produced from the extract was produced using a reference medium consisting of glucose, a trace solution containing magnesium, iron, manganese, zinc, copper and calcium, and yeast extract as a nitrogen source. The pH of the medium was adjusted to 6.5 using a phosphate buffer, and the flask was incubated at 30 °C for 5 days.

[0221] Table 9. Summary of the extraction conditions of the brewing residue.

[0222]

Table 10

[0223] The solid lignocellulosic material recovered after the thermal extraction of the brewing residue of Example 1 (brewing residue in this specification) was treated according to the method described in the section "Hydrolysis of Lignocellulosic Materials". The recovery rate of the C6-polysaccharide fraction determined as described in "Analysis of Sugar and Furfural Contents", and the biomass obtained from the fermentation test using these extracts conducted as described in the protocol "Cultivation of Fungal Biomass" (expressed per solid % input for comparison purposes) are summarized in Table 10.

[0224] Hydrolysis of Lignocellulosic Materials The dry matter of the recovered lignocellulosic material after being processed as described in the section "Thermal Extraction of Lignocellulosic Materials" was determined using a moisture analyzer (incubated at 160 °C until a constant weight was reached), and the input was adjusted as described herein for the heat treatment. Subsequently, the material was charged into a reaction vessel, and an enzyme cocktail (Ctec2 and Ctec3 from Novozymes) was added according to the cellulose content in the material and following the manufacturer's guidelines. Then, the mixture of the enzyme and the cellulose material was incubated at a temperature of 60 °C and a pH of 6.0 for 10 to 200 hours. The homogeneity of the mixture was ensured either by using a stirrer or by incubating the vessel in an incubator. After incubation, the reaction mixture was rapidly cooled using either an ice bath, a cooling jacket of the reaction vessel, or a heat exchanger in the case of a continuous process. Finally, the liquid fraction and the solid fraction were separated by pressure filtration or centrifugation. The solid fraction rich in lignin was stored at 4 °C before analysis, and the clarified liquid fraction was used for the fermentation experiment.

[0225] Table 10. Summary of the hydrolysis experiments conducted on the pretreated BSG.

[0226] [Table 11]

[0227] Example 7: Production of a fungal fermentation medium from brewing lees using steam pre-hydrolysis As described in the protocol "Thermal Pretreatment and Extraction of Lignocellulosic Biomass Using Steam", extraction experiments by steam pre-hydrolysis were conducted on brewing lees.

[0228] Thermal Pretreatment and Extraction of Lignocellulosic Biomass Using Steam Before the operation, the reactor is preheated with steam until the operating temperature is reached. During the preheating stage, the condensate generated from the steam coming into contact with the cold reactor must be removed.

[0229] Weigh the materials and feed them into the reactor using either a metal cartridge (batch process) or a screw feeder (continuous process). Batch extraction is carried out in a closed system, and the brewing lees and water are fed into the reactor only once at first. When the reactor is pressurized, the system remains closed until the reaction mixture is cooled to at least 40°C. On the other hand, continuous extraction refers to the continuous supply and recovery of raw materials at the same rate. When the materials are supplied, there is no pressure drop inside the reactor, and when the materials are recovered, a sudden pressure drop occurs, resulting in steam explosion. However, in a semi - continuous process, the feed and the reaction mixture continuously enter and leave the system. The supply and recovery of raw materials are not at the same rate because the raw materials are supplied periodically.

[0230] The materials are pretreated for a desired residence time while injecting steam constantly, and the temperature inside the reactor is controlled by setting the pressure control device to the steam pressure corresponding to the desired temperature. In the case of continuous processing, the residence time is controlled by the rotation speed of the screw conveyor reactor.

[0231] After the residence time is achieved, the materials are recovered from the reactor, their weight and moisture content are recorded, and the next processing step (extraction) is continued.

[0232] Extraction is carried out in a separate unit, and the steam - pretreated materials are mixed with warm water (40 - 60°C, preferably 40 - 50°C) at a desired ratio to achieve a specific solid input. Constant mixing is provided by a stirring tank for at least about 20 minutes before the liquid fraction and the solid fraction are separated.

[0233] Pump the solid - liquid mixture to a press, where the two fractions are separated at a constant pressure of about 4 bar. The liquid hydrolyzate (rich in C5 sugars) and the solid fraction (rich in cellulose and lignin) are recovered for further processing.

[0234] Next, the solid fraction can be further treated with enzymes to convert cellulose to C6 sugars.

[0235] Analysis of sugar and furfural content For the C5 extract, i.e., the extract that can be obtained according to the section "Thermal extraction of lignocellulosic materials", before the analysis that can quantify the recovered C5 sugars as monosaccharides, the sample was hydrolyzed with hydrochloric acid (final concentration 4% w / w) at 121 °C for 60 minutes. After hydrolysis, the pH of the extract was set to 5.5 to avoid damage to the HPLC column. Next, the monosaccharides derived from the C6 extract that can be obtained according to the section "Hydrolysis of lignocellulosic materials", and the monosaccharides derived from the above-mentioned C5 extract, were measured with an Agilent HPLC 1200 system using a Metacarb 87C column (300×7.8 mm, Varian Inc, Paolo Alto, CA, USA) as the stationary phase and ultrapure water as the mobile phase. The measurement was carried out at a temperature of 85 °C and an isocratic flow of 0.6 mL min -1 -1. The analytes were detected by a refractive index detector, except for furfural which was measured using a UV detector at a wavelength of 270 nm after column separation.

[0236] The experimental conditions for pre-hydrolysis are listed in Table 11. The results of the analysis of sugar and furfural (protocol "Analysis of sugar and furfural content") and the biomass obtained from the fermentation using the resulting extract (protocol "Culture of fungal biomass") (expressed as % of the solid by-product input for comparison purposes) are shown in Table 12.

[0237] Table 11. Summary of the extraction conditions of brewing lees using steam pre-hydrolysis.

[0238]

Table 12

[0239] Table 12. Summary of the extraction experiment using brewing lees with steam pre-hydrolysis.

[0240]

Table 13

[0241] Example 8 - Hydrolysis of Cellulose Derived from Pretreated Brewing Residue from Example 7 The pretreated solid material remaining after pre - hydrolysis with steam and recovery with water was recovered, subjected to a second pre - hydrolysis, and finally subjected to enzymatic hydrolysis as described in "Hydrolysis of Lignocellulosic Materials". The tested conditions are listed in Table 13.

[0242] Table 13. Summary of the extraction conditions of the pretreated brewing residue using pre - hydrolysis with steam.

[0243] [Table 14]

[0244] Data on the recovery rate of C6 sugars and the available biomass (measurements carried out as in the general protocols "Analysis of Sugar and Furfural Content" and "Culture of Fungal Biomass" included above) are summarized in Table 14. For comparison purposes, they are normalized with respect to the solid input used in the experiment.

[0245] Table 14. Summary of the extraction experiment using brewing residue with pre - hydrolysis with steam.

[0246] [Table 15]

[0247] Example 9. Sensory Evaluation of Meatballs Produced Using Mycelia of Different Origins Form meatballs from the mycelium biomass and fry them in a pan. Blindfold each trained panelist and consecutively serve meatballs prepared from either the mycelium derived from the standard medium or the mycelium derived from the brewing lees. During this first session, the panelists define the sensory attributes recognized in the two meatballs. Then, the panelists discuss the attributes together, select the common attributes that all panelists can associate with the same taste and aroma of the meatballs, and compare them. Next, start the second session, and the panelists need to evaluate the meatballs according to the selected attributes and assign a score from 0 to 5 for each attribute. Repeat this session on different days to increase the statistical relevance of the data, calculate the average of the scores, and plot them on a spider web.

[0248] Use an RGB system and a color measuring instrument to determine the color at 20 different positions on the meatball. The positions used for the 10 measurements are the same for all meatballs. Then, use the average value of these measurements to compare the colors of the meatballs.

[0249] Example 10. Amino acid content analysis of the obtained biomass Using the medium obtained in the process of extracting the beer brewing lees with liquid water, the amino acid content of the biomass obtained as described in Table 9 was determined as follows.

[0250] The amino acid profile of the protein extracted from the lignocellulosic material was determined by hydrolyzing the sample of the extract with 6M HCl at 105 °C for 24 hours before HPLC measurement. Subsequently, the hydrolysate was evaporated under a nitrogen stream and resuspended in 200 μM α-aminobutyrate, which was used as an internal standard. The amino acid concentration in the prepared sample was finally measured by fluorescence detection using an Agilent 1200 HPLC system (Agilent technologies, Waldbronn, Germany) equipped with a reversed-phase column Gemini 5μ C18 110 A (150×4.6 mm, Phenomenex, Aschaffenburg, Germany) as the stationary phase. The separation of different protein constituent amino acids relied on separately mixing eluent A (40 mM NaH2PO4, pH 7.8) and eluent B (45% methanol, 45% acetonitrile, 10% water) according to a well-defined gradient profile and gradually changing the mobile phase composition throughout the measurement. Furthermore, a pre-column (Gemini C18, MAX, RP, 4×3 mm, Phenomenex, Aschaffenburg, Germany) was used to extend the column life. Fluorescence detection was achieved by pre-column derivatization with o-phthalaldehyde (OPA) and 9-fluorenylmethyloxycarbonyl (FMOC) and modification of the excitation and emission wavelengths (Table 15). -1 The column separation was carried out at a flow rate of 1 mL min

[0251] Table 15. Methods used for the separation and quantification of amino acids - Separation by gradient elution was achieved by changing the composition of the mobile phase during the measurement. Eluent A: 40 mM NaH2PO4, pH 7.8; Eluent B: 45% methanol, 45% acetonitrile, 10% water.

[0252]

Table 16

[0253] The results were summarized in Table 16 below.

[0254] Table 16. Amino acid content of the obtained biomass, expressed in % w / w (YE - yeast extract, CSL - corn steep liquor).

[0255] [Table 17]

[0256] Example 11: Production of a fungal fermentation medium from wheat bran The extraction experiments were conducted on wheat bran according to the general protocol outlined in the section "Thermal extraction of lignocellulosic materials" using four different conditions defined in Table 24 below. The pH of the water was adjusted to approximately 13.5 using NaOH to extract sugars from the wheat bran, achieving a sugar recovery rate of at least 80%.

[0257] Table 17. Summary of the extraction conditions of wheat bran and the related fermentation results

[0258] [Table 18]

[0259] Further examples and / or embodiments of the present invention are disclosed in the following numbered clauses. 1. A method for producing a fungal fermentation medium from brewing spent grain (BSG). 2. The method comprises (a1) extracting C5 - sugars from the lignocellulosic material contained in BSG by steam pretreatment, followed by a washing step with liquid water at a temperature of 50 °C or lower, and (b) combining the extract thus obtained with at least one non - carbohydrate nutrient for fungal culture, the method according to clause 1. 3. During steam pretreatment, the lignocellulosic material contained in BSG is brought into contact with steam at a temperature of 130°C to 180°C, preferably 160°C to 180°C, more preferably 165°C to 175°C, and / or for a time up to a maximum of 30 minutes, preferably up to a maximum of 15 minutes, and / or In step (a1), the washing step with liquid water is carried out at a temperature of 40°C or lower, preferably 30°C or lower, more preferably 25°C or lower, according to the method described in clause 2. 4. BSG is characterized by a final moisture content of 50% to 75% by weight. Preferably, the method is a step of dehydrating BSG by pressing the material to a final moisture content of 50 to 75% by weight. The dehydration step further includes a step that precedes step (a1), and / or In step (b), the extract of step (a1) is not substantially diluted. Preferably, the extract of step (a1) constitutes 90 to 99% v / v of the entire medium obtained in step (b). More preferably, the extract of step (a1) constitutes 94 to 98% v / v of the entire medium obtained in step (b), and / or The medium can support fungal growth without a further dilution step to reduce the concentration of the fungal growth inhibitor, according to the method described in clause 2 or 3. 5. The method is (a2) Extracting C5 sugars from the lignocellulosic material contained in BSG by liquid extraction with water at a temperature of 145°C to 155°C and / or for a time up to a maximum of 70 minutes, preferably up to a maximum of 50 minutes, preferably at a pressure of 30 to 50 bar, and (b) Combining the extract thus obtained with at least one non-carbohydrate nutrient for fungal culture, Preferably, the lignocellulosic material contained in BSG is untreated. More preferably, the material is not dehydrated, according to the method described in clause 1. 6. Step (a1) or step (a2) is carried out with a severity factor of 2.9 to 3.3, according to any one of the methods described in clauses 2 to 5. 7. The method according to any one of clauses 1 to 6 further comprises step (a') of enzymatically hydrolyzing the solid lignocellulosic residue obtained in step (a1) or step (a2) with cellulase, separating the liquid product of the hydrolysis from the solid residue, and mixing the liquid product with the extract of step (a1) or the extract of step (a2). 8. Sugars other than those present in the extract of step (a1) or step (a2) or in the liquid product of the hydrolysis of step (a') are not substantially added, and / or step (a1) or step (a2) is characterized in that the solid input to the reactor is 4 to 14% w / w, and / or the complex C5-polysaccharide constitutes at least 50% of the total sugars in the extract of step (a1) or step (a2), preferably, the complex C5-polysaccharide constitutes at least 65% of the total sugars in the extract of step (a1) or step (a2), more preferably, the complex C5-polysaccharide constitutes at least 80% of the total sugars in the extract of step (a1) or step (a2), and / or The medium is suitable for culturing at least one fungal strain selected from P. pulmonarius and M. rufobrunnea, the method according to any one of clauses 1 to 7. 9. A fungal fermentation medium obtainable according to the method according to any one of clauses 1 to 8. 10. Substantially all sugars are derived from BSG, and / or The complex C5-polysaccharide constitutes at least 50% of the total sugars in the medium, preferably, the complex C5-polysaccharide constitutes at least 65% of the total sugars in the medium, more preferably, the complex C5-polysaccharide constitutes at least 80% of the total sugars in the medium, the fungal fermentation medium according to clause 9. 11. Use of the medium according to clause 9 or 10 in fungal culture. 12. A method for producing fungal biomass, the method comprising a step of submerged fermentation of at least one fungal strain using the fungal fermentation medium according to clause 9 or 10, preferably, at least one fungal strain is an edible fungus, more preferably, at least one fungal strain is P. pulmonarius, P. ostreatus, P. citrinopileatus or P. salmonostramineus, or at least one fungal strain is M. rufobrunnea, M. esculenta, M. angusticeps or M. deliciosa, the method. 13. Fungal biomass produced according to the method of clause 12, preferably characterized by an Asp / Asn content of at least 20% of the total amino acid content, the fungal biomass. 14. Use of the fungal biomass according to clause 13 in the production of food, preferably fungus-based food. 15. Food prepared using the fungal biomass according to clause 14, preferably fungus-based food.

Claims

1. A method for producing a fungal fermentation medium from beer lees (BSG), (a2) A step of extracting C5 sugars from lignocellulose material contained in BSG by liquid extraction treatment using water at a temperature of 130°C to 140°C, wherein the water used in the liquid extraction treatment contains H₂SO₄ in a concentration range of 0.5% w / w to 1.6% w / w, and (b) A method comprising combining the extract thus obtained with at least one non-carbohydrate nutrient for fungal culture.

2. The method according to claim 1, wherein the BSG is characterized by a final moisture content of 50% to 75% by weight.

3. The method according to claim 2, further comprising the step of dehydrating the BSG by pressing the material to a final moisture content of 50 to 75% by weight, wherein the dehydration step precedes step (a2).

4. The method according to claim 1, wherein the lignocellulose material contained in BSG is untreated.

5. The method according to claim 4, wherein the material is not dehydrated.

6. In the step (a2), H ２ SO ４ The method according to claim 1, wherein the method is carried out using water containing the substance at a concentration in the range of 0.5% w / w to 0.9% w / w.

7. The method according to claim 6, wherein step (a2) is carried out using water containing H₂SO₄ at a concentration in the range of 0.5% w / w to 0.6% w / w.

8. The method according to claim 1, wherein the extraction in step (a2) is performed for a maximum of 10 minutes, preferably for a maximum of 5 minutes, and more preferably for about 5 minutes.

9. The method according to claim 1, wherein the liquid extraction is performed at a pH of 1.0 to 3.

0.

10. The method according to claim 1, wherein the extraction is performed at a pressure of 5 to 10 bar.

11. The method according to claim 1, further comprising step (a') of enzymatically hydrolyzing the solid lignocellulose residue obtained in step (a2) with cellulase, separating the liquid product of the hydrolysis from the solid residue, and mixing the liquid product with the extract from step (a2).

12. The method according to claim 1, wherein no sugars other than those present in the extract of step (a2) or in the liquid product of the hydrolysis of step (a') are substantially added.

13. The method according to claim 1, wherein step (a2) is characterized by adding 4 to 14% w / w of solids to the reactor.

14. The method according to any one of claims 1 to 13, wherein the complex C5-polysaccharide constitutes at least 50% of the total sugars in the extract of step (a2).

15. The method according to claim 14, wherein the complex C5-polysaccharide constitutes at least 65% of the total sugars in the extract of step (a2).

16. The method according to claim 15, wherein the complex C5-polysaccharide constitutes at least 80% of the total sugars of the extract in step (a2).

17. The method according to claim 1, wherein the culture medium is suitable for culturing at least one fungal strain selected from P. pulmonarius and M. rufobrunnea.

18. A fungal fermentation medium that can be obtained according to the method of claim 1.

19. The fungal fermentation medium according to claim 18, wherein substantially all of the aforementioned sugars are derived from BSG.

20. The fungal fermentation medium according to claim 18 or 19, wherein the complex C5-polysaccharide constitutes at least 50% of the total sugars in the medium.

21. The fungal fermentation medium according to claim 20, wherein the complex C5-polysaccharide constitutes at least 65% of the total sugars in the medium.

22. The fungal fermentation medium according to claim 21, wherein the complex C5-polysaccharide constitutes at least 80% of the total sugars in the medium.

23. Use of the culture medium according to claim 18 in fungal culture.

24. A method for producing fungal biomass, the method comprising a step of deep fermentation of at least one fungal strain using the fungal fermentation medium described in claim 18.

25. The method according to claim 24, wherein the at least one fungal strain is an edible fungus.

26. The method according to claim 25, 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. rufobrunnea, M. esculenta, M. angustieps, or M. deliciosa.

27. Fungal biomass produced according to the method of claim 24.

28. The fungal biomass according to claim 27, characterized by an Asp / Asn content of at least 20% of the total amino acid content.

29. Use of fungal biomass according to claim 27 or 28 in the production of food, preferably fungal-based food.

30. A food prepared using fungal biomass according to claim 27 or 28, preferably a fungal-based food.

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