Fermentation medium for producing fungi from brewer's grains

Abstract

The invention relates to a method for producing a fungal fermentation medium from brewer's grains (BSG) and to a correspondingly obtainable fermentation medium, to a method for producing a fungal biomass by submerged fermentation of at least one fungal strain and to a correspondingly obtainable fungal biomass, and to food products obtainable by using the instant fungal biomass of the invention.

Description

Technical Field

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

[0003] In recent years, the production of food from animals has attracted attention due to its unsustainability and increasing concern for animal welfare. In the context of climate change, many plant-based meat substitutes have emerged with the aim of reducing CO2 emissions and reducing animal suffering. However, these products are currently produced from three major monocrops (soybeans, peas and rice), the cultivation of which requires a large amount of land and water, relies heavily on chemical reagents (pesticides and fertilizers) and produces a large amount of waste, because the protein isolated only from these crops is used to produce meat substitutes. In addition, these isolates have a strong bitter taste and do not have an inherent texture, so their use in food requires further processing steps and the addition of additional ingredients, including but not limited to flavorings, conditioning agents and / or coloring agents. Therefore, plant-based substitutes are not necessarily healthy, and their production induces other environmental problems, such as felling, a significant reduction in biodiversity, soil pollution and / or water pollution. It should be noted that the inventors can use waste side streams as described above in the methods described herein, and therefore contribute to improving the sustainability of these plant-based methods.

[0004] Producing food using fermentation methods seems to solve several of these shortcomings. It enables better use of land, because fermentation tanks can be vertically scaled and allow food to be produced locally in cities or villages. In addition, the water required for their per kilogram of product is less than plant protein, and along with the continuous development and improvement of filtration and treatment technology, these waters can be recycled in the process. This paper discloses the use of novel fermentation methods to produce fungal mycelium as a new food, wherein lignocellulosic materials (such as industrial and / or agricultural side streams) are used as raw materials to at least partially produce growth medium and final product. In this sense, the method described herein contributes to efforts to build a circular economy, wherein industrial, food and agricultural waste are reduced to a minimum and resources are used to their maximum extent. Another advantage of fungal fermentation relative to conventional plant isolate production is the raw material-fungal biomass obtained, which itself has a desired fiber texture and has brought a balanced nutritional profile, not only with complete protein, but also with dietary fiber, vitamins and micronutrients, which provides healthy products for consumers. In particular, the use of mycelium isolated from fruiting bodies from known edible mushrooms additionally brings out the typical mushroom umami taste characteristic of this group, slightly varying between species (such as morels, truffles or Agaricus bisporus), and enables the production of a clean and tasty product with a very short ingredient list.

[0005] The increasing demand for sustainable food supply chains and managing GHG emissions by finding alternative foods are major challenges facing the world. Using renewable and waste lignocellulosic biomass as raw materials for producing food creates a greener roadmap towards environmentally and economically sustainable development.

[0006] Brewer's spent grains (BSG) are lignocellulosic waste generated as a by-product of the brewing industry. BSG is obtained as a solid residue after the production of wort 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 is a lignocellulosic material rich in carbohydrates (hexoses, pentoses and lignin), so it is worthwhile to exploit the sugars locked within its structure. Using BSG as a by-product alleviates the problems associated with its disposal in the environment and contributes to the circular economy.

[0007] It is apparent to those skilled in the art that the available carbohydrates in BSG are in the form of fiber, cellulose and hemicellulose, and thus obtaining the carbohydrates from BSG is considered challenging.

[0008] DE10201410884 describes a method for deodorizing lignin, which method comprises the step of extracting a lignocellulosic substrate with a supercritical fluid or a supercritical fluid mixture.

[0009] DE102016110653 relates to food / fermented products comprising fungal mycelium.

[0010] CN101838673A discloses fermenting fungi of the Basidomycota family in a liquid fermentation medium supplemented with rice wine grains.

[0011] CN1078872A discloses a method for preparing a beverage, which comprises cultivating a fungus in a fermentation medium which comprises, among other components, wine lees.

[0012] WO 2017 / 208255 A1 relates to a method for preparing edible fungi (fungi of the phylum Ascomycota) by culturing in a culture medium comprising wine lees.

[0013] WO2002090527A1 relates to a method for preparing edible fungi, such as Fusarium.

[0014] WO2017 / 181085A1 discloses certain methods for producing fungal mycelium.

[0015] WO 2019 / 046480A1 relates to the preparation of edible filamentous fungal preparations by growing filamentous fungal biomats.

[0016] RU 2006 / 126554 relates to a method for producing food and feed biomass based on waste from brewery stillage production on a nutrient medium, the method comprising the sequential cultivation of baker's yeast Saccharomyces cerevisiae and an edible basidiomycete (e.g. selected from the group comprising Pleurotus ostreatus, Pleurotus pulmonaria etc.).

[0017] US 5846787 discloses a method for treating cellulose-containing material.

[0018] Papadaki (doi: 10.3390 / microorganisms7070207) discloses the cultivation of Pleurotus (P. pulmonarius and P. ostreatus) by solid-state fermentation and semi-liquid fermentation using grape pomace as a side stream.

[0019] Kim Min-Keun et al. (Korean Journal of Mycology, DOI: 10.4489 / KJM.2012.40.1.049) disclosed the development of an optimal medium for mycelial culture of Pleurotus eryngii using hydrothermal extracts of raw materials. The method is a solid-state fermentation for producing fruiting bodies, in which steam is not used.

[0020] Platt MW et al. (Eur. J. Appl. Microbiol. Biotechnol, Vol. 17, pp. 140-142, 1983) disclose increased straw degradation by Pleurotus ostreatus "Pleurotus florida". The method disclosed is solid state fermentation.

[0021] Beltran-Garcia MJ et al. (Revista de la Sociedad Quimica de Mexico, Vol. 45, pp. 77-81, 2001) disclosed that lignin degradation products from corn stover significantly enhanced the radial growth of basidiomycete mushroom mycelium, which was performed as a solid-state fermentation.

[0022] Document CN 108 203 693A discloses certain Rhizopus oryzae seed culture media.

[0023] Document KR 2013 / 0057507 discloses certain methods for cultivating Cordyceps militaris.

[0024] Document ES2'370'215 discloses certain means for cultivating fungi.

[0025] Sidana Arushdeep et al. (Chinese Journal of Biology, Vol. 2014, pp. 1-5) disclosed sugarcane bagasse as a potential culture medium for fungal cultures.

[0026] Document US 9206446 discloses certain extraction methods from plant biomass.

[0027] Kemppainen et al., Appl. Biochem. Biotechnology, April 16, 2016, doi: 10.1007 / s12010-016-2085-9) discloses certain aspects of steam explosion of brewer's spent grains.

[0028] Parchami et al. (Bioresource Technology 337(2021)125409–doi:10.1016 / j.biortech.2021.125409) disclose certain aspects of recovering starch and protein from brewer's spent grains using hydrothermal pretreatment and converting them into edible filamentous fungi. Summary of the invention

[0029] Particularly desirable are means and methods utilizing lignocellulosic materials, preferably agricultural and / or industrial wastes, industrial and / or agricultural side streams herein, particularly brewer's grains, because they are cost effective and more sustainable. Further particularly desirable are methods leading to obtaining fungal biomass and therefore food products having an amino acid composition reflecting the amino acids of a complete protein according to the FAO definition (www.fao.org, https: / / en.wikipedia.org / wiki / Complete_protein).

[0030] Further particularly desirable is a method for producing fungal biomass that is resistant to contamination by other microorganisms (e.g., bacteria). Thus, a fungal fermentation medium that is resistant to bacterial contamination that can be obtained by using lignocellulosic materials, preferably industrial and / or agricultural side streams (i.e., waste products), in particular brewer's grains, is particularly desirable.

[0031] The object of the present invention is to provide improved means and methods for the production of fungus-based foods, methods for producing fungal fermentation media from lignocellulosic materials, preferably from agricultural and / or industrial side streams, and methods and means for producing fungal biomass for the production of fungus-based foods.

[0032] The inventors have surprisingly found that brewer's grains can be extracted according to the methods disclosed herein to produce a fungal fermentation medium with a high sugar content (if necessary, the extract is supplemented with at least one non-carbohydrate nutrient for fungal culture), wherein the sugar content is mainly C5 complex sugars. Therefore, the medium is fungus-specific because C5 complex sugars cannot support the growth of most bacteria and are therefore resistant to contamination. It is worth noting that the available carbohydrates in BSG can be extracted according to the methods of the present invention, although they are present in the form of fiber, cellulose and hemicellulose, which makes the extraction process more difficult.

[0033] Therefore, the problems described herein are solved by the embodiments described below and characterized according to the claims.

[0034] The present invention is summarized in the following embodiments.

[0035] In a first embodiment, the present invention relates to a method for producing a fungal fermentation medium from brewer's spent grains (BSG), the method comprising: (a1) extracting C5 sugars from lignocellulosic material contained in BSG via steam pretreatment, followed by a washing step with liquid water at a temperature not exceeding 50°C, and (b) combining the extract thus obtained with at least one non-carbohydrate nutrient for fungal cultivation.

[0036] In a second embodiment, the present invention relates to a method for producing a fungal fermentation medium from brewer's spent grains (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 between 145°C and 155°C and / or for a period of up to 70 minutes, preferably for a period of up to 50 minutes, preferably at a pressure of 30 bar to 50 bar, and (b) combining the extract thus obtained with at least one non-carbohydrate nutrient for fungal cultivation.

[0037] In a third embodiment, the present invention relates to a fungal fermentation medium obtainable by the method for producing a fungal fermentation medium from brewer's grains according to the present invention.

[0038] In a fourth embodiment, the present invention relates to the use of the culture medium of the present invention in the culture of fungi.

[0039] In a fifth embodiment, the present invention relates to a method for producing fungal biomass, comprising the step of submerged fermentation of at least one fungal strain using the fungal fermentation medium of the present invention.

[0040] In a sixth embodiment, the present invention relates to a fungal biomass produced according to the method for producing a fungal biomass according to the present invention.

[0041] In a seventh embodiment, the present invention relates to the use of the fungal biomass of the present invention in the production of fungus-based food products.

[0042] In an eighth embodiment, the present invention is directed to a fungus-based food product prepared using the fungal biomass of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The workflow of a method for producing a fungal fermentation medium from BSG is shown, the method comprising step (a1) steam pretreatment followed by washing with liquid water. In this context, thermal hydrolysis followed by extraction may also be referred to as steam pretreatment followed by washing with liquid water.

[0044] Figure 2A workflow is shown for a method for producing a fungal fermentation medium from BSG, the method comprising step (a2) hot liquid water treatment (indicated as thermal hydrolysis / extraction in the figure).

[0045] Figure 3 An overview of contamination experiments performed with the fungal fermentation medium of the present invention is shown.

[0046] Figure 4 Shown are the concentrations of extracted sugars obtained under the same steam treatment conditions, but at different wash water temperatures. DETAILED DESCRIPTION

[0047] The present invention will be described in detail below. It should be understood that, unless otherwise stated, all disclosed features can be combined with each other.

[0048] In one embodiment, the present invention relates to a method for producing a fungal fermentation medium from brewer's spent grains (BSG).

[0049] Spent grains are preferably understood as residues or by-products of the brewing industry. Preferably, spent grains are the material remaining after the mashing step and have a dry matter content of preferably between 10% and 30%. However, the dry matter content as described herein is not meant to be limiting, as it is clear to the skilled person that the dry matter content can be increased in pre-processing, for example by pressing, by drying or by other methods known to the skilled person. In addition, spent grains originating from other industries (for example spent grains obtainable as by-products of food production) can also be used within the scope of the present invention.

[0050] Preferably, the spent grains suitable for the process of the invention are characterized in that the particle size distribution comprises a maximum value between 0.3 mm and 1 mm, preferably a particle size referred to by a person skilled in the art as a coarse particle size, preferably a particle size distribution with a maximum value between 0.4 mm and 0.8 mm. Preferably, the particle size of the spent grains is determined during the screening process leading to the production of the spent grains. Preferably, without any pretreatment, the particle size distribution comprises a maximum value between 2.0 mm and 4.0 mm and a second maximum value between 1.0 mm and 2.0 mm. In the case of mechanical treatment, a particle size distribution comprising a single maximum value not exceeding 1.0 mm is used. Thus, as discussed in the literature (Ozturk et al., Journal of the Brewing Society (J. Inst. Brew. 108(1):23–27, 2002), the spent grains can be sieved after grinding through a series of sieves with openings of 850 μm, 425 μm and 212 μm. Based on the fractions, the spent grain product can be considered as a coarse fraction (425 μm-850 μm), a medium fraction (212 μm-425 μm) and a fine fraction (<212 μm). As demonstrated in the Examples section, a person skilled in the art will be able to determine the particle size by using a set of sieves different from those disclosed above. Size distribution. Preferably a set of sieves conforming to ASTM standards is used. It will also be appreciated that a person skilled in the art will be able to implement the method of the invention also for other particle size distributions, for example a particle size distribution comprising a maximum value between 2.0 mm and 4.0 mm. A person skilled in the art will also recognize that spent grains without any further mechanical (pre)treatment have a particle size distribution comprising a maximum value between 2.0 mm and 4.0 mm and a second maximum value between 1.0 mm and 2.0 mm. Upon mechanical treatment, the particle size distribution typically obtained will include a single maximum value not exceeding 1.0 mm.

[0051] Preferably, BSG comprises between 20% w / w and 25% w / w cellulose (preferably 22%), between 23% w / w and 28% w / w hemicellulose (preferably 25.8%), and / or between 20% w / w and 30% w / w protein (preferably 25% w / w protein). These figures are understood to refer to the content of BSG relative to its dry mass.

[0052] The method of the present invention is disclosed and described herein in the context of brewer's grains. However, it will be appreciated by those skilled in the art that the method may also be applicable to any other lignocellulosic material. Lignocellulosic material is preferably defined herein as a material comprising dry plant matter. Preferably, the lignocellulosic material comprises cellulose, hemicellulose and lignin. Preferably, the at least one lignocellulosic material is at least one industrial and / or agricultural side stream as defined herein. Further preferably, the lignocellulosic material is preferably solid.

[0053] Examples of lignocellulosic materials include spent grains, cereal bran, cotton, cottonseed hulls, bagasse, cocoa shells, cocoa, cocoa pods; cotton and oil cake from sunflower, peanut, hazelnut, palm oil, olives; husks and skins from nuts, grass and leaf waste; wood chips, coffee grounds, coffee husks, coffee silver husks, rapeseed and by-products from the soybean industry such as soy pulp ("okara").

[0054] Preferred lignocellulosic materials include spent grains, cereal bran (also known as wheat bran) and oil cake. In a specific embodiment, a particularly preferred lignocellulosic material is cereal bran. It is particularly preferred that the water used for pre-hydrolyzing the cereal bran with steam contains a dilute alkali, for example not more than 1% w / w of the alkali. Particularly suitable is NaOH in a concentration of up to 0.2% w / w, especially a concentration of 0.2% w / w. Alternatively, particularly suitable is NaOH in a concentration between 0.2% w / w and 0.8% w / w, more preferably between 0.6% w / w and 0.8% w / w, especially between 0.7% w / w and 0.8% w / w. Particularly suitable is 0.7% w / w NaOH, more preferably particularly suitable is 0.8% w / w NaOH. In an alternative embodiment, particularly suitable is 0.2% w / w NaOH.

[0055] Thus, it will be appreciated by those skilled in the art that in the process of the present invention, brewer's spent grains may be replaced by a side stream selected from the group consisting of spent spent grains, spent cereals (from industries other than breweries), cereal bran, bagasse, cotton and oil cake from sunflower, hazelnuts, shells and husks from nuts, grass and leaf waste, sawdust, coffee grounds, coffee husks, coffee silver skins, rapeseed and by-products from the soy industry such as okara ("residue"), banana leaves, banana peels, chicory roots, cassava peels, citrus pulp, cocoa, cocoa bean shells, cocoa mucilage, cocoa pod shells, coconut fiber, coconut shells, coconut shells , coffee pulp, corn cobs, corn stover, cotton, cottonseed meal, cottonseed, hemp, spent hops, pea by-products, peanut shells, peanut meal, peanuts, raw potato peels, potato tubers, eucalyptus bark, lantana grass, switchgrass, rice bran, rice husks, rice straw, waste sugar beets, sugar beet pulp, sawdust, wet sugarcane bagasse, walnut shells, wheat bran, wheat distiller's grains, malt extract, wheat straw, lupin seeds, chickpea bran, chickpea pod husks, chickpea straw, olive waste, wine pomace, pear pulp, sorghum bran, sorghum germ, sorghum stalks, sorghum stover, sunflower waste, and tea waste. In addition, it will be appreciated by those skilled in the art that in the process of the present invention, the spent grains may be replaced by the peels or waste or pulp or pomace of the following side streams: oats, pine, dates, apples, apricots, spent barley, broccoli, cabbage, carrots, radishes, eggplant, kiwi, melon, alfalfa, pineapple, pomegranate, plum, watermelon, zucchini, asparagus, beetroot, cauliflower, garlic, onions, squash, pumpkin and / or tomatoes.

[0056] In one embodiment, a method for producing a fungal fermentation medium from brewer's spent grains (BSG) comprises the following steps: (a1) extracting C5 sugars from lignocellulosic material contained in BSG via steam pretreatment, followed by a washing step with liquid water at a temperature not exceeding 50°C, and (b) combining the extract thus obtained with at least one non-carbohydrate nutrient for fungal cultivation.

[0057] C5 sugars as defined herein preferably refer to fractions in which at least 80% w / w of the total sugar content constitutes pentoses (sugars including polysaccharides consisting of sugar subunits of five carbon atoms). Note that C5 sugars defined herein as fractions containing sugars may contain other sugars, in particular C6 sugars (sugars having 6 carbon atoms, also called hexoses) as monomers and / or contained in polysaccharides and / or oligosaccharides. Therefore, sugars other than pentoses may also be extracted in step (a1) or step (a2) of the method of the present invention.

[0058] According to the invention, the extraction conditions, in particular the temperature, pressure and time of the extraction, are set so 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 sugars contained in the lignocellulosic material in the spent grains are recovered in the aqueous extraction. As will be appreciated by those skilled in the art, the time of the extraction may depend on further conditions, in particular on the reactor used (in particular in the case of available stirring, as discussed herein), and on the specific type of material used in the extraction.

[0059] In step (a1 ), preferably, the steam pretreatment is performed at a severity factor between 2.9 and 3.3.

[0060] The severity factor is also called log 10 (Ro), where Ro is defined herein as:

[0061]

[0062] where t R is the retention time (expressed in minutes), also understood to be the time of the process, and T is the temperature expressed in °C.

[0063] The steam pretreatment in step (a1) can be carried out at a severity factor between 2.9 and 3.0, a severity factor between 3.0 and 3.1, a severity factor between 3.1 and 3.2, and / or a severity factor between 3.2 and 3.3. Preferably, the steam pretreatment in step (a1) can be carried out at a severity factor between 3.1 and 3.3, more preferably, the steam pretreatment in step (a1) can be carried out at a severity factor between 3.1 and 3.2.

[0064] In one embodiment, the steam pretreatment in step (a1 ) may be performed at a severity factor between 2.0 and 4.0, more preferably at a severity factor between 2.5 and 3.3, even more preferably at a severity factor between 2.9 and 3.3.

[0065] In one embodiment, the steam pretreatment in step (a1 ) may be performed at a severity factor between 2.6 and 3.0 or between 3.1 and 3.3.

[0066] During the steam pretreatment in step (a1), the lignocellulosic material contained in the BSG may be contacted with steam at a temperature between 130°C and 180°C. Preferably, the steam pretreatment in step (a1) is carried out at a temperature between 160°C and 180°C, more preferably at a temperature between 165°C and 175°C. Thus, the steam pretreatment in step (a1) of the present invention may be carried out at 130°C, 131°C, 132°C, 133°C, 134°C, 135°C, 136°C, 137°C, 138°C, 139°C, 140°C, 141°C, 142°C, 143°C, 144°C, 145°C, 146°C, 147°C, 148°C, 149°C, 150°C, 151°C, 152°C, 153°C, 154°C, 155°C, 156°C, 157°C, 158°C, 159°C, 160°C, 161°C, 162°C, 163°C, 164°C, 165°C, 166°C, 167°C 140°C, 141°C, 142°C, 143°C, 144°C, 145°C, 146°C, 147°C, 148°C, 149°C, 150°C, 151°C, 152°C, 153°C, 154°C, 155°C, 156°C, 157°C, 158°C, 159°C, 160°C, 161°C, 162°C, 163°C, 164°C, 165°C, 166°C, 167°C, 168°C, 169°C, 170°C, 171°C, 172°C, 173°C, 174°C, 175°C, 176°C, 177°C, 178°C, 179°C or 180°C. Preferably, during the steam pretreatment in step (a1), the lignocellulosic material contained in BSG is contacted with steam for a time of up to 30 minutes, preferably a time of up to 15 minutes. Preferably, the steam pretreatment in step (a1) is carried out for at least 5 minutes. Preferably, during the steam pretreatment in step (a1), the lignocellulosic material contained in BSG is contacted with steam at a temperature between 165°C and 175°C, and / or is contacted for a time of up to 15 minutes, preferably a time of up to 12.5 minutes, more preferably a time of up to 10 minutes, even more preferably a time of up to 7.5 minutes, even more preferably a time of up to 5 minutes, even more preferably a time of up to 2.5 minutes, even more preferably a time of up to 1 minute. In the present invention, the extraction of step (a1) can therefore 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 contacted with steam at a temperature between 165°C and 175°C for a time of up to 15 minutes. More preferably, during the steam pretreatment in step (a1), the lignocellulosic material contained in the BSG is contacted with steam at a temperature between 165°C and 175°C for a time of up to 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 between 165°C and 175°C for a time of up to 7.5 minutes, even more preferably for a time of up to 5 minutes, even more preferably for a time of up to 2.5 minutes, even more preferably for a time of up to 1 minute. Therefore, as described herein, the extraction of step (a1) can therefore preferably be carried out for about 7.5 minutes, about 5 minutes, about 2.5 minutes or about 1 minute.Preferably, the temperature between 165° C. and 175° C. relates to a temperature of about 170° C., more preferably, the temperature between 165° C. and 175° C. relates to a temperature of 170° C. Preferably, step (a1) is performed at a pressure not exceeding 10 bar.

[0067] In one embodiment of the invention, the water used for prehydrolysis with steam may contain a dilute acid, for example no more than 1% w / w of said acid, 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. Particularly suitable are H2SO4 preparations of 0.2% w / w or 0.4% w / w, or 0.8% w / w or 0.9% w / w. However, the present invention also encompasses embodiments in which the water contains up to 2% w / w of said acid, for example H2SO4. Particularly suitable are H2SO4 preparations ranging between 0.4% w / w and 1.6% w / w, preferably ranging between 0.6% w / w and 1.6% w / w, more preferably ranging between 0.7% w / w and 1.5% w / w. In a specific embodiment, the concentration of the acid (preferably H2SO4) is between 0.4% w / w and 1% w / w, more preferably between 0.4% w / w and 0.9% w / w, still more preferably between 0.5% w / w and 0.9% w / w. In a specific embodiment, the concentration of the acid (preferably H2SO4) is between 1.1% w / w and 1.6% w / w. This is particularly applicable to embodiments in which the lignocellulosic material is brewer's grains.

[0068] In one embodiment of the invention, the water used for prehydrolysis with steam may contain a dilute alkali, for example not more than 1% w / w of said alkali. Particularly suitable is NaOH in a concentration between 0.2% w / w and 0.8% w / w, more preferably between 0.6% w / w and 0.8% w / w, in particular between 0.7% w / w and 0.8% w / w. Particularly suitable is 0.7% w / w NaOH, more preferably particularly suitable is 0.8% w / w NaOH. In an alternative embodiment, particularly suitable is 0.2% w / w NaOH.

[0069] In an alternative embodiment of the present invention, the water used for pre-hydrolysis with steam is replaced by a phosphate buffer having a pH range between 4 and 6, more preferably a pH range between 4.5 and 5.5.

[0070] In step (a1), the steam pretreatment is followed by a washing step with liquid water at a temperature not exceeding 50°C. Preferably, the washing step with liquid water is carried out with water at a temperature not exceeding 40°C, preferably not exceeding 30°C, more preferably not exceeding 25°C. Preferably, the liquid water used for the washing step is at room temperature, i.e. between 20°C and 25°C, preferably at a temperature of about 22°C, more preferably at a temperature of 22°C. In an alternative preferred embodiment, the liquid water used for the washing step ranges between 30°C and 50°C (particularly between 30°C and 49°C, preferably between 30°C and 48°C), even more preferably between 40°C and 50°C (particularly between 40°C and 49°C, preferably between 40°C and 48°C). Thus, the washing step can be carried out with liquid water at a temperature of 1° C., 2° C., 3° ​​C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 11° C., 12° C., 13° C., 14° C., 15° C., 16° C., 17° C., 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C. or 50° C. The washing time is preferably at least 15 minutes, more preferably at least 20 minutes, more preferably at least 25 minutes, even more preferably at least 30 minutes. Preferably, the time of the washing step or the scope of the duration is between 15 minutes and 30 minutes, even more preferably between 20 minutes and 30 minutes. Therefore, the duration of the washing step can be about 20 minutes, about 25 minutes or about 30 minutes. Depending on the desired result, it is also possible to be lower than the time of 15 minutes, and it is also considered to be covered by the present invention.

[0071] Preferably, as described above, the washing step with liquid water at a temperature not exceeding 50° C. can be performed simultaneously or sequentially with the ultrasonic treatment.

[0072] Preferably, the washing step according to the invention is carried out with water at a pH between 1 and 8, preferably between 1 and 5, more preferably between 1 and 4, even more preferably between 1 and 3, still more preferably between 1 and 2. Alternatively, the washing step may be carried out with water at a pH between 10 and 14, more preferably between 11 and 13.5, even more preferably between 12 and 13.5, yet more preferably between 12.5 and 13.5. It is well known to those skilled in the art how to calculate the pH to reach a specific H3O + or OH - Concentration, that is, the corresponding amount of a particular acid or base to be added required for a specific pH value.

[0073] Preferably, in a method for producing a fungal fermentation medium from brewer's grains (BSG), the BSG is characterized by a final moisture content of between 50% and 75% by weight. Thus, the final moisture content can be achieved by dehydrating the BSG using methods known to those skilled in the art by pressing the material to a final moisture content of between 50% and 75% by weight. However, this is not meant to be construed as restrictive, as it will be more apparent to those skilled in the art that other means and methods for dehydrating the BSG may also be applied herein.

[0074] Therefore, in a preferred embodiment, the method for producing a fungal fermentation medium from brewer's grains (BSG) also includes a step of dehydrating the BSG by pressing the material to a final moisture content between 50% by weight and 75% by weight, wherein the dehydration step is before step (a1). This step is conventional and therefore known to those skilled in the art, and can be performed, for example, by pressing a side stream using a screw press (or any other suitable press). Alternatively, the lignocellulosic material, preferably lignocellulose, can be dried, for example, by using a fluidized bed dryer. A convection oven can also be used for this purpose. Preferably, in the method of the present invention, the water thus obtained is discarded and is not further used in the method of the present invention.

[0075] The method for producing a fungal fermentation medium may also include a further step of pre-processing the brewer's grains. Preferably, the pre-processing of the brewer's grains includes changing the particle size or other mechanical properties of the spent grains. For example, the pre-processing of the spent grains may include grinding of the spent grains. This step is conventional and is known to those skilled in the art.

[0076] It is further obvious to the skilled person that the residue of the brewer's grains obtained after step (a1) can be further pretreated, for example by, for example, breaking the particles, for example by grinding, crushing, pulverizing, etc. to increase its available surface area. This step disclosed herein as an optional step is known to the skilled person. The pretreatment step may also be referred to as mechanical pretreatment. In particular, the pretreatment step involving breaking the particles (e.g. by grinding, crushing, pulverizing) may also be referred to as mechanical treatment or mechanical pretreatment. Although the pretreatment step is described herein as after step (a1), it may also be implemented at other stages of the method, for example the brewer's grains may undergo the mechanical pretreatment steps discussed herein before performing step (a1). Preferably, each of these mechanical pretreatment procedures can be completed using a shredder or a disc grinder. Those skilled in the art will recognize that such a shredder or disc grinder is preferably located at the extractor outlet (extractor is understood herein as the reactor in which the extraction process, as described herein, will take place; extractor outlet is defined as the opening in the cavity for removing solid residues after extraction), wherein the shredder or the disc grinder is configured to process the spent grains so that the BSG surface area is further increased (i.e., the particle size is further reduced) for higher sugar extraction at the washing step.

[0077] In step (b) of the process for producing a fungal fermentation medium of the present invention, the extract obtained in step (a1) is combined with at least one non-carbohydrate nutrient for fungal cultivation.

[0078] Therefore, the aqueous extract of step (a1) obtained according to the present invention can be further supplemented with nitrogen source, carbon source, trace elements, vitamins and / or protein composition. 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. 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, in addition to the carbon source derived from BSG, no carbon source is added to the culture medium of the present invention. Trace elements as defined herein may include, for example, iron (III) salts, copper (II) salts, zinc salts, manganese (II) salts, molybdenum salts and / or cobalt (II) salts. Vitamins as defined herein preferably include vitamins that are beneficial to fungi growing on culture medium obtainable according to the method of the present invention, such as folic acid, riboflavin, pantothenic acid or biotin. Protein composition can be further used to supplement the aqueous extract of the present invention (a1). Preferably, a protein composition obtainable from proteins isolated from the aqueous extract of (a1) of the process of the invention, preferably by flocculation or by precipitation with CO2, is preferably used in the process of the invention.

[0079] Further preferably, according to step (b), the extract of step (a1 ) is preferably supplemented with at least one nitrogen source, as described above.

[0080] It is further preferred that in step (b), the extract of step (a1) is substantially undiluted. In this article, the term "substantially undiluted" preferably means a dilution of no more than 15% (preferably understood as a dilution of no more than 15% in which the final concentration of the extract at point (a1) in the culture medium of step (b) is reduced after step (b) by no more than 15%, more preferably no more than 10%. Therefore, preferably, the extract of step (a1) accounts for at least 85% v / v of the total culture medium obtained in step (b), preferably, the extract of step (a1) accounts for at least 90% of the total culture medium obtained in step (b), more preferably, the extract of step (a1) accounts for 90% v / v to 99% v / v of the total culture medium obtained in step (b), even more preferably, wherein the extract of step (a1) accounts for 94% v / v to 98% v / v of the total culture medium obtained in step (b).

[0081] Preferably, in the method of producing a fungal fermentation medium of the present invention, step (b) comprises adding solid matter, preferably adding a solid nitrogen source, as known to those skilled in the art.

[0082] The inventors have found that preferably, the culture medium obtainable in the method of the present invention, wherein the method comprises step (a1), is substantially free of fungal growth inhibitors, such as hydroxymethylfurfural or furfural. Thus, the culture medium obtainable as described herein is preferably capable of supporting fungal growth without the need for a further dilution step to reduce the concentration of fungal growth inhibitors.

[0083] The person skilled in the art realizes that if the culture medium obtainable according to the invention is post-processed by removing the fungal growth inhibitor, the use of a dilution step aimed at reducing its concentration to an acceptable level can be avoided.

[0084] Further examples of fungal growth inhibitors that may be formed during 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, acetylvanillin, feruloylamide, coumaroylamide).

[0085] As mentioned above, the undesirable presence of furfural and / or hydroxymethylfurfural and / or other undesirable compounds can be avoided. In addition, undesirable furfural and / or hydroxymethylfurfural and / or other undesirable compounds can be removed, for example, by gas stripping, by isoazeotropic distillation or by liquid-liquid extraction. For example, in one embodiment, furfural and / or other fungal growth inhibitors can be removed by using a suitable membrane. Optionally, furfural can be recovered and used for other industrial applications. Preferably, when extracting as described herein, the concentration of furfural does not exceed 0.6g / l, preferably the concentration of furfural does not exceed 0.2g / L, preferably the concentration of furfural does not exceed 0.15g / L, more preferably the concentration of furfural does not exceed 0.1g / L, even more preferably the concentration of furfural does not exceed 0.05g / L, and even more preferably the concentration of furfural does not exceed 0.01g / L. Preferably, when extracting according to the method of the present invention, the concentration of furfural in the final extract is as described herein.

[0086] Therefore, the present invention further relates to an embodiment, wherein in the medium obtained before step (b), the concentration of furfural does not exceed 0.6 g / L, preferably the concentration of furfural does not exceed 0.2 g / L, preferably the concentration of furfural does not exceed 0.15 g / L, more preferably the concentration of furfural does not exceed 0.1 g / L, even more preferably the concentration of furfural does not exceed 0.05 g / L, and even more preferably the concentration of furfural does not exceed 0.01 g / L.

[0087] In one embodiment of the method for producing a fungal fermentation medium from brewer's spent grains (BSG) of the present invention, the method comprises step (a2) comprising subjecting the BSG to a liquid extraction treatment with water.

[0088] Therefore, in step (a2) of the process of the present invention, C5 sugars in the lignocellulosic material contained in the BSG are extracted via a liquid extraction treatment with water.

[0089] In the method of the present invention, the extraction to obtain C5 in step (a2) may be carried out at a temperature between 140° C. and 180° C. Preferably, the extraction in step (a2) is carried out at a temperature between 145° C. and 175° C., more preferably at a temperature between 145° C. and 170° C., even more preferably at a temperature between 145° C. and 160° C., even more preferably at a temperature between 145° C. and 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 present invention, the extraction in step (a2) can be carried out at 140°C, 141°C, 142°C, 143°C, 144°C, 145°C, 146°C, 147°C, 148°C, 149°C, 150°C, 151°C, 152°C, 153°C, 154°C, 155°C, 156°C, 157°C, 158°C, 159°C, 160°C, 161°C, 162°C, 163°C, 164°C, 165°C, 166°C, 167°C, 168°C, 169°C, 170°C, 171°C, 172°C, 173°C, 174°C, 175°C, 176°C, 177°C, 178°C, 179°C or 180°C.

[0090] As known to those skilled in the art, temperature and pressure are linearly related. In the present invention, the extraction of obtaining C5 sugars in step (a2) can be carried out under a pressure of 30 bar to 50 bar. Therefore, the extraction of step (a2) can be carried out at 30 bar, 31 bar, 32 bar, 33 bar, 34 bar, 35 bar, 36 bar, 37 bar, 38 bar, 39 bar, 40 bar, 41 bar, 42 bar, 43 bar, 44 bar, 45 bar, 46 bar, 47 bar, 48 bar, 49 bar or 50 bar. Preferably, the extraction of step (a2) is carried out at 50 bar.

[0091] In the method of the present invention including step (a2), extraction is carried out to obtain C5 sugars from the lignocellulosic material contained in BSG. Therefore, extraction is carried out for the shortest time sufficient to obtain C5 sugars. In the present invention, extraction is carried out for up to 70 minutes. Preferably, the extraction of step (a2) is carried out for up to 50 minutes, more preferably up to 30 minutes, more preferably up to 25 minutes, more preferably up to 15 minutes, more preferably up to 10 minutes. Most preferably, the extraction of step (a2) is carried out for up to 5 minutes to obtain C5 sugars. In the present invention, the extraction of step (a2) can therefore preferably be carried out for 5 minutes to 15 minutes, more preferably about 15 minutes, about 10 minutes or about 5 minutes. Preferably, the extraction of step (a2) is carried out for at least 5 minutes. In addition, the extraction can be carried out at a pressure of 5 bars to 50 bars. Preferably, the extraction is carried out at a pressure of 10 bars to 50 bars, more preferably at a pressure of 30 bars to 50 bars. Preferably, pressure and temperature are selected so that under the extraction conditions in step (a2), water remains liquid.

[0092] Preferably, step (a2) of the method of the present invention is carried out at a severity factor between 2.9 and 3.3. Thus, step (a2) may be carried out at a severity factor between 2.9 and 3.0, at a severity factor between 3.0 and 3.1, at a severity factor between 3.1 and 3.2 and / or at a severity factor between 3.2 and 3.3. Preferably, the steam pretreatment in step (a2) may be carried out at a severity factor between 3.1 and 3.3, more preferably, the steam pretreatment in step (a2) may be carried out at a severity factor between 3.1 and 3.2.

[0093] In one embodiment, step (a2) of the process of the present invention may be carried out at a severity factor between 2.0 and 4.0, more preferably at a severity factor between 2.5 and 3.3, even more preferably at a severity factor between 2.9 and 3.3. A particularly preferred embodiment is one in which step (a2) is carried out at a severity factor between 2.9 and 3.3 and at a temperature between 145°C and 175°C, preferably at a temperature between 145°C and 170°C, more preferably at a temperature between 145°C and 160°C, even more preferably at a temperature between 145°C and 155°C, even more preferably at a temperature of about 150°C. Thus, in this particularly preferred embodiment, step (a2) of the present invention may be carried out at a temperature of 145°C, 146°C, 147°C, 148°C, 149°C, 150°C, 151°C, 152°C, 153°C, 154°C or 155°C. In an alternative embodiment, step (a2) of the process of the present invention may be carried out between 130°C and 140°C. In this embodiment, the pH of the water used in step (a2) is preferably between 1.0 and 3.0.

[0094] Alternatively, it is preferred that step (a2) of the method of the present invention is performed at a severity factor between 2.0 and 3.0.

[0095] In one embodiment of the invention, the water used for the liquid extraction treatment with water may contain a dilute acid, such as no more than 1% w / w of the acid, 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. Particularly suitable are 0.2% w / w or 0.4% w / w, or 0.8% w / w or 0.9% w / w H2SO4 preparations. However, the present invention also encompasses embodiments in which the water contains up to 2% w / w of the acid, such as H2SO4. This is particularly applicable to embodiments in which the lignocellulosic material is brewer's grains. Particularly suitable are acids ranging from 0.4% w / w to 1.6% w / w, preferably ranging from 0.6% w / w to 1.6% w / w, more preferably ranging from 0.7% w / w to 1.5% w / w, preferably H2SO4 preparations. In a specific embodiment, the concentration of the acid (preferably H2SO4) is between 0.4% w / w and 1% w / w, more preferably between 0.4% w / w and 0.9% w / w, and still more preferably between 0.5% w / w and 0.9% w / w. In a specific embodiment, the concentration of the acid (preferably H2SO4) is between 1.1% w / w and 1.6% w / w. This is particularly applicable to embodiments in which the lignocellulosic material is brewer's grains. It should be understood that this embodiment can be combined with the embodiments of the previous three paragraphs listing preferred temperatures and severity factors for step (a2).

[0096] In one embodiment of the invention, the water used for the liquid extraction treatment with water may contain a dilute alkali, for example not more than 1% w / w of said alkali. Particularly suitable is NaOH in a concentration between 0.2% w / w and 0.8% w / w, more preferably between 0.6% w / w and 0.8% w / w, in particular between 0.7% w / w and 0.8% w / w. Particularly suitable is 0.7% w / w NaOH, more preferably particularly suitable is 0.8% w / w NaOH. Alternatively, particularly suitable is 0.2% w / w NaOH.

[0097] In an alternative embodiment of the present invention, the water used for the liquid extraction process is replaced by a phosphate buffer having a pH range between 4 and 6, more preferably a pH range between 4.5 and 5.5.

[0098] Preferably, in the process of the present invention, the lignocellulosic material comprised in the BSG to be extracted in step (a2) of the process is untreated, preferably wherein said material has not been dewatered.

[0099] Preferably, the step (a2) of aqueous extraction of lignocellulosic material, preferably industrial and / or agricultural side streams according to the present invention is carried out with water at a pH between 2.0 and 12.0, preferably between 3.0 and 10.0, more preferably between 4.0 and 8.0, even more preferably between 5.0 and 8.0. However, it is also preferred that the step (a2) of aqueous extraction of lignocellulosic material, preferably industrial and / or agricultural side streams according to the present invention is carried out with water at a pH between 1.0 and 12.0, preferably between 1.0 and 10.0, more preferably between 1.0 and 8.0, even more preferably between 1.0 and 8.0. As understood herein, the pH value 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 contacted with at least one lignocellulosic material, preferably industrial and / or agricultural side stream. It should also be understood herein that the addition of acid or base to water to a final concentration exceeding 1% w / w as described herein is preferably avoided.

[0100] More preferably, step (a2) of aqueous extraction of the lignocellulosic material, preferably industrial and / or agricultural side streams, requiring acid treatment according to the present invention is carried out with water at a pH between 1 and 5, more preferably at a pH between 1 and 4, even more preferably at a pH between 1 and 3, still more preferably at a pH between 1 and 2. This applies in particular to embodiments in which the lignocellulosic material is brewer's grains. Alternatively, in case of an alkali treatment, it is preferred that step (a2) of aqueous extraction of the lignocellulosic material, preferably industrial and / or agricultural side streams, requiring alkali treatment according to the present invention is carried out with water at a pH between 10 and 14, more preferably at a pH between 11 and 13.5, even more preferably at a pH between 12 and 13.5, still more preferably at a pH between 12.5 and 13.5. This applies in particular to embodiments in which the lignocellulosic material is wheat bran. A person skilled in the art is well aware of how to calculate the pH required to reach a specific H3O + or OH - Concentration, that is, the corresponding amount of a particular acid or base to be added required for a specific pH value.

[0101] 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 step) can be carried out in a batch process. However, preferably, the aqueous extraction step (a2) of the method of the present invention is carried out in a continuous extraction process, as known to those skilled in the art.

[0102] Preferably, the batch extraction is carried out in a closed system, where the spent grain and water are loaded only once at the beginning. The reactor is preferably pressurized and remains closed until the mixture cools to at least 40°C. The continuous extraction is preferably carried out in a manner where the continuous feed and withdrawal of the raw material are at the same rate, resulting only in a pressure drop when withdrawing the material in the steam explosion. In a semi-continuous process, the feed and reaction mixture are continuously fed in and out of the system. The feed and withdrawal of the raw material are not at the same rate, and the raw material is fed in a cyclic manner.

[0103] As will be understood herein, as described in step (a1), the step of washing with water may also be performed as a batch process or a semi-continuous process. Thus, in one embodiment, as described herein, the step of washing with water in step (a1) is performed as a batch process, as described herein. Preferably, in a 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, wherein the step of washing with water in step (a1) is performed 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 performed as a semi-continuous process, as described herein. Preferably, in a semi-continuous process, at least 80% of the C5 sugars are recovered, more preferably at least 90% of the C5 sugars are recovered, even more preferably at least 96% of the C5 sugars are recovered.

[0104] The method of the invention for producing a fungal fermentation medium from brewer's grains (BSG) comprising step (a2) further comprises a step (b) of combining the extract so obtained with at least one non-carbohydrate nutrient for fungal cultivation. Step (b) in the embodiment of the method described herein is as described above, i.e., with respect to the method of the invention for producing a fungal fermentation medium from brewer's grains comprising step (a1).

[0105] Preferably, in the method of the present invention, substantially no sugar is added to the culture medium, except for the sugar present in the extract of step (a1) or the extract of step (a2), as applicable. "Substantially" preferably means in the present context that the added sugar does not exceed 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 that the added sugar does not exceed 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, where applicable, no additional sugar is added to the sugar present in the extract of step (a1) or the extract of step (a2). Therefore and preferably, in the method of the present invention, no sugar is added to the culture medium, except for the sugar present in the extract of step (a1) or the extract of step (a2), as applicable.

[0106] 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 brewer's grains, may be subjected to a further pretreatment step before being subjected to the method of the present invention for producing a fungal fermentation medium. Thus, the lignocellulosic material as used herein (e.g., as described herein, contained in brewer's grains) may be subjected to a pretreatment according to a method selected from the group consisting of washing, solvent extraction, solvent swelling, crushing, grinding, dilute acid pretreatment, hot water pretreatment, alkaline pretreatment, lime pretreatment, wet oxidation, wet explosion, ammonia fiber explosion, organic solvent pretreatment, biological pretreatment, ammonia percolation, ultrasound, electroporation, microwave, supercritical CO2, supercritical H2O, ozone and gamma radiation. Preferably, no further preprocessing steps are included in the method of the present invention for producing a fungal fermentation medium, except as described in step (a).

[0107] As understood herein, in the method of the present invention comprising step (a1) or in the method of the present invention comprising step (a2), the extraction step (a1) or (a2) (where applicable) of extracting the lignocellulosic material contained in the brewer's grains is carried out in a suitable reactor known to those skilled in the art. At least one lignocellulosic material, preferably an industrial and / or agricultural side stream, is preferably loaded into the reactor at a solid loading of between 5% weight / volume (w / w) and 70% weight / volume (w / w), preferably between 10% (w / w) and 55% (w / w), and treated with water or with steam. The solid loading as understood herein is defined as the weight of the dry solid side stream (the loaded material) to the complete reaction volume (including the water and at least one solid side stream used for extraction), and is preferably expressed as a percentage. The weight of the loaded material is understood herein as dry weight. The solid loading required in the present invention depends on the loaded material and the reactor characteristics. As understood by those skilled in the art, the amount of the loaded material preferably should not affect the stirring and heat transfer inside the reactor. It also depends on the amount of liquid extract that must be recovered and its composition. As understood herein, the reactor may preferably be loaded with up to 55% w / w of dry lignocellulosic biomass (also referred to as dry solid sidestream). It should be noted that in certain embodiments, alternatively, the reactor may preferably be loaded with wet lignocellulosic biomass. As known to those skilled in the art, the preferred loading depends on the material, i.e., lignocellulosic material, preferably industrial and / or agricultural sidestream.

[0108] Optionally, in the process of the invention comprising step (a1) or in the process of the invention comprising step (a2), the aqueous extract obtained in step (a1) or step (a2) may undergo further processing. Processing the aqueous extract obtained in (a1) / (a2) before step (b) may preferably comprise separating proteins from the aqueous extract of (a1) / (a2). Any separation method suitable for this purpose and known to those skilled in the art may be used in the process of the invention. Preferably, proteins are separated from the aqueous extract, preferably by flocculation or by precipitation with CO2, preferably followed by mechanical separation, for example with a decanter centrifuge. Optionally, the protein thus obtained may be further prepared as a solution comprising not less than 50% w / w of polypeptides and / or amino acids. Further optionally, the protein thus obtained is preferably hydrolyzed by using a proteolytic enzyme, in particular selected from alkaline protease, papain, proteinase K and trypsin. The proteolytic enzymes may be used at a concentration between 0.01% (w / w) and 5% (w / w) (which is understood herein as the total concentration of all enzymes used herein) and / or at a temperature between 15° C. and 100° C. and / or for a period between 0.5 hours and 96 hours. The solution thus obtained containing hydrolyzed proteins (i.e. polypeptides and amino acids) may optionally be further used in step (b) of the process of the invention. It is also noted that further processing of the aqueous extract obtained in step (a1) or step (a2) may remove any contaminants and / or fungal growth inhibitors.

[0109] As used herein, the term "polypeptide" encompasses proteins, peptides and polypeptides, wherein the protein, peptide or polypeptide may or may not be post-translationally modified. The post-translational modification may be, for example, phosphorylation, methylation and / or glycosylation. However, other post-translational modifications recognizable by those skilled in the art are also encompassed.

[0110] Preferably, in the method for producing a fungal fermentation medium according to the invention, no protein is isolated from the extract, i.e. the product of the method. The inventors have found that it is beneficial to keep a protein as defined herein in the culture medium as a nitrogen source. As understood herein, a protein may include amino acids, peptides and / or proteins.

[0111] Preferably, step (a1) or step (a2) in the method for producing a fungal fermentation medium is characterized in that the solid loading of the reactor is between 1% and 25%, preferably 1% and 15%, more preferably 4% w / w and 14% w / w, even more preferably 8% and 14% w / w, even more preferably 10% w / w and 14% w / w, even more preferably 12% w / w and 14% w / w. In a specific embodiment, the solid loading of the reactor is between 5% and 15%.

[0112] As understood herein, the product of step (b) of the inventive method may be further processed. Optionally, the water contained in the product of step (b) may be removed by, for example, spray drying, drum drying, belt drying or freeze drying to produce a dry fungal fermentation medium, which, as known to those skilled in the art, may have an improved storage time. Optionally, within the scope of the inventive method, the fungal fermentation medium of the present invention may be further sterilized or pasteurized. Optionally, the fungal fermentation medium obtainable according to the inventive method may be further supplemented with, for example, salt (preferably sodium chloride, sodium nitrate, magnesium sulfate, calcium chloride, calcium carbonate, ammonium chloride, diammonium hydrogen phosphate, ammonium sulfate, potassium phosphate, disodium phosphate and / or monosodium phosphate), antibiotics or water. Water as used herein is preferably used to optimize the concentration of each component in the culture medium.

[0113] Further preferably, the pH of the fungal fermentation medium obtainable in the method of the invention for producing a fungal fermentation medium from at least one lignocellulosic material, preferably an industrial and / or agricultural side stream, can be set to a desired value, preferably by adding a buffer. Particularly useful for the present invention are citrate or phosphate buffer systems. Furthermore, in the fermenter, the pH can be adjusted by adding appropriate amounts of urea, NaOH, ammonia, sulfuric acid, phosphoric acid or hydrochloric acid.

[0114] Preferably, the fungal fermentation medium of the present invention comprises complex C5 sugars (also called complex C5 polysaccharides). As understood herein, complex C5 sugars are C5 sugars of which preferably at least 60%, at least 70%, at least 80% or at least 90% are composed of molecules comprising two or more sugar units.

[0115] Therefore and preferably, complex C5 polysaccharides account for at least 50% of all sugars in the culture medium, preferably, wherein complex C5 polysaccharides account for at least 65% of all sugars in the culture medium, more preferably, wherein complex C5 polysaccharides account for at least 80% of all sugars in the culture medium. It should be understood that the culture medium obtainable in the method of the present invention (wherein the method includes step (a')) does not necessarily meet this condition, because the product of step (a') contains C6 sugars. Therefore and preferably, complex C5 polysaccharides account for at least 50% of all sugars in the extract produced in step (a1) or step (a2), preferably, wherein complex C5 polysaccharides account for at least 65% of all sugars in the extract, more preferably, wherein complex C5 polysaccharides account for at least 80% of all sugars in the extract.

[0116] Preferably, the culture medium obtainable according to the method of the present invention is suitable for culturing at least one fungal strain selected from the orders of Peliciales, Boletales, Chanterelles, Agaricales, Polyporaceae, Russulares, Auriculariales, Sordoriales and Hypocreales, preferably at least one fungal strain selected from the group consisting of Pleurotus pulmonatus, Pleurotus ostreatus, Pleurotus rosus, Pleurotus rosus, Morchella rubra ...

[0117] The present invention also encompasses such an embodiment, wherein in step (a1) steam treatment is followed by washing with liquid water, then the solid residue obtained is subjected to liquid water treatment as in step (a2), and then step (b). Alternatively, step (a2) may be followed by step (a1) performed on the solid residue obtained after said step (a2), followed by step (b). The present invention also encompasses 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 present inventors, more than one step is performed to the BSG selected from step (a1) and step (a2) (or respectively the residue obtained in the previous step), as described herein, allowing more sugar to be recovered and avoiding the mass production of fungal growth inhibitors.

[0118] In one embodiment, the step (a1) of steam treatment followed by washing with liquid water is followed by another step (a1) of steam treatment of the solid residue obtained in the first step (a1) followed by washing with liquid water, and then step (b). Alternatively, step (a') may be performed before step (b).

[0119] In one embodiment, step (a2) of liquid water treatment is followed by another step (a2) of liquid water treatment of the solid residue obtained in the first step (a2) and then step (b). Alternatively, step (a') may be performed before step (b).

[0120] In one embodiment, the step (a1) of steam treatment followed by washing with liquid water treatment is followed by another step (a2) of liquid water treatment of the solid residue obtained in the first step (a1) and then step (b). Alternatively, step (a') may be carried out before step (b).

[0121] In one embodiment, the step (a2) of liquid water is followed by another step (a1) of steam treatment of the solid residue obtained in the first step (a2) followed by washing with liquid water, and then step (b). Alternatively, step (a') may be performed before step (b).

[0122] In the method of the present invention, in step (a1) or step (a2), a solid lignocellulosic residue is generated. The residue can be further used in the method of the present invention. Therefore and preferably, the method for producing a fungal fermentation medium from brewer's grains can also include enzymatic hydrolysis of the solid lignocellulosic residue obtained in step (a1) or step (a2) with cellulase, and a step (a') of separating the hydrolyzed liquid product from the solid residue. Therefore, the lignocellulosic residue is loaded into a second reactor (preferably at a loading between 5% w / w and 50% w / w), and the lignocellulosic residue is enzymatically hydrolyzed with cellulase in the second reactor. Preferably, step (a') is carried out at a temperature between 15°C and 100°C, more preferably at a temperature between 40°C and 80°C. Preferably, step (a') is carried out at a pH between 3.0 and 8.0, more preferably at a pH between 4.0 and 7.0. Preferably, step (a') is carried out for a time between 10 minutes and 200 hours.

[0123] Preferably, the solid lignocellulosic residue produced in step (a1 ) or step (a2) does not undergo further processing before it is subjected to step (a').

[0124] Preferably, the extract obtained in step (a') comprises C6 sugars. C6 sugars are preferably defined as hexoses (ie sugars comprising saccharide units of six carbon atoms) or oligosaccharides and polysaccharides consisting of hexose units.

[0125] 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 mixed extract in step (a1) to the extract in step (a2) is not particularly limited. The ratio may depend on the requirements of the specific culture medium production and will be a result of the reactor solids loading applied to each of these steps.

[0126] In another embodiment, the present invention relates to a fungal fermentation medium, which can be obtained in the method for producing a fungal fermentation medium of the present invention. The fungal fermentation medium of the present invention obtainable in the method for producing a fungal fermentation medium from at least one lignocellulosic material, preferably industrial and / or agricultural side streams of the present invention may optionally further include 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, except for those carbon sources obtained by extracting BSG, no carbon source is added herein. Therefore, except for those sugars obtained in any one of the steps (a1), (a2) and / or (a') of the present method, the present invention preferably encompasses not adding additional sugars. Trace elements as defined herein may include, for example, iron (III) salts, copper (II) salts, zinc salts, manganese (II) salts, molybdenum salts and / or cobalt (II) salts. Vitamins as defined herein preferably include vitamins that are beneficial for the growth of fungi on a culture medium obtainable according to the inventive method as defined herein.

[0127] Preferably, in the fungal fermentation medium of the present invention, substantially all sugars are derived from BSG. "Substantially" in this context preferably means that the added sugar does not exceed the amount corresponding to 5% w / w of the sugar present in the extract of step (a1) and / or the extract of step (a2) and / or the extract of step (a'), more preferably the added sugar does not exceed the amount corresponding to 1% w / w of the sugar 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, where applicable, no additional sugar is added to the sugar present in the extract of step (a1) and / or the extract of step (a2) and / or the extract of step (a').

[0128] In the fungal fermentation medium of the present invention, complex C5 polysaccharides account for at least 50% of all sugars in the medium, preferably, complex C5 polysaccharides account for at least 65% of all sugars in the medium, and more preferably, complex C5 polysaccharides account for at least 80% of all sugars in the medium. It should be understood that the culture medium obtainable in the method of the present invention (wherein the method includes step (a')) does not necessarily meet this condition because the product of step (a') contains C6 sugars. Therefore and preferably, complex C5 polysaccharides account for at least 50% of all sugars in the extract produced in step (a1) or step (a2), preferably, wherein complex C5 polysaccharides account for at least 65% of all sugars in the extract, and more preferably, wherein complex C5 polysaccharides account for at least 80% of all sugars in the extract.

[0129] In one embodiment of the present invention, wherein the extract of step (a1) or the extract of step (a2) is mixed with the extract of step (a'), the complex polysaccharide may account for less than 50% of all sugars in the culture medium.

[0130] 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 as described herein may be further processed into a dry form. To this end, the water contained in the fungal fermentation medium obtainable according to the present invention may be removed by, for example, spray drying, belt drying, drum drying or freeze drying, thereby producing a dry fungal fermentation medium, which, as known to those skilled in the art, may have an improved shelf life. Optionally, within the scope of the inventive method, the fungal fermentation medium of the present invention may be further sterilized.

[0131] In another embodiment, the present invention relates to the use of the fungal fermentation medium of the present invention in the production of fungal biomass. Therefore, the fungal fermentation medium of the present invention, i.e. the medium as described above and obtainable according to the method disclosed above, can be used in the method of the present invention for producing fungal biomass.

[0132] Therefore, in another embodiment, the present invention relates to a method for producing fungal biomass, comprising the step of submerged fermentation of at least one fungal strain using the fungal fermentation medium of the present invention.

[0133] According to the present invention, submerged fermentation can be operated as a batch, fed-batch or continuous process. These three main fermentation methods are known to those skilled in the art and differ in the outflow and inflow of materials to / from the fermentation vessel.

[0134] As understood herein, submerged fermentation or submerged fungal fermentation is defined as cultivating fungi in a liquid culture medium. As known to those skilled in the art, an alternative to submerged fungal fermentation is surface fungal fermentation, also referred to as solid-state fungal fermentation. According to methods known to those skilled in the art, a liquid fungal fermentation medium (herein understood as a fungal fermentation medium of the present invention in a solution or suspension (optionally pH adjusted)) is placed in a closed container (herein preferably a fermentor), which is usually sterilized to kill organisms that may interfere with fungal growth. An inoculum of at least one fungal strain as defined herein is introduced into a container (herein preferably a fermentor), and at least in the case of aerobic fungi, air is blown into the container. The contents of the container (herein a fermentor) are preferably stirred according to methods known to those skilled in the art, and are preferably integrated into the fermentor design. Stirring allows nutrients and oxygen present in the culture medium to be continuously contacted with the fermented material (herein at least one fungal strain), and preferably, temperature and pH are controlled at a level suitable for fungi. After a certain period of time, usually between 1 day and 12 days (this depends on the type of fermentation, fungi and precise fermentation conditions, etc.), fungal biomass can be harvested. (As will be noted by those skilled in the art, the timings given herein may not necessarily apply to continuous fermentation situations.) However, as will be known to those skilled in the art, mixing may also be achieved by methods other than stirring, which may also affect the morphology of the fungal cells, as well as causing the fungal cells to be subjected to shear stress. As will be understood herein, the mixing method is not meant to be limiting, and any applicable method known to those skilled in the art falls within the scope of the present invention.

[0135] Batch process is characterized in that no material flows into the fermentation vessel. In batch process, all nutrients are provided at the beginning of cultivation, and no nutrients are added in the subsequent bioprocess. During the whole bioprocess, except gas, acid and alkali, no additional nutrients are added. Then the bioprocess continues until nutrients are consumed. This strategy is suitable for rapid experiments, such as optimization of strain characterization or nutrient medium. The shortcoming of this convenient method is that biomass and product yields are limited. Because carbon source and / or oxygen transfer are usually limiting factors, microorganisms are not in exponential growth phase for a long time. After the bioprocess running in batch mode ends, only biomass or culture medium are harvested and appropriately treated to obtain desired products. From the perspective of bioreactor, the process is repeatedly interrupted by cleaning and sterilization steps, and biomass is only produced in stages.

[0136] In the fed-batch process, substrate, nutrients and other materials can be added to the fermentation vessel to extend possible culture time or increase productivity, etc. The advantage of feeding during the culture period is that it allows to achieve a higher overall product amount. Under specific growth conditions, microorganisms and / or cells constantly double and therefore follow an exponential growth curve. Therefore, in certain embodiments, the feed rate can also increase exponentially. Usually, for example, substrate is pumped to the culture vessel from a supply bottle by a silicone tube. The user can manually set the feeding (linear, exponential, pulse mode) at any time, or add nutrients when meeting specific conditions (such as when reaching a specific biomass concentration or when nutrients are exhausted). The fed-batch process provides a wide range of control strategies, and is also suitable for highly specialized applications. However, it can increase processing time and potentially lead to inhibition by the accumulation of toxic byproducts.

[0137] Preferably, in the method for the present invention, submerged fermentation is operated as a continuous process. After the batch growth period, a balance (also referred to as steady state) about a particular component is established. Under these conditions, when fresh culture medium is removed, the same fresh culture medium (chemostat) is added. These biological processes are called continuous culture, and are particularly suitable when excessive nutrients will cause inhibition due to, for example, acid or ethanol accumulation or overheating. Other advantages of the method include reduced product inhibition and improved space-time yield. When removing culture medium, harvested cells, here is the reason why the inflow and outflow rate must be less than the doubling time of microorganisms. Alternatively, cells can be retained in a variety of ways (for example, in a rotary filter), which is called perfusion. In a continuous process, compared with a fed-batch process, the space-time yield of a bioreactor can even be further improved.

[0138] Therefore, the fungal fermentation medium obtainable according to the method of the present invention is provided to a fermenter suitable for growing fungal mycelium. Suitable fermenters are known to those skilled in the art. For example, suitable stirred tanks or airlift fermenters with specific agitators for reducing shear stress are useful within the scope of the present invention. Fungal fermentation medium should be understood as a medium obtainable according to the present invention and the method disclosed herein. As understood herein, in certain embodiments of the present invention, the fungal fermentation medium may be further sterilized. As known to those skilled in the art, sterilization may be performed by exposing the culture medium to an elevated temperature for a period of time. Typically, it is performed at a temperature between 150°C and 200°C for a time between 30 seconds and 10 minutes. However, the conditions as described herein are not meant to limit the scope of the present invention. For example, the process may be performed at a temperature between 120°C and 200°C, preferably between 150°C and 200°C, and / or for a time between 30 seconds and 20 minutes, preferably between 30 seconds and 20 minutes.

[0139] Preferably, fungal growth is carried out at a temperature between 15°C and 40°C. Typically, a constant temperature is maintained throughout the process, which temperature can be selected for optimal growth of a particular fungal strain, as known to those skilled in the art. For example, in the case of Pleurotus ostreatus, growth is preferably carried out at a temperature between 25°C and 30°C. Further preferably, growth is carried out at a pH between 3.0 and 8.5. The pH of the culture medium can be adjusted within the scope of the method for producing a fungal fermentation medium of the present invention. As will be appreciated by those skilled in the art, the choice of pH may depend on the fungal strain to be cultivated, or on potential contaminating strains to be excluded from the growth. Further preferably, growth is carried out for a time between 12 hours and 240 hours. However, as will be appreciated by those skilled in the art, if growth is carried out as a continuous process, it is preferably not limited to 240 hours.

[0140] As understood herein, the selection of growth conditions (including, for example, 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 a mass or mycelium.

[0141] In 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 as food by mammals (preferably humans) without causing any adverse reactions. Adverse reactions are defined herein as food poisoning or undesirable taste properties that hinder consumption. Edible fungi are not limited to their fruiting bodies (mushrooms) herein, but other parts of the fungi (e.g., mycelium) may also be considered as edible mushrooms.

[0142] 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 the phylum Basidiomycota and Ascomycota.

[0143] According to the present invention, the at least one fungal strain may be selected from the phylum Basidiomycota. Preferably, the at least one fungal strain selected from the phylum Basidiomycota may be a fungal strain selected from the subphylum Agaricida. As defined herein, the fungal strain selected from the subphylum Agaricida may be a fungal strain selected from the class Agaricales. Preferably, the fungal strain selected from the class Agaricales may be a fungal strain selected from the orders Boletales, Chanterelles, Agaricales, Polyporales, Russulares and Auriculariales.

[0144] As defined herein, the fungal strain selected from the family Boletaceae is preferably Boletus edulis. In certain embodiments, the fungal strain selected from the class Agariciformis may be a fungal strain selected from the order Polyporaceae. Preferably, as defined herein, the fungal strain selected from the order Polyporaceae may be a fungal strain selected from the family Subgridiaceae, Polyporaceae, Ganodermaceae, Hydrangeaceae.

[0145] As defined herein, the fungal strain selected from the family Sub-Grifola frondosa is preferably Grifola frondosa. As defined herein, the fungal strain selected from the family Polyporaceae is preferably selected from the family Polyporaceae P. umbellatus and L. sulphureus. As defined herein, the fungal strain selected from the family Hydrangeaceae is preferably S. crispa.

[0146] Preferably, the fungal strain selected from the class Agaricomycetes may be a fungal strain selected from the order Canthareales. Further preferably, the fungal strain selected from the order Canthareales may be a strain selected from the family Cantharellaceae and the family Odontaceae. As defined herein, the strain selected from the family Cantharellaceae may be C. cornucopioides or C. cibarius, preferably C. cibarius. As further defined herein, the strain selected from the family Odontaceae may be H. repandum.

[0147] Alternatively, the fungal strain selected from the class Agaricomycetes may be a fungal strain selected from the order Boletales.Preferably, as defined herein, the fungal strain selected from the order Boletales may be a fungal strain selected from the family Boletaceae and the family Scleroderma.

[0148] Alternatively, the fungal strain selected from the class Agaricomycetes may be a fungal strain selected from the order Russulares. Further preferably, as defined herein, the fungal strain selected from the order Russulares may be a fungal strain selected from the family Hericium and the family Polyporaceae. Preferably, the fungal strain selected from the order Russulares is a fungal strain selected from the family Hericium, preferably selected from the family H.erinaceus and H.coralloides. Further preferably, the fungal strain selected from the family Polyporaceae is B.berkeleyi.

[0149] Alternatively, the fungal strain selected from the class Agaricus may be a fungal strain selected from the order Auriculariales, more preferably a fungal strain selected from the family Auriculariaceae. Preferably, the fungal strain selected from the family Auriculariaceae is A. auricula-judae.

[0150] Preferably, in the method of the present invention, the at least one fungal strain is selected from the order Agaricales. Thus, the at least one fungal strain selected from the order Agaricales may be selected from the group consisting of Truffleaceae, Pleurotus eryngii, Agaricaceae, Microhylidae, Strophariaceae, Hylomycetaceae, Tricholomataceae, Omphalaceae, Bubbleheadaceae, Schizophyllaceae and Bolognathaceae.

[0151] As defined herein, the fungal strain selected from the family of Agrocybe is preferably L. edodes. As further defined herein, the fungal strain selected from the family of Strophariaceae is preferably a fungal strain selected from the family of Agrocybeae and H. capnoides. As further defined herein, the fungal strain selected from the family of Agrocybeae is preferably a fungal strain selected from the family of C. indica. As further defined herein, the fungal strain selected from the family of Tricholomataceae is preferably a fungal strain selected from the family of H. tesselatus and C. nuda. As further defined herein, the fungal strain selected from the family of Omphalomaceae is preferably C. gigantean. As further defined herein, the fungal strain selected from the family of Aspergillus is preferably F. velutipes. As further defined herein, the fungal strain selected from the family of Schizophyllum is preferably S. commune. As further defined herein, the fungal strain selected from the family of Bolognathaceae is preferably F. hepatica.

[0152] At least one fungal strain according to the invention selected from the order Agaricales may be selected from the family Tuberaceae. Preferably, the fungal strain according to the invention selected from the family Tuberaceae is white truffle (T. magnatum), summer truffle (T. estivum), hooked truffle (T. uncinatum), Indian truffle (T. indicum), brown red truffle (T. rufum) or black cell truffle (T. melanosporum), more preferably black cell truffle and white truffle.

[0153] More preferably, at least one fungal strain selected from the order Agaricales may be a fungal strain selected from the family Pleurotus. Even more preferably, at least one fungal strain of the present invention is a fungal strain selected from the group consisting of Pleurotus pulmonatus, Pleurotus ostreatus, Pleurotus aurantii and Pleurotus rosaceus, more preferably selected from the group consisting of Pleurotus pulmonatus or Pleurotus ostreatus, most preferably Pleurotus pulmonatus.

[0154] According to the present invention at least one fungal strain selected from the order Agaricales may be selected from the family Agaricaceae. Preferably, the fungal strain selected from the family Agaricaceae as defined herein is A. bisporus or A. blazei, more preferably A. bisporus.

[0155] According to the present invention, the at least one fungal strain may be selected from the phylum Ascomycota. Preferably, the at least one fungal strain selected from the phylum Ascomycota may be a fungal strain selected from the subphylum Pezizomycotina. As defined herein, the fungal strain selected from the subphylum Pezizomycotina may be selected from the class Pezizomycotina. Preferably, within the scope of the present invention, the fungal strain selected from the class Pezizomycotina may be a fungal strain selected from the order Peziales.

[0156] Further preferably, at least one fungal strain as defined in the method for producing fungal biomass of the present invention can be selected from the order Pyriculariales. Preferably, the fungal strain selected from the order Pyriculariales can be selected from the family Morchellaceae. Preferably, the fungal strain selected from the family Morchellaceae is Morchella fusca, Morchella fusca or Morchella fusca.

[0157] Alternatively, at least one fungal strain selected from the Ascomycota can be a fungal strain selected from the class of dung mycotoxins. Preferably, at least one strain selected from the class of dung mycotoxins as defined herein can be a fungal strain selected from the order of fleshy mycotoxins. Further preferably, the fungal strain selected from the order of fleshy mycotoxins can be a fungal strain selected from the family of Cordyceps. Even further preferably, the fungal strain selected from the family of Cordyceps is a fungal strain selected from Cordyceps militaris and Cordyceps sinensis (C.sinensis). Alternatively, the fungal strain selected from the order of fleshy mycotoxins can be a fungal strain preferably selected from the family of plexylocystis. Further preferably, the fungal strain selected from the family of plexylocystis can be a Fusarium strain. In another embodiment, the fungal strain selected from the class of dung mycotoxins can be a fungal strain selected from the family of dung mycotoxins. Further preferably, the fungal strain selected from the family of dung mycotoxins can be a Neurospora strain.

[0158] As disclosed herein, 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 may be selected from the subdivision Pezizomycotina and the subdivision Agaricomycotina.

[0159] As further disclosed herein, in the process 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 the class Pechomycetes, Agaricomycetes and Sphaerotheca.

[0160] 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 the order Pesciales, Boletales, Chanterelles, Agaricales, Polyporales, Russulares, Auriculariales, Sordales and Hypocreales.

[0161] 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 the group consisting of Morelaceae, Truffleaceae, Pleurotus eryngii, Agaricaceae, Agaricaceae, Cantharellaceae, Odontaceae, Boletaceae, Sub-Grifolaceae, Polyporaceae, Strophariaceae, Pleurotus eryngii, Tricholomataceae, Omphalaceae, Bubbleheadaceae, Schizophyllaceae, Sclerodermaceae, Ganodermaceae, Hydrangeaceae, Hericium erinaceae, Polyporaceae, Cordyceps, Auriculariaceae and Bolognese.

[0162] 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 Pleurotus pulmononis, Pleurotus ostreatus, Pleurotus junceus or Pleurotus roxburghii, even more preferably Pleurotus pulmononis or Pleurotus ostreatus.

[0163] 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 Morchella edulis, Morchella nigra, or Morchella serrata.

[0164] As described herein, co-fermentation of more than one fungal strain is also encompassed by the present invention. The selection of strains for co-fermentation depends on their compatibility and can be performed by one skilled in the art.

[0165] It should be understood that according to the method of the present invention, the yield is at most 100%, preferably at most 99% (based on the conversion of the available carbon source in the culture medium), more preferably 85% to 95%. It should be understood that the biomass yield is calculated based on the amount of biomass produced and the ratio of the amount of substrate (e.g., C5 sugars) consumed.

[0166] In another 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 the family Pleurotus, in particular wherein the fungal strain is Pleurotus pulmonatus, Pleurotus ostreatus, Pleurotus rufipogon or Pleurotus rosaceus, more preferably selected from Pleurotus pulmonatus or Pleurotus ostreatus. In another embodiment, preferably, the fungal biomass comprises fungal cells of a fungal strain selected from the family Morchellaceae, wherein the fungal strain is Morchella fusca, Morchella fusca or Morchella edulis. However, the fungal biomass of the present invention is not limited to a single fungal strain. As described herein, co-fermentation of more than one fungal strain to produce the fungal biomass of the present invention is also included in the present invention. The selection of strains for co-fermentation depends on their compatibility and can be performed by those skilled in the art. In addition, the selection of strains for co-contained in the fungal biomass of the present invention depends on their properties and envisioned applications, as well as their growth rate, as disclosed herein.

[0167] The fungal biomass of the present invention preferably has a protein content between 10% and 60% (w / w).As further disclosed herein, the fungal biomass of the present invention preferably has a fiber content between 20% and 60% (w / w).

[0168] 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. Therefore, the present invention relates to a fungal biomass of the present invention, characterized in that the Asp / Asn content accounts for 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.

[0169] In another embodiment, the present invention relates to the use of the fungal biomass of the present invention in the production of fungi-based foods. Therefore, the present invention also relates to fungi-based foods that can be obtained as described herein. The fungi-based foods of the present invention can be prepared in any form known to those skilled in the art. For example, the fungi-based foods of the present 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.

[0170] The food according to the present invention can be, for example, a nutritional supplement. The nutritional supplement can be in the form of a liquid or solid, such as a pill, lozenge or tablet. For example, the nutritional supplement of the present invention can be a protein supplement and / or a carbohydrate supplement.

[0171] Food as understood herein may be a dairy product, such as yoghurt, milk beverage and ice cream. Food as understood herein may also relate to different embodiments of seafood products, such as crab cakes, fish cakes, tuna, salmon or shrimp.

[0172] The food can be an organized food or a textured food. Therefore, the food of the present invention contains all amino acids that are necessary for daily intake by people and cannot be resynthesized. In addition, the textured food product of the present invention is preferably heat-resistant, boiling-resistant and suitable for cooking. For example, as described herein, the fungus-based food of the present invention can be a meat substitute product. It should be noted that, preferably, the meat substitute product is an organized food or a textured food. It is also noted that the structure of the textured food improves the acceptability of the textured food by consumers. It is further noted that the inherent fiber structure of the fungal biomass of the present invention may be beneficial for producing textured foods or organized foods without using conventional texturing methods (such as extrusion).

[0173] According to the inventors, the food product of the present invention is characterized by enhanced taste.The taste properties of the food product can be determined for the examples as shown in the Examples section.

[0174] As understood herein, when referring to temperature, the term "about" preferably means ±3° C., more preferably ±1° C. When referring to other numerical values, the term "about" preferably means ±10% of the value, more preferably ±5% of the value, even more preferably ±1% of the value.

[0175] Other examples and / or implementations are disclosed in the following numbered items.

[0176] 1. A method for producing a fungal fermentation medium from brewer's spent grains (BSG), the method comprising:

[0177] (a1) extracting C5 sugars from lignocellulosic material contained in BSG via steam pretreatment followed by a washing step with liquid water at a temperature not exceeding 50°C, and (b) combining the extract thus obtained with at least one non-carbohydrate nutrient for fungal cultivation.

[0178] 2. The method according to claim 1, wherein the steam pretreatment is carried out at a severity factor between 2.9 and 3.3.

[0179] 3. A method according to item 1 or 2, wherein during the steam pretreatment, the lignocellulosic material contained in BSG is contacted with steam at a temperature between 130°C and 180°C, preferably between 160°C and 180°C, more preferably between 165°C and 175°C, and / or for a period of up to 30 minutes, preferably for a period of up to 15 minutes.

[0180] 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 not exceeding 40°C, preferably not exceeding 30°C, more preferably not exceeding 25°C.

[0181] 5. A method according to any one of items 1 to 4, wherein the BSG is characterized by a final moisture content between 50 wt% and 75 wt%, preferably, wherein the method further comprises a step of dehydrating the BSG by pressing the material to a final moisture content between 50 wt% and 75 wt%, wherein the dehydration step precedes step (a1).

[0182] 6. The method according to any one of items 1 to 5, wherein in step (b), the extract of step (a1) is substantially undiluted, preferably, wherein the extract of step (a1) accounts for 90% v / v to 99% v / v of the total culture medium obtained in step (b), more preferably, wherein the extract of step (a1) accounts for 94% v / v to 98% v / v of the total culture medium obtained in step (b).

[0183] 7. A method according to any one of items 1 to 6, wherein the culture medium is capable of supporting fungal growth without the need for a further dilution step to reduce the concentration of a fungal growth inhibitor.

[0184] 8. A method for producing a fungal fermentation medium from brewer's spent grains (BSG), the method comprising:

[0185] (a2) extracting C5 sugars from the lignocellulosic material contained in the BSG via a liquid extraction treatment with water at a temperature between 145° C. and 155° C. and / or for a period of up to 70 minutes, preferably for a period of up to 50 minutes, preferably at a pressure of 30 to 50 bar, and

[0186] (b) combining the extract thus obtained with at least one non-carbohydrate nutrient for fungal cultivation.

[0187] 9. The method according to claim 8, wherein step (a2) is carried out at a severity factor between 2.9 and 3.3

[0188] 10. The method according to item 8 or 9, wherein the lignocellulosic material contained in the BSG is untreated, preferably, wherein the material has not been dewatered.

[0189] 11. A method according to any one of items 1 to 10, wherein the method further comprises a step (a') of enzymatically hydrolyzing the solid lignocellulosic residue obtained in step (a1) or step (a2) with cellulase, separating the hydrolyzed liquid product from the solid residue, and mixing the liquid product with the extract of step (a1) or the extract of step (a2).

[0190] 12. The method according to any one of items 1 to 11, wherein substantially no sugar is added other than the sugar present in the extract of step (a1) or step (a2) or in the liquid product of the hydrolysis of step (a').

[0191] 13. The process according to any one of items 1 to 12, wherein step (a1) or step (a2) is characterized in that the solid loading of the reactor is between 4% w / w and 14% w / w.

[0192] 14. The method according to any one of items 1 to 13, wherein complex C5 polysaccharides account for at least 50% of all sugars in the extract of step (a1) or step (a2), preferably, wherein complex C5 polysaccharides account for at least 65% of all sugars in the extract of step (a1) or step (a2), more preferably, wherein complex C5 polysaccharides account for at least 80% of all sugars in the extract of step (a1) or step (a2).

[0193] 15. The method according to any one of items 1 to 14, wherein the culture medium is suitable for culturing at least one fungal strain selected from Pleurotus pulmonaria and Morchella rubrum.

[0194] 16. A fungal fermentation medium, which is obtainable according to the method of any one of items 1 to 15.

[0195] 17. The fungal fermentation medium according to item 16, wherein substantially all sugars are derived from BSG.

[0196] 18. A fungal fermentation medium according to item 16 or 17, wherein complex C5 polysaccharides account for at least 50% of all sugars in the medium, preferably, wherein complex C5 polysaccharides account for at least 65% of all sugars in the medium, more preferably, wherein complex C5 polysaccharides account for at least 80% of all sugars in the medium.

[0197] 19. Use of the culture medium according to any one of items 16 to 18 in fungal culture.

[0198] 20. A method for producing fungal biomass, 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.

[0199] 21. The method according to item 20, wherein the at least one fungal strain is an edible fungus.

[0200] 22. The method according to item 20 or 21, wherein the at least one fungal strain is Pleurotus ostreatus, Pleurotus eryngii or Pleurotus rosa, or wherein the at least one fungal strain is Morchella rubra, Morchella foetida, Morchella black vein or Morchella edulis.

[0201] 23. A fungal biomass produced according to the method of any one of items 20 to 22.

[0202] 24. Fungal biomass according to item 23, characterized in that the Asp / Asn content is at least 20% of the total amino acid content.

[0203] 25. Use of the fungal biomass according to item 23 or 24 in the production of food, preferably fungus-based food.

[0204] 26. A food, preferably a fungus-based food, prepared using the fungal biomass according to item 23 or 24.

[0205] 27. The food product of claim 26, characterized in that it has enhanced taste.

[0206] 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 conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. In fact, the present invention is intended to encompass various modifications of the described modes for implementing the present invention that are apparent to those skilled in the relevant art.

[0207] The following examples are merely illustrative of the present invention and should not be construed as limiting in any way the scope of the present invention as defined by the appended claims.

[0208] Example

[0209] Example 1: Steam treatment (extraction with hot water at 50°C or 22°C)

[0210] Brewer's grains (BSG) are first subjected to hydrothermal treatment under pressure to release pentoses (C5 sugars), i.e., mainly xylose and arabinose. The pretreated biomass is then subjected to enzymatic hydrolysis (EH) to release glucose (C6 sugars). Hydrothermal treatment refers to a thermochemical process in which biomass is decomposed with water under high temperature and pressure conditions. Enzymatic hydrolysis (EH) or saccharification is the process of converting complex carbohydrates into more common sugars. EH is performed by using cellulolytic enzymes. Cellulolytic enzymes mainly decompose cellulose and, to a certain extent, hemicellulose into hexoses (mainly glucose) and pentoses (mainly xylose). These studies are conducted at different scales to screen effective processing conditions, so as to obtain the maximum amount of sugar in the extract with less or no toxic compounds (mycelial growth inhibitors) to provide conditions for maximum mycelial growth. In the following example, Pleurotus pulmonarius is grown on sugar extracted from BSG.

[0211] The composition of the BSG used was determined, where cellulose was found to be 22%, hemicellulose to be 25.8%, and protein to be 25% (based on dry mass), with a moisture content of about 80% by weight.

[0212] In order to reduce the water content of BSG and enhance heat transfer during steam treatment, fresh BSG with a water content of about 80% was treated with a hydraulic press at a pressure of 3 bar to dehydrate to a final water content of about 70%.

[0213] Several experiments were performed under different process conditions to identify the most promising set of temperatures and retention times. To recover C5 sugars, experiments were performed with a one-step treatment with steam. To recover C6 sugars, a two-step steam treatment cascade was performed before saccharification to improve the enzymatic digestibility of BSG.

[0214] Process conditions were tested in the range of 150-180°C and short retention times (5-20 minutes) to limit energy losses. Different solid loadings ranging from 4% w / w to 14% w / w were evaluated.

[0215] After steam treatment, extraction is 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., percentage of dry matter of the pretreated spent grains), warm water (i.e., at 50°C) or water around room temperature (i.e., at 22°C) is added in a given ratio to achieve the desired solid loading during extraction. Stirring is continued for at least 20 minutes. To separate the liquid fraction and the solid fraction, a hydraulic press is used. The liquid fraction and the solid fraction are recovered for further processing by fermentation or further enzymatic hydrolysis, respectively.

[0216] After the steam treatment, the material is collected for subsequent extraction or further steam treatment. At the end of the treatment, the material exhibits a uniform dark color, indicating that the steam is effectively diffused throughout the mass.

[0217] Sample analysis showed that C5 sugars (xylose and arabinose) were effectively dissolved from BSG after steam treatment. In addition, small amounts of glucose (1 g / L to 3.5 g / L) were detected in the samples.

[0218] After this value, the highest concentrations of total sugars were at treatment intensities of LogRo = 3.23 and LogRo = 2.94. Therefore, the best candidates for scale-up correspond to those process conditions with severity factors ranging between 2.9 and 3.2. In addition, the results show that at treatment intensities above (LogRo = 3.5), a decrease in sugar concentration was detected due to thermal degradation of sugars to furfural. Furfural is an undesirable compound as it is a known potential inhibitor of mycelial growth.

[0219] Several fermentation experiments were performed to determine the optimal conditions for mycelial growth. Prior to fermentation, the liquid hydrolysate was centrifuged to reduce the amount of fines that would interfere with the process. The screening portion was performed by fermenting 50 mL of the liquid hydrolysate in a conical flask.

[0220] The results show that during fermentation, the growth of biomass can be limited by some inhibitors present in the extract. Therefore, diluting the extract of the fermentation medium is considered as an option to reduce the inhibitor concentration and examine the effect on the growth of biomass. When washed in water at 22°C, the mycelial growth observed was similar when the extract was diluted or undiluted, that is, it was proportional to the amount of extract added. Therefore, the inventors surprisingly found that by controlling the washing temperature, the production of undesirable growth inhibitors such as furfural can be reduced. In addition, it is obvious to a person skilled in the art that the inhibitors can be separated from the extract obtained using methods known to a person skilled in the art. The following table shows some of the highest mycelial concentrations achieved.

[0221] The experimental setup is shown in Figure 1 The results are summarized in Tables 1 and 2 below.

[0222] Table 1.1. Experimental results of BSG steam treatment followed by water extraction at 22°C and 50°C in batch settings. The values ​​obtained were comparable to those of biomass grown using culture medium containing the diluted (50%) and undiluted (100%) extracts from step (a1). The C5 recovery range is based on triplicate .

[0223]

[0224] The C5 recovery defined herein refers to the ratio of C5 sugars to the total amount of hemicellulose, determined based on the solid loading of the reactor and the composition of the spent grains. The maximum achieved recovery of C5 sugars obtained for the conditions performed under the following conditions was 53% (i.e., up to 60%): at 170°C, a retention time of 7.5 minutes, a pressure of 8 bar, a solid loading percentage of 14% (w / w), and extracted with water at 50°C. In the 50% diluted extract and the 100% extract used for fermentation, the total concentration of sugars was 19.2 g / L, and the biomass concentrations were 0.73 [g / L per % loading] and 0.95 [g / L per % loading], respectively. It was found that a wash temperature of 22°C was effective for conditions with a solid loading percentage of 8 and 14, but not as effective as when a wash temperature of 50°C was used. Therefore, this example clearly shows that higher wash temperatures are more effective for the preparations disclosed in the present invention, which is also surprising in terms of mycelial growth using sugars extracted at higher temperatures.

[0225] In addition, after studying the effects of solid loading and washing temperature on the extract and mycelial growth, the extractor setup was changed from batch to semi-continuous. Condition 4 in Table 1.2 shows a C5 sugar recovery of up to 96%, while the same conditions in Table 1.1 show a C5 sugar recovery of up to 60%. This shows the effect of the extractor configuration on the extraction yield. In a semi-continuous configuration, the feed and reaction mixture are continuously fed into and out of the system, wherein the feed and withdrawal of the raw material are not at the same rate, i.e., the raw material is fed in a cyclic manner and is not stationary in the extractor as is the case in a batch system, which increases the surface area of ​​the BSG exposed to the steam treatment. In addition, a shredder or a disc grinder may be present or placed at the extractor outlet, wherein the shredder or the disc grinder is configured to process the brewer's grains so that the BSG surface area is further increased (i.e., the particle size is further reduced) for higher sugar extraction at the washing step.

[0226] Table 1.2. BSG steam treatment with 14% (w / w) solids loading followed by heating at 50 °C in a semi-continuous setup. The values ​​indicated below are comparable to those obtained using a culture medium comprising the extract of step (a1) and 50% dilution. Biomass growth related .

[0227]

[0228] In order to reduce energy demand and process cost, the retention time was reduced from 7.5 minutes to 5 minutes. As shown in Table 2 below, for the conditions performed, the BSG solid loading was 14% (w / w). The results of C5 sugar recovery, biomass growth and furfural concentration are listed in Table 2. When the retention time was 7.5 minutes, it was found that the recovery percentage of C5 sugar was higher. Compared with 5 minutes, it was found that the biomass concentration in the extract processed at a retention time of 7.5 minutes was higher. It was observed that the change in retention time had no effect on furfural concentration.

[0229] Table 2. BSG steam treatment at 14% solids loading (w / w) followed by hot water extraction at 50 °C in a batch setting. The values ​​indicated below are consistent with the results of experiments with a retention time of 5 minutes and 7.5 minutes. Biomass growth of the culture medium of the diluted extract .

[0230]

[0231] In a further step, in order to improve the energy consumption and the overall economy of the process, extraction of sugars was carried out with water at three different temperatures, namely at 22°C, 40°C and 50°C, wherein the BSG batch was treated at 170°C for 7.5 minutes at a solids loading of 14% (w / w) in a batch setting. Figure 4The C5 sugar concentrations (g / L) of the extracts obtained at three different washing temperatures are shown. The concentration of sugars decreases as the temperature of the water used for extraction decreases. The maximum concentrations of extracted C5 sugars were found for the extraction conditions with water at 50°C (19.2 g / L), 40°C (14.5 g / L) and 22°C (10.82 g / L). Therefore, this example also clearly shows that higher washing temperatures are more effective for the preparation disclosed in the present invention. Depending on the desired extract composition, a washing temperature range between 40°C and 50°C is preferred, and even more preferably, a washing temperature range between 40°C and 48°C is preferred.

[0232] Example 2: Formation of a fungal growth inhibitor in a process involving steam pretreatment and washing with liquid water .

[0233] Furfural has been shown to inhibit mycelial growth at concentrations as low as 0.2 g / L. This compound is a product of thermal degradation of hemicellulose. Even at higher furfural concentrations, biomass growth still occurs due to the possible influence of the presence of other inhibitors (described above).

[0234] The production of furfural can be partially avoided by carefully controlling the process conditions. In general, sugars are more susceptible to degradation to furfural when the processing conditions are harsh. However, even under mild processing conditions, the production of furfural cannot be completely avoided. Furfural formation is highly dependent not only on the process conditions, but also on the material composition. The more free sugars a material has, the easier it is to degrade those sugars to furfural.

[0235] BSG is a material with a certain composition that is similar every time it leaves the filtration process during beer production. However, BSG is not a stable material and its composition changes rapidly after leaving the filtration process. In this case, from one batch to another, the delivered material can contain more or less free sugars. As a result, the formation of furfural is difficult to control, but its occurrence is greatly reduced when BSG is treated under fairly mild conditions. The results are summarized in Table 3 below. The furfural concentration was determined by HPLC. Furfural can also be isolated or separated according to methods known in the art.

[0236] The results are summarized in Table 3 below.

[0237] Table 3. Furfural concentration range observed under different steam pretreatment and scrubbing conditions .

[0238]

[0239] Example 3: Liquid Hot Water Treatment

[0240] The experimental setup of this process is shown in Figure 2The pressure was set constant at 50 bar. The extraction (dissolution of C5 sugars) was carried out in the same unit and the separation of the liquid and solid fractions was carried out by filter pressing. In this specific example, the temperature was tested in the range of 110°C to 210°C and the retention time was increased from 10 minutes to 120 minutes in 10 minute increments to find the optimal conditions for maximum sugar extraction and maximum mycelial growth. An excerpt of the results is summarized in Table 4. Table 5 shows another set of conditions on a larger scale, comparing the extraction results in the presence / absence of acid, highlighting that the use of acid increases the efficiency of the liquid extraction.

[0241] Table 4. Experimental results of small-scale (30 mL extraction volume) hot water treatment of BSG liquid, showing the lung Dry cell weight of Pleurotus eryngii (in g) .

[0242]

[0243] Table 5. Extract of some experimental results for hot water treatment of BSG liquid on a larger scale. The values ​​indicated below are consistent with those obtained using The biomass growth of the medium containing the undiluted extract of step (a2) was related to the biomass growth of the medium containing the undiluted extract of step (a2). of H2SO4, and in condition 6, 0.5% w / w of H2SO4 was added. If condition 6 is modified to have the same The acid concentration was 82%, and the sugar recovery rate was 82%. .

[0244] condition 1 2 3 4 5 6 7 Temperature(℃) 110 140 150 180 140 145 150 Pressure (bar) 50 50 50 50 50 50 50 Retention time (minutes) 60 60 50 20 5 5 5 Severity Factor 2.07 2.96 3.17 3.66 1.88 2.02 2.17 Total sugars [g / L per % loading] 0.575 1.2125 1.575 2.3125 2.84 2.83 3.46 Biomass [g / L per % loading] 0.5075 1.16 1.78 0 1.08 0.97 1.45 Furfural [g / L per % loading] 0 0 0 0.07 0.04 0.08 0.08 C5 recovery rate [%] 7.585 19.275 29.07 45.8 77 73 92

[0245] Example 4: Contamination Test

[0246] All chemicals required for media preparation were purchased from Carl Roth (Karlsruhe, Germany), VWR (Darmstadt, Germany), Merck KGaA (Darmstadt, Germany), or Sigma-Aldrich (Steinheim, Germany).

[0247] The culture medium for preparing agar plates was prepared by weighing the different compounds according to Table 6 and thereafter dissolving them in water. The culture medium according to the invention described as C5 extract was prepared as in Example 6 Condition 2. C5 complete refers to a medium further supplemented with 5.9 g / l K2HPO4, 9.0 g / L KH2PO4, 12.0·10 -2 g / L MgSO4, 8.10·10 -3 FeCl3 and 10.10·10 -3CaCl2 as described herein C5 extract. The mixture was then autoclaved at 121°C for 20 minutes, then aseptically poured under a sterile clean bench and allowed to solidify completely. Subsequently, 100 μL of a 1:1000 diluted E. coli suspension was inoculated with a plate prepared completely with LB medium, malt extract, M9 classic medium, C5 extract and C5, the suspension being obtained from a culture in LB medium incubated overnight at 37°C. The plates were then incubated overnight at 37°C and photographed the next day. In another experiment, only the lids of the plates prepared completely with malt extract, M9 classic medium, C5 extract and C5 were removed and exposed to air contamination to compare growth on different media. In this case, photographs of the plates were taken 10 days later (see for an overview). Figure 3 ).

[0248] Table 6. Composition of culture medium used for contamination testing .

[0249]

[0250]

[0251] Example 5: Production of fungal fermentation medium from spent grain using acid-added liquid extraction

[0252] The spent grains were subjected to extraction experiments using liquid water according to the general protocol described above or in Table 5, where H2SO4 was added to the water used for extraction at different concentrations. The experiments were conducted according to the conditions discussed in Table 7.

[0253] Table 7. Summary of conditions for extraction of spent grains with dilute acid .

[0254]

[0255] Similar to the previous examples, the recovery of C5 sugars and furfural concentration were quantified using the protocol "Analysis of sugar and furfural content", after which the extract obtained was fermented using the protocol "Cultivation of fungal biomass". The results of the extraction and fermentation are summarized in Table 8.

[0256] Table 8. Summary of extraction experiments with spent grain using dilute acid .

[0257]

[0258]

[0259] Example 6. Hydrolysis of cellulose from pretreated spent corn

[0260] Extraction experiments were performed on spent grains according to the general protocol as outlined in the section "Thermal extraction of lignocellulosic material" using ten different conditions as defined in Table 9 below, and subsequently biomass was produced using the protocol "Cultivation of fungal biomass".

[0261] Thermal extraction of lignocellulosic materials

[0262] For thermal extraction, the dry matter of lignocellulosic material was determined using a 3 g material moisture analyzer (160 ° C until constant weight). Based on the obtained dry matter, the reactor load was adjusted by weighing the required amount of material and introducing it into the reactor thereafter. Water was then added to fill the reactor to its final working volume, and a rubber sealing ring was placed on the top of the reactor shell. The lid equipped with a pressure gauge and a temperature probe was then tightly closed to avoid leakage, and the reactor was then pressurized with nitrogen until the desired operating pressure was reached. Afterwards, the desired temperature, pressure and retention time were set in the software, and extraction was started. During the experiment, the temperature and pressure were constantly monitored and adjusted.

[0263] At the end of the extraction, the reactor is cooled by immersing it in cold water or, for larger reactors, using a cooling jacket. Once the temperature drops below 30°C, the reactor is depressurized by slowly opening the exhaust valve and the lid is opened once the pressure reaches atmospheric pressure. In the case of a continuous extraction process, a heat exchanger is used to cool the mixture after extraction from the reactor. The solid and liquid phases (extracts) are then separated by filter pressing or with a decanter centrifuge. The liquid extract is then stored at 4°C for a short time or frozen at -20°C for long-term storage. The remaining treated solid lignocellulosic material is further subjected to enzymatic hydrolysis to recover glucose from cellulose.

[0264] Cultivation of fungal biomass

[0265] The liquid extract obtained after heat extraction was used directly as growth medium and mixed with additional compounds (5.9 g / L K2HPO4, 9.0 g / L KH2PO4, 12.0·10 -2 g / L MgSO4, 8.10·10 -3 FeCl3, 10.10·10 -3CaCl2) and other trace elements are mixed and / or supplemented with the additional compound according to a typical M9 medium (Miller, 1972, Experiments in Molecular Genetics, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, New York). The resulting mixture is then placed in an appropriate container and sterilized (121°C, 20 minutes). After sterilization, the culture medium is cooled to room temperature and inoculated with spores or mycelium from a suspension prepared from a fully grown mycelium agar plate. The broth is then incubated at a temperature of 30°C for a period of 5 days. During fermentation, acid and alkali are used to adjust the pH to maintain it at an optimal pH of 6.5 for the culture strain of Pleurotus pulmonarius. Thereafter, the biomass is harvested by centrifugation, washed with water, and finally the dry matter is determined using a moisture analyzer (160°C until constant weight).

[0266] Use the broth volume to convert dry matter to biomass concentration.

[0267] Reference biomass for comparison with biomass produced from extract was produced using a reference medium consisting of glucose, trace solutions 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 phosphate buffer and the flasks were incubated at 30°C for 5 days.

[0268] Table 9. Summary of extraction conditions for spent grains

[0269]

[0270] The solid lignocellulosic material recovered after the thermal extraction of the spent grains (herein spent grains) of Example 1 was treated according to the method described in the section "Hydrolysis of lignocellulosic material". The recovery of the C6-polysaccharide fraction determined as described in "Analysis of sugar and furfural content" and the biomass obtained from the fermentation tests using these extracts performed as described in the protocol "Cultivation of fungal biomass" (expressed per % solids loaded for comparison purposes) are summarized in Table 10.

[0271] Hydrolysis of lignocellulosic materials

[0272] The dry matter of the lignocellulosic material recovered after the treatment as described in the part "heat extraction of lignocellulosic material" is determined using a moisture analyzer (incubated at 160°C until constant weight), and the load is adjusted as described herein for thermal treatment. The material is then loaded into a reaction vessel, and an enzyme mixture (Ctec2 and Ctec3 from Novozymes) is added according to the cellulose content in the material and following the guidance of the manufacturer. The mixture of enzyme and cellulose material is then incubated between 10 hours and 200 hours at a temperature of 60°C and a pH of 6.0. The homogeneity of the mixture is ensured by agitator or by incubating the container in an incubator. After incubation, the reaction mixture is rapidly cooled using an ice bath, the cooling jacket of the reaction vessel or using a heat exchanger in the case of a continuous process. Finally, by press filtration or centrifugation liquid and solid fraction. The solid fraction rich in lignin is stored at 4°C before analysis, and the clarified liquid fraction is used for fermentation experiments.

[0273] Table 10. Summary of hydrolysis experiments performed on pretreated BSG .

[0274]

[0275] Example 7: Production of fungal fermentation medium from spent grain using steam prehydrolysis

[0276] Extraction experiments were performed on spent grains using steam prehydrolysis as described in the protocol “Thermal pretreatment and extraction of lignocellulosic biomass using steam”.

[0277] Thermal pretreatment and extraction of lignocellulosic biomass using steam

[0278] Prior to operation, the reactor is preheated with steam until the operating temperature is reached. During the preheating phase, the condensate produced by the steam coming into contact with the cold reactor must be removed.

[0279] The material is weighed and loaded into a metal drum (batch process) or into a reactor using a screw feeder (continuous process). Batch extraction is carried out in a closed system where the spent grain and water are loaded only once at the beginning in the reactor. Once the reactor is pressurized, the system remains closed until the reaction mixture cools to at least 40°C. Whereas continuous extraction refers to continuous feeding and withdrawal of raw materials at the same rate. When feeding the material, there is no pressure drop in the reactor, and when withdrawing the material, the pressure drops suddenly, resulting in steam explosion. However, in a semi-continuous process, the feed and reaction mixture are continuously fed in and out of the system. The feeding and withdrawal of raw materials are not at the same rate because the raw materials are fed in a cyclic manner.

[0280] The material is pre-treated with constant steam injection for the desired residence time, and the temperature in the reactor is controlled by setting the pressure controller 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.

[0281] After reaching the residence time, the material was recovered from the reactor and its weight and water content were recorded for further processing (extraction).

[0282] Extraction is carried out in a separate unit, mixing the steam pretreated material with warm water (between 40°C and 60°C, preferably between 40°C and 50°C) in a desired ratio to achieve a certain solid loading. Mixing is continued by stirring the tank for at least about 20 minutes before separating the liquid and solid fractions.

[0283] The solid-liquid mixture is pumped to a press where the two fractions are separated at a constant pressure of about 4 bar. The liquid hydrolysate (rich in C5 sugars) as well as the solid fraction (rich in cellulose and lignin) are recovered for further processing.

[0284] The solid fraction may then be further treated with enzymes to convert the cellulose to C6 sugars.

[0285] Analysis of sugar and furfural content

[0286] For the C5 extract, i.e. as obtained according to the section "Thermal extraction of lignocellulosic material", the sample was hydrolyzed with hydrochloric acid (final concentration 4% w / w) at 121°C for 60 minutes before analysis to enable quantification of the recovered C5 sugars as monosaccharides. After hydrolysis, the pH of the extract was then set to 5.5 to avoid damaging the HPLC column. The monosaccharides from the C6 extract as obtained according to the section "Hydrolysis of lignocellulosic material" and the monosaccharides from the C5 extract as described above were then measured with an Agilent HPLC 1200 system using a Metacarb 87C column (300 mm×7.8 mm, Varian Inc, Paolo Alto, CA, USA) as the stationary phase and ultrapure water as the mobile phase. The monosaccharides from the C6 extract as obtained according to the section "Hydrolysis of lignocellulosic material" were measured at a temperature of 85°C at 0.6 mL min -1 After column separation, the analytes were detected by a refractive index detector, except in the case of furfural where a UV detector was used at a wavelength of 270 nm.

[0287] The experimental conditions for the prehydrolysis are listed in Table 11. The results of the sugar and furfural analysis (Protocol "Analysis of sugar and furfural content") as well as the biomass obtained from the fermentation using the obtained extract (Protocol "Cultivation of fungal biomass") (expressed as % of solid sidestream per loading for comparison purposes) are provided in Table 12.

[0288] Table 11. Summary of extraction conditions for spent grains using steam prehydrolysis

[0289]

[0290]

[0291] Table 12. Summary of extraction experiments using steam prehydrolyzed spent grains

[0292]

[0293] Example 8 - Hydrolysis of Cellulose from the Pretreated Spent Cereals of Example 7

[0294] The pretreated solid material remaining after steam prehydrolysis and water recovery was recovered, subjected to a second prehydrolysis, and finally to enzymatic hydrolysis as described in "Hydrolysis of lignocellulosic material". The conditions tested are listed in Table 13.

[0295] Table 13. Summary of extraction conditions for pretreated spent grain using steam prehydrolysis

[0296]

[0297] Data on the recovery of C6 sugars and the biomass available (measured as performed in the general protocols "Analysis of sugar and furfural content" and "Cultivation of fungal biomass" included above) are summarized in Table 14. For comparison purposes, they were normalized to the solid loading used for the experiments.

[0298] Table 14. Summary of extraction experiments using steam prehydrolyzed spent grains

[0299]

[0300] Example 9. Sensory evaluation of meatballs produced using mycelium from different sources

[0301] Meatballs are formed from mycelial biomass and fried in a pan. Each trained panelist is blinded and continuously receives meatballs prepared from mycelium from standard culture medium and meatballs from spent grains. During this first phase, they define the sensory attributes they identify in the two meatballs. Subsequently, they discuss these attributes together and select common attributes that each panelist can associate with the same taste and fragrance of these meatballs to compare them. Then the second phase begins, with panelists evaluating meatballs based on the selected attributes and scoring between 0 and 5 for each attribute. This phase is repeated on different days to increase the statistical relevance of the data, and the mean value of the scores is calculated and plotted on a spider web graph.

[0302] The color was determined at 20 different locations on the meatballs using an RGB system and a color analyzer. The locations used for the 10 measurements were the same for all meatballs. The average of these measurements was then used to compare the color of the meatballs.

[0303] Example 10. Analysis of amino acid content of the obtained biomass

[0304] Using the culture medium obtained during the extraction of brewer's grains with liquid water, the amino acid content of the biomass obtained as described in Table 9 has been determined in the following manner.

[0305] The amino acid profile of the protein extracted from the lignocellulosic material is determined by hydrolyzing the extract sample with 6M HCl at 105°C for 24 hours before HPLC measurement. The hydrolyzate is then evaporated under a nitrogen stream and resuspended in 200 μM α-aminobutyrate, which is used as an internal standard. The amino acid concentration in the prepared sample is measured by fluorescence detection using an Agilent 1200 HPLC system (Agilent technologies, Waldbronn, Germany) equipped with a reversed-phase column Gemini 5 μC18 110A (150 mm × 4.6 mm, Phenomenex, Aschaffenburg, Germany) as a stationary phase. The separation of different proteomic amino acids relies on a gradual change in the mobile phase composition throughout the measurement by mixing eluent A (40 mM NaH2PO4, pH 7.8) and eluent B (45% methanol, 45% acetonitrile, 10% water) differently according to a well-defined gradient profile. In addition, the eluent was diluted to 1 mL min at 40 °C. -1 The column was separated at a flow rate of 1.5 Å. In addition, a precolumn (Gemini C18, MAX, RP, 4 mm × 3 mm, Phenom, Aschaffenburg, Germany) was used to increase the column life. Fluorescence detection was achieved by pre-column derivatization with o-phthalaldehyde (OPA) and 9-fluorenylmethoxycarbonyl (FMOC) and changing the excitation and emission wavelengths (Table 15).

[0306] Table 15. Method for separation and quantification of amino acids - the composition of the mobile phase was varied during the measurement to Separation was achieved by gradient elution. Eluent A: 40 mM NaH2PO4, pH 7.8; Eluent B: 45% methanol, 45% acetonitrile, 10% water .

[0307]

[0308] The results are shown in Table 16 below.

[0309] Table 16. Amino acid content of the obtained biomass, expressed in % w / w (YE - yeast extract, CSL - corn Pulp) .

[0310]

[0311]

[0312] Example 11: Production of fungal fermentation medium from wheat bran

[0313] Extraction experiments were performed on wheat bran according to the general protocol as outlined in the section "Thermal extraction of lignocellulosic material" using 4 different conditions as defined below in Table 24. The pH of the water was adjusted to about 13.5 using NaOH to extract sugars from the wheat bran, achieving at least 80% sugar recovery.

[0314] Table 17. Summary of wheat bran extraction conditions and related fermentation results

[0315]

[0316] Other examples and / or embodiments of the invention are disclosed in the following numbered clauses.

[0317] 1. A method for producing a fungal fermentation medium from brewer's spent grains (BSG).

[0318] 2. The method according to clause 1, comprising:

[0319] (a1) extracting C5 sugars from the lignocellulosic material contained in BSG via steam pretreatment followed by a washing step with liquid water at a temperature not exceeding 50° C., and

[0320] (b) combining the extract thus obtained with at least one non-carbohydrate nutrient for fungal cultivation.

[0321] 3. A method according to clause 2, wherein during the steam pretreatment, the lignocellulosic material contained in the BSG is contacted with steam at a temperature between 130°C and 180°C, preferably between 160°C and 180°C, more preferably between 165°C and 175°C, and / or for a period of up to 30 minutes, preferably for a period of up to 15 minutes, and / or

[0322] Wherein in step (a1), the washing step with liquid water is carried out at a temperature not exceeding 40°C, preferably not exceeding 30°C, more preferably not exceeding 25°C.

[0323] 4. A method according to clause 2 or 3, wherein the BSG is characterized by a final moisture content between 50 wt% and 75 wt%, preferably wherein the method further comprises a step of dehydrating the BSG by pressing the material to a final moisture content between 50 wt% and 75 wt%, wherein the dehydrating step precedes step (a1), and / or

[0324] wherein in step (b), the extract of step (a1) is substantially undiluted, preferably, wherein the extract of step (a1) accounts for 90% v / v to 99% v / v of the total culture medium obtained in step (b), more preferably, wherein the extract of step (a1) accounts for 94% v / v to 98% v / v of the total culture medium obtained in step (b), and / or

[0325] The culture medium is capable of supporting fungal growth without the need for further dilution steps to reduce the concentration of fungal growth inhibitors.

[0326] 5. The method according to clause 1, comprising:

[0327] (a2) extracting C5 sugars from the lignocellulosic material contained in the BSG via a liquid extraction treatment with water at a temperature between 145° C. and 155° C. and / or for a period of up to 70 minutes, preferably for a period of up to 50 minutes, preferably at a pressure of 30 to 50 bar, and

[0328] (b) combining the extract thus obtained with at least one non-carbohydrate nutrient for fungal cultivation,

[0329] Preferably, wherein said lignocellulosic material comprised in the BSG is untreated, more preferably wherein said material has not been dewatered.

[0330] 6. The method according to any one of clauses 2 to 5, wherein step (a1) or step (a2) is performed at a severity factor between 2.9 and 3.3.

[0331] 7. A method according to any one of clauses 1 to 6, wherein the method further comprises a step (a') of enzymatically hydrolyzing the solid lignocellulosic residue obtained in step (a1) or step (a2) with cellulase, separating the hydrolyzed liquid product from the solid residue, and mixing the liquid product with the extract of step (a1) or the extract of step (a2).

[0332] 8. A method according to any one of clauses 1 to 7, wherein substantially no sugar is added other than the sugar present in the extract of step (a1) or step (a2) or in the liquid product of the hydrolysis of step (a'), and / or

[0333] wherein step (a1) or step (a2) is characterized in that the solids loading of the reactor is between 4% w / w and 14% w / w, and / or

[0334] wherein complex C5 polysaccharides account for at least 50% of all sugars in the extract of step (a1) or step (a2), preferably, wherein complex C5 polysaccharides account for at least 65% of all sugars in the extract of step (a1) or step (a2), more preferably, wherein complex C5 polysaccharides account for at least 80% of all sugars in the extract of step (a1) or step (a2), and / or

[0335] The culture medium is suitable for culturing at least one fungal strain selected from Pleurotus pulmonaria and Morchella rubrum.

[0336] 9. A fungal fermentation medium obtainable according to the method of any one of clauses 1 to 8.

[0337] 10. A fungal fermentation medium according to clause 9, wherein substantially all sugars are derived from BSG, and / or

[0338] wherein complex C5 polysaccharides account for at least 50% of all sugars in the culture medium, preferably, wherein complex C5 polysaccharides account for at least 65% of all sugars in the culture medium, more preferably, wherein complex C5 polysaccharides account for at least 80% of all sugars in the culture medium.

[0339] 11. Use of a culture medium according to item 9 or 10 in fungal culture.

[0340] 12. A method for producing fungal biomass, the method comprising the step of submerging at least one fungal strain using the fungal fermentation medium according to clause 9 or 10, preferably, wherein the at least one fungal strain is an edible fungus, more preferably, wherein the at least one fungal strain is Pleurotus ostreatus, Pleurotus eryngii or Pleurotus rosaceae, or wherein the at least one fungal strain is Morchella rubra, Morchella foetida, Morchella black vein or Morchella edulis.

[0341] 13. A 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.

[0342] 14. Use of fungal biomass according to clause 13 for producing food, preferably fungus-based food.

[0343] 15. A food, preferably a fungus-based food, prepared using the fungal biomass according to clause 14.

Claims

1. A method for producing a fungal fermentation medium from brewer's spent grains (BSG).

2. The method according to claim 1, comprising: (a1) extracting C5 sugars from lignocellulosic material contained in BSG via steam pretreatment, This is followed by a washing step with liquid water at a temperature not exceeding 50° C., and (b) combining the extract thus obtained with at least one non-carbohydrate nutrient for fungal cultivation.

3. The method according to claim 2, wherein during the steam pretreatment, the lignocellulosic material contained in BSG is contacted with steam at a temperature between 130°C and 180°C.

4. The method according to claim 3, wherein during the steam pretreatment, the lignocellulosic material contained in BSG is contacted with steam at a temperature between 160°C and 180°C.

5. The method according to claim 3 or 4, wherein during the steam pretreatment, the lignocellulosic material contained in BSG is contacted with steam at a temperature between 165°C and 175°C.

6. The method according to any one of claims 3 to 5, wherein during the steam pretreatment the lignocellulosic material contained in BSG is contacted with steam for a time of at most 30 minutes.

7. The method according to any one of claims 3 to 6, wherein during the steam pretreatment the lignocellulosic material contained in BSG is contacted with steam for a time of at most 15 minutes.

8. The method according to any one of claims 3 to 7, wherein in step (a1), the washing step with liquid water is carried out at a temperature not exceeding 40°C, preferably not exceeding 30°C, more preferably not exceeding 25°C.

9. The process of any one of claims 2 to 8, wherein the BSG is characterized by a final moisture content between 50% and 75% by weight.

10. The method of claim 9, wherein the method further comprises a step of dehydrating the BSG by pressing the material to a final moisture content between 50% and 75% by weight, wherein the dehydrating step precedes step (a1).

11. The method of claim 9 or 10, wherein in step (b), the extract of step (a1) is substantially undiluted.

12. The method according to claim 9 or 10, wherein in step (b), the extract of step (a1) is diluted.

13. The method according to claim 11 or 12, wherein the extract of step (a1) accounts for 90% v / v to 99% v / v of the whole culture medium obtained in step (b).

14. The method according to any one of claims 11 to 13, wherein the extract of step (a1) accounts for 94% v / v to 98% v / v of the whole culture medium obtained in step (b).

15. A method according to any one of claims 11 to 14, wherein the culture medium is capable of supporting fungal growth without the need for a further dilution step to reduce the concentration of a fungal growth inhibitor.

16. The method according to claim 1, comprising: (a2) extracting C5 sugars from the lignocellulosic material contained in the BSG via a liquid extraction treatment with water at a temperature between 145° C. and 155° C. and / or for a period of up to 70 minutes, preferably for a period of 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 cultivation.

17. The method of claim 16, wherein the lignocellulosic material contained in the BSG is untreated.

18. The method of claim 17, wherein the material has not been dehydrated.

19. The method according to any one of claims 16 to 18, wherein the (a2) step is carried out using water comprising an acid, preferably H2SO4, in a concentration ranging between 0.4% w / w and 1.6% w / w.

20. The method according to claim 19, wherein the (a2) step is carried out using water comprising an acid, preferably H2SO4, in a concentration range between 0.5% w / w and 0.9% w / w.

21. The method according to any one of claims 16 to 20, wherein the liquid extraction is performed for a period of 5 minutes to 15 minutes.

22. The method of any one of claims 16 to 21, wherein the liquid extraction is performed at a pH between 1.0 and 3.

0.

23. The method according to any one of claims 2 to 22, wherein step (a1) or step (a2) is performed at a severity factor between 2.9 and 3.

3.

24. The method according to any one of claims 1 to 23, wherein the method further comprises a step (a') of enzymatically hydrolyzing the solid lignocellulosic residue obtained in step (a1) or step (a2) with cellulase, separating the hydrolyzed liquid product from the solid residue, and mixing the liquid product with the extract of step (a1) or the extract of step (a2).

25. The method according to any one of claims 1 to 24, wherein substantially no sugar is added other than the sugar present in the extract of step (a1) or step (a2) or in the liquid product of the hydrolysis of step (a').

26. The process according to any one of claims 1 to 25, wherein step (a1) or step (a2) is characterized in that the solids loading of the reactor is between 4% w / w and 14% w / w.

27. The method of any one of claims 1 to 26, wherein complex C5 polysaccharides comprise at least 50% of all sugars in the extract of step (a1) or step (a2).

28. The method of claim 27, wherein complex C5 polysaccharides comprise at least 65% of all sugars in the extract of step (a1) or step (a2).

29. The method of claim 28, wherein complex C5 polysaccharides comprise at least 80% of all sugars in the extract of step (a1) or step (a2).

30. The method according to any one of claims 1 to 29, wherein the culture medium is suitable for culturing at least one fungal strain selected from Pleurotus pulmonaria and Morchella rubrum.

31. A fungal fermentation medium obtainable by the method according to any one of claims 1 to 30.

32. The fungal fermentation medium of claim 31 , wherein substantially all of the sugars are derived from BSG.

33. A fungal fermentation medium according to claim 31 or 32, wherein complex C5 polysaccharides comprise at least 50% of all sugars in the medium.

34. The fungal fermentation medium of claim 33, wherein complex C5 polysaccharides comprise at least 65% of all sugars in the medium.

35. The fungal fermentation medium of claim 34, wherein complex C5 polysaccharides comprise at least 80% of all sugars in the medium.

36. Use of a culture medium according to any one of claims 31 to 35 in fungal culture.

37. A method for producing fungal biomass, comprising the step of submerged fermentation of at least one fungal strain using the fungal fermentation medium according to any one of claims 31 to 35.

38. The method of claim 37, wherein the at least one fungal strain is an edible fungus.

39. The method of claim 38, wherein the at least one fungal strain is Pleurotus pulmonarius, Pleurotus ostreatus, Pleurotus rufus, or Pleurotus rosa-red, or wherein the at least one fungal strain is Morchella rubra, Morchella foetida, Morchella black vein, or Morchella foetida.

40. A fungal biomass produced according to the method of any one of claims 37 to 39.

41. The fungal biomass according to claim 40, characterized in that The Asp / Asn content is at least 20% of the total amino acid content.

42. Use of a fungal biomass according to claim 40 or 41 in the production of a food, preferably a fungus-based food.

43. A food, preferably a fungus-based food, prepared using the fungal biomass according to claim 40 or 41.

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