SE2151533A1 - Fungal biomass food product - Google Patents

Abstract

The present document relates to fungal biomass food products which have properties making them more appealable as meat and protein replacement products. The document discloses methods for the production of such products.

Description

FUNGAL BIOMASS FOOD PRODUCT TECHNICAL FIELD The present document is directed to food products comprising fungal biomass as a protein source and/or animal protein replacement product and methods for producing such food products.

BACKGROUND ln recent years, the excessive use of meat as a dietary protein source has come under close scrutiny and received significant negative criticism. Several factors are at play, but the root cause of this movement can be narrowed down to two key components. First, it is apparent that production, distribution and consumption of meat leads to substantial negative climate impact.

Livestock rearing not only emits massive quantities of greenhouse gases due to its excessive use of land, water and resources, but also contributes to deforestation, biodiversity loss, eutrophication, and a range of other climate-related issues. Second, excessive consumption of animal-based protein is associated with a range of detriments to health and wellbeing that include but are not limited to higher prevalence of obesity, and elevated risks of cancer and cardiovascular disease. ln addition, the unsustainable practices that prevail in many parts of meat and dairy manufacturing contribute to increased risks of zoonosis as well as antibiotic resistance. ln recent years, these issues have led to a heavily increased demand for meat resembling food products ('meat replacements') comprised of protein sources of non-animal origin ('alternative protein'). These forces have spilled over into the segment for fish as well.

Consumers are increasingly looking for fish replacements based on alternative protein, despite the fact that production and consumption of fish is arguably not as harmful for the climate or for individual health as meat. The food manufacturing industry has responded by innovating heavily within the area, outputting large quantities of products that are perceived as capable of meeting the emerging needs of the market. Typically, these products are made using plant-based protein SOU FCGS.

While it is apparent that plant-based protein sources have the potential to perform significantly better than meat and fish on factors relating to both nutrition and climate impact, achieving appealing palatability is a challenge. On one hand, creating non- meat and non-fish products that have taste profiles similar to those of meat and fish is difficult. More pressing, however, is the issue of texture. Most raw materials of alternative protein are provided in non-texturized (e.g. as powder) form such as plant protein isolates or concentrates, meaning that several advanced processing steps and extensive use of additives is required to acquire a meat- or fish-like texture.

One suggested solution has been to use mycoprotein, i.e. protein derived from fungi that are produced for the purpose of human consumption. Mycoprotein and its various fermentation based production methods have a range of advantages and characteristics that make them highly suitable for solving present challenges related to poor nutrition, food security and climate change. Consumption of mycoprotein is associated with a range of benefits to health and wellbeing, attributable to its beneficial nutritional composition.

The potential of using biomass from filamentous fungi as a protein source has therefore garnered positive attention in recent years, as it offers high quality nutrition benefits, low allergenicity, and the biomass is naturally texturized. This texture is due partly to: (a) its morphology, growing as filaments in a highly structured network, and partly to; (b) its naturally high content ofdietary fiber, which is located on the fungi cell walls and contributes to a resistant structure.

One alternative to solving one or more of the objects described above is thus to use species of filamentous fungi for the production of fungi biomass to be used in food production. Many species, however, have morphologies and rigid fibre levels that create an issue with regards to excessive toughness, chewiness, and pulpy mouthfeel, making products too chewy to the extent that they are not acceptable for consumers. Certain other fungi biomass products are mixed with egg albumin to achieve a meat- or fish-like structure, but this makes the product unsuitable for vegans and there have been complaints from consumers that such food products are "too soft", "mushy", and "Iacking chewiness" instead.

There is thus still need for a food product with a non-animal based, high-nutritious and environmentally friendly food product which has a taste and texture resembling a wide variety of meat- and fish-based food product types.

SUMMARY The present document is directed to a method for modifying the texture, such as the hardness, adhesiveness, springiness and/or chewiness, and/or nutritiona| value of a fungal biomass food product, said method comprising growing food-safe fi|amentous fungi on a vegetable substrate, wherein said vegetable substrate comprises, for said food-safe fi|amentous fungi, indigestible substance(s), such as fibers, and admitting at least part of said indigestible substances to remain in said fungal biomass food product. The vegetable substrate may be a plant fiber substrate, bread, and/or dough, such as oat, potato, corn, wheat flour, rye flour, seaweed, wheat bran or any combination thereof. The vegetable substrate may be a food waste substrate and/or a side stream substrate.

The vegetable substrate may have a particle size of from about 100 to about 4000 um, such as from about 100 to about 3000 um, such as from about 100 to about 500 um or from about 500 to about 3000 um (mean diameter).

The method according to the present document may comprise the steps of: i) growing said food-safe fi|amentous fungi on said plant substrate to produce a fungal biomass; ii) harvesting, such as by dewatering, the fungal biomass of step i); and iii) optionally further dewatering, such as by pressing, the harvested fungal biomass of step ii), to obtain a fungal biomass.

The food-safe filamentous fungi may be grown in step i) using liquid aerobic fermentation.

The vegetable substrate may or may not be supplemented with nutrients, such as a nitrogen and/or phosphate source, trace meta|(s), vitamin(s) and/or sulphate.

The vegetable substrate may be pre-treated, such as by reducing the size of the particles constituting the vegetable substrate, hydrolysing the vegetable substrate with enzyme(s) and/or microorganisms(s).

The food-safe filamentous fungi may be of the Zygomycota and/orAscomycota phylum, excluding yeasts, such as fungi of the genera Rhizopus, Neurospora, Aspergillus, Trichoderma, Pleurotus, Ganoderma, lnonotus, Cordyceps, Ustilago, Tuber, Fusarium, Pennicillium, Xylaria, Trametes, or any combination thereof.

The food-safe filamentous fungi may e.g. be of the species Aspergillus oryzae, Rhizopus oryzae, Fusarium graminareum, Cordyceps militaris, Cordyceps sinensis, Tuber melanosporum, Tuber magnatum, Pennicillium camemberti, Neurospora intermedia, Neurospora sitophila, Xylaria hypoxion, or any combination thereof.

The amount of fungi in fungal biomass after fermentation and optional dewatering may e.g. from about 40 to about 78 wt%.

The present document is also directed to a fungal biomass food product obtained or obtainable by the method of any one of the preceding claims.

The fungal biomass food product may for example be a meat-replacement product, such as minced meat, meat slices, meat cubes, whole cut meat, shredded fish or spreads The present document is also directed to the use of a vegetable substrate comprising, for filamentous food-safe fungi, indigestible substances for modifying the texture, such as the hardness, adhesiveness, springiness and/or chewiness, and/or nutritional value of a fungal biomass food product by admitting at least part of said indigestible substances to remain in said fungal biomass food product.

Other features and advantages of the invention will be apparent from the following detailed description, drawings, examples, and from the claims.

DEFINITIONS Singular references do not exclude a plurality, i.e. terms such as "a", "an", "first", "second" etc. do not preclude a plurality.

By "fungal biomass food product" is in the context of the present document intended a food product comprising fungal biomass obtained from food-safe filamentous fungi.

"Texture" as used in the context of the present document refers to the sensory profile of a product, such as the hardness, toughness, springiness and/or chewiness of the product. The term can also refer to the visual appearance of the product.

By "indigestible substance" is in the context of the present document intended a substance that cannot be completely digested by the fungi growing on it.

By "undigested" substance is in the context of the present document intended a substance that is digestible by the fungi, but that has not been digested during the growth of the fungi, e.g. because the growth time has not been enough to digest all digestible substances.

"Side stream" is a term commonly used in the food industry to refer to byproducts generated during the production of food products that are usually discarded. This term can also be applied to the final food product that is discarded for any reason, e.g. bread.

"Fungal biomass" in the context of the present document refers to the biomass of fungi produced by fermentation (growth) of the fungi on a vegetable substrate.

"Fungal biomass food product" in the context of the present document refers to the final food product, where the fungal biomass has been formed, processed and the like to form a food product ready for consumption with or without a cooking step. ln the context of the present document the terms "growth", "growing" and the like and "fermentation", "fermenting" and the like are used interchangeably and refer to the process where fungi are propagated on a substrate/growth medium to produce a fungal biomass.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Microscopy images of mycoprotein grown in synthetic media (A) or with dough (B). The images in B show fragments of the substrate as part of the final product.

Figure 2. Textural evaluation of mycoprotein grown with dough (20 or 30 g/L) or bread (20 or 40 g/L) in comparison with boiled chicken or boiled pork. Hardness (g), adhesiveness (g*s), springiness or chewiness were measured.

Figure 3. Microscopy pictures of fungi grown with wheat f|our substrate. Fungal filaments can be observed together with fibrous cellular structures.

Figure 4. Evaluation of the fungal growth in substrates with different particle sizes. The substrates with larger particles were more prominent in the final product (on the right).

Figure 5. Measurement of filtered solid content, pH values and biomass quality from fungi grown in different substrates. Solid content indicates amount of product (biomass + indigestible substances and undigested vegetable substrate from the sidestream), while pH drop indicates fungal growth.

Figure 6. Fungi was grown in oat residues ofdifferent sizes. The final product pressed and treated under same conditions showed different toughness levels when analyzed with a blade cut test (texture analysis).

Figure 7. Fungal mycelium bound to plant fiber structure. Figure 8. Process drawing. DETAILED DESCRIPTION ln the production of meat-replacement products, the addition of different kinds of additives, such as emulsifiers, stabilisers etc., often have to be used in order to provide a product with an appealing texture. The present inventors have surprisingly found that the texture of a fungal biomass food product can be modified by growing filamentous food safe fungi (herein also referred to simply as "fungi" or as "mycoprotein") on a substrate comprising, for the fungi, indigestible substances and admitting these indigestible substances to remain in the final fungal biomass food product. Thereby a fungal biomass food product can be provided that has an appealing texture without necessarily adding texture modifying additives to the product. Further, as the indigestible substances do not have to be removed from the fungal biomass, the production process is facilitated and thus e.g. cheaper and faster. Also, the remaining indigestible substances may provide the fungal biomass food product with an enhanced nutritional content and/or substances othen/vise beneficial for the consumer, such as fibers.

The present document is thus directed to a method for modifying the texture and/or the nutritional value of a fungal biomass food product. The method comprises growing food-safe filamentous fungi on a vegetable substrate that contains substances that are indigestible for the fungi and admitting at least part of these indigestible substances to remain in the fungal biomass food product. By allowing at least part of the indigestible substances to remain in the final fungal biomass food product, the texture of the product can be modified so that the desired texture is obtained. Improvement of the texture may also be considered as an improvement of the sensory profile of the fungal biomass food product.

By allowing at least part of or all of the indigestible substances to remain in the final fungal biomass food product, the texture of the food product can be modified. For example, the hardness, adhesiveness, springiness and/or chewiness of the food product may be modified. Thereby a fungal biomass food product having an appealing texture can be provided. For example, by the present method, the fungal biomass food product may be modified to have texture resembling meat or fish, such as for example minced meat, meat slices and cubes, whole cut meat, shredded fish or spreads.

The texture of a fungal biomass food product can in accordance with the method of the present document be modified by modifying e.g. the type, amount and/or particle size of vegetable substrate containing indigestible substances that is used and/or by modifying the amount of indigestible (and optionally undigested) substances that are allowed to remain in the fungal biomass food product. lndigestible and, if present, undigested substances may e.g. be removed from the fungal biomass after growth by skimming them off before harvesting the fungal biomass.

Further, even if the indigestible substances in the vegetable substrate are not digested by the fungi, the fungi may make them more bioavailable for the consumer of the fungal biomass food product. Thus, the nutritiona| value of the fungal biomass food product can be increased.

The vegetable substrate that the fungi are grown on may e.g. a plant fiber substrate, bread, and/or dough. For example the vegetable substrate may comprise or consist of oat, potato, corn, wheat flour, rye flour, wheat bran, seaweed or parts of these (e.g. potato peel) or any combination thereof. One advantage of the present method is that the vegetable substrate may be a food waste substrate and/or a side stream substrate. Thus, vegetable substrates that were othen/vise to be discarded can be used for producing fungal biomass food products, thus recycling such substrates.

An example of an indigestible substance include, but is not limited to, fat, polysaccharides, gluten and fibers. Fibers may be soluble or insoluble. Soluble fibers include, but are not limited to pectin, beta glucans, gums. Soluble fibers may be digested in part, but not completely and may thus remain in the final fungal biomass product. Examples of insoluble fibers include, but are not limited to cellulose, hemicellulose, lignin, glutenin, gliadin.

The vegetable substrate may preferably be pre-treated, such as by reducing the size of the particles constituting the vegetable substrate, or by hydrolysing the vegetable substrate with enzymes and/or microorganisms. One example of an enzyme that can be used to hydrolyse the vegetable substrate is amylase. ln this way the availability of the vegetable substrate as a growth substrate for the fungi may be improved. Further such pretreatment may improve the texture of the final fungal food product. For example, the vegetable substrate may be treated to reduce the size of substrate particles to 1 mm or less in diameter. By modifying the size of the particles of the vegetable substrate, the texture of the final fungal biomass food product may be modified to the desired one. The particle size can be reduced by e.g. blending the substrates into smaller pieces. Heat-drying or freeze-drying the vegetable substrates and then creating a powder using a mortar and pestle is another way of reducing the particle size of the vegetable substrate. Depending on what type of fungal biomass food product is to be formed, the texture may have to be modified in order to create a product with an appealing texture and look. The vegetable substrate typically has a particle size in the range of 100-4000 um, such as 100-3000 um, such as 100-500 um or 500-3000 um (mean diameter). For example, if a fungal biomass food product with less tough texture is desired, then the particle size should not be reduced to much. Thus, if a more homogenous but also more fibrous fungal biomass food product is desired, it may be preferable to reduce the size of the vegetable substrate particles more. Typically, for a softer fungal biomass food product, a vegetable substrate particle size of from about 500 um to about 3000 um may be used. Typically, for a tougher fungal biomass food product a substrate particle size of 500 um or less may be used.

Before the vegetable substrate is used as a substrate for growth of fungi, it is preferably treated to inactivate or kill other microorganisms, e.g. by sterilizing the vegetable substrate. Liquid, such as water, may be added before or after this treatment, in the latter case, sterile liquid has to be used. The treatment may e.g. comprise heat treating the vegetable substrate. For example, the substrate may be heated up to about 121 °C for about 20 min or more, preferably at an elevated pressure, such as 1 bar overpressure. This is preferably performed directly in the bioreactor the fungi are to be grown in or in an autoclave before addition of the substrate to the bioreactor. The vegetable substrate may also be heated up in a separate vessel before being transferred to the bioreactor where the fungi are to be g rown.

Thereafter a fungal starting culture comprising the filamentous food-safe fungi is added to the vegetable substrate and the fungi allowed to grow until a satisfactory concentration of fungi has been obtained, such as for about 20-96 hours or about 20- 72 hours. This may e.g. be the point when the growth slows down or stops. The fungi are preferably grown using liquid aerobic fermentation, preferably under agitation and/or stirring. The growth may typically take place in a stirred-tank bioreactor, airlift reactor or bubble column reactor. The temperature during growth is typically about 30 to about 45 °C, such as about 35 °C. The vegetable substrate may optionally be supplemented with nutrients, such as a nitrogen and/or phosphate source, trace metal(s), vitamin(s) and/or sulphate. Alternatively, the vegetable substrate may not be supplemented with any such additional nutrients.

The process could also be performed in a fed-batch process where a separate tank with concentrated substrate is used to feed the main bioreactor during the fermentation process, until further addition of substrate does not result in a significant or satisfactory growth of the fungi or the oxygen supply is not enough to keep the fungi in aerobic conditions. The fungal biomass is then harvested. This may e.g. be performed by filtering the fungal biomass to remove excess liquid or by centrifuging the fungal biomass. lf necessary, a further dewatering step may be applied to obtain a dryer fungal biomass. Such a dewatering step may e.g. be performed by pressing the fungal biomass. The water content of the fungal biomass after harvesting and optionally a dewatering step is performed is typically about from about 50 wt% to about 85 wt%, such as from about 60 to about 90 wt%, from about 70 wt% to about 90 wt%, such as about 75 wt%.

The amount of fungi in fungal biomass after fermentation and optional dewatering is typically from about 40 to about 78 wt%. The amount of fungi in the fungal biomass may e.g. be determined by determining the protein content as is done in Example 1.

The procedure could also be performed in a continuous process where a separate tank with concentrated substrate is used to feed the main bioreactor during the fermentation process, and simultaneously the fungi is harvested at the same rate in a way that the biomass concentration is kept constant. The harvested biomass can be processed in a continuous manner or as a batch.

Before or after the harvesting and/or dewatering step, the fungal biomass may be heat-treated to inactivate or kill the fungi and reduce RNA content in the biomass. Such a heat treatment step may e.g. take place at a temperature of about 50°C to about 95°C, such as about 60°C to about 85°C, or it may take place using two different temperatures of the above range, one for RNA reduction and one for inactivation. lt is also possible to include a washing step of washing the fungal biomass with water, such as sterile or tap water, optionally containing 0-5% of salt (e.g. NaCl). 11 After the fungal biomass is produced and harvested, including any additional treatments according to the above, the fungal biomass food product is formed. ln this process, additives further improving the texture, taste, nutritional content etc. may be added to the fungal biomass. Examples of such additives include, but are not limited to, hydrocolloids, starches, salts, antioxidants, fibers, colorants, flavorings, and fats.

Such additives may also include growth inhibitors inhibiting the growth of the fungi or other microorganisms in the fungal biomass food product. The additives are preferably of non-animal origin. Also, it is possible to add more of a (preferably sterilized) vegetable substrate to the fungal biomass to further improve the texture, taste, nutritional content etc. of the fungal biomass food product. The addition of additives may in addition or alternatively take place at the time before harvesting or dewatering the fungal biomass.

The fungal biomass food product may e.g. be a cut, pressed, shredded or grinded product or formed into a shaped product such as a hamburger, kebab or meatball like structure. lt is also possible to dry the fungal biomass to produce a dried fungal biomass food product. Such a dried fungal biomass can optionally be grinded to form a fungal biomass food product in the form of a powder. Drying may for example be done by heat (oven drying or dry spray) or freeze-drying. Depending on the type of drying, the final product is different. ln freeze-drying the functionality of the powder is kept intact, so the resulting dried product is more versatile and can be used in different formulations. Drying by heat is on the other hand easier and more cost- effective. Fluidized bed drying may also be used to dry the fungal biomass food product.

After production, the fungal biomass and the fungal biomass food product are preferably kept cold, such as at a temperature of from about 0°C to about 17°C, such as from about 0°C to about 15°C or from about 0°C to about 12°C.

As mentioned above, the fungal biomass food product comprises indigestible substances remaining from the vegetable substrate the fungi were grown on. Also, the fungal biomass food product may comprise substances that are not digested during the growth of the fungi, e.g. due to the time for growth not being long enough for them to get digested. These undigested substances may also help improving the 12 texture and/or nutritional content of the fungal biomass food product. The amount of indigestible and optionally undigested substances admitted to remain in the fungal biomass after growth is stopped may e.g. be 4 wt%, such as about 3-90 wt%, about 4-90 wt%, about 7-50 wt%, about 7-30 wt%, about 7-20 wt% or about 13-20 wt% of total amount of carbohydrates originally present in the vegetable substrate.

Food-safe filamentous fungi are well-known in the art and include, but are not limited to fungi of the Zygomycota and/or Ascomycota phylum, excluding yeasts, such as fungi of the genera Rhizopus, Neurospora, Aspergillus, Trichoderma, Pleurotus, Ganoderma, lnonotus, Cordyceps, Ustilago, Tuber, Fusarium, Pennicillium, Xylaria, Trametes, or any combination thereof. Examples of fungal species that may be used according to the present document include, but are not limited to fungi of the species Aspergillus oryzae, Rhizopus oryzae, Fusarium graminareum, Cordyceps militaris, Cordyceps sinensis, Tuber melanosporum, Tuber magnatum, Pennicillium camemberti, Neurospora intermedia, Neurospora sitophila, X ylaria hypoxion, or any combination thereof. Further examples of fungi that can be used according to the present document include fungi described in WO2020061502.

The present document is also directed to a fungal biomass food product obtained or obtainable by the method described in the present document. Examples of such fungal biomass food products are disclosed elsewhere herein.

The present document is also directed to the use of a vegetable substrate as disclosed herein comprising, for filamentous food-safe fungi, indigestible substances for modifying the texture, such as the hardness, adhesiveness, springiness and/or chewiness, and/or nutritional value of a fungal biomass food product by admitting at least part of said indigestible substances to remain in said fungal biomass food product as disclosed elsewhere herein.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. 13 EXAMPLES Table 1. Composition of the growth medium.

Growth medium (NH4)2SO4 0 to 1 g/L KH2PO4 0 to 0.4 g/L Side-stream 10 to 40 g/L Example 1: Growing fungi in bread and dough side-streams: Textural, microscopy, nutritional content + % biomass and side-stream Bread or dough at 20 g/L of dry weight was submerged in water for 10 minutes prior mixing with a industrial mixer for 3 min. Ammonium sulphate ((NH4)2SO4) and potassium phosphate (KH2PO4) were added at 1 g/L and 0.4 g/L respectively. Sterilization in a 250L working volume reactor was performed for 20 min at 100°C. A preculture was prepared with either bread or dough (20 g/L dry weight) containing the same chemicals and Rhizopus spores (0.7% of total volume) were grown at 35°C for 24h prior inoculation. lnoculation was performed by transferring the preculture to the sterilized bioreactor (5% of total volume). Fermentation in the reactor was performed for 24h under the following conditions: temperature 35°C, pH 7, airflow 70L/ min and stirrer 150 rpm. Ammonia (NHg) was added continuously during the fermentation process. The resulting biomass was dewatered and washed with water, then it was heat treated at 70°C for 10 min. A final step of dewatering was performed by pressing the biomass in a Hydropress (9060149, P-Lindberg) at 2 bar for 10 min. Microscopy pictures were taken using a Olympus CK2 inverted microscope and a RS Pro C microscope camera (MicFi).

Additionally, 20 g/ L of oats were either blended with a commercial blender (oat fragments >1 mm) or freeze dried and then powdered by mortar and pestle (particles <1 mm). The substrates were autoclaved together with the chemicals shown before at 120°C for 20 min. Then, the media was added to shake flasks (0.5 L) and inoculated with spore suspensions made out of Rhizopus plates (0.7% of total volume) and allowed to ferment for 22 - 43 h at 35°C while shaking (120 rpm). The resulting biomass was dewatered, washed and heat treated at 70°C for 10 min prior pressing using a 14 Hydropress (9060149, P-Lindberg) at 2 bar for 10 min. As a control, biomass was grown in the same conditions but using sucrose instead of oats.

The nutritional profiles of the different mycoproteins generated in the previous experiments were determined by GC-FID at Eurofins Food and Feed Testing Sweden AB, Lidköping, together with the intact substrates (bread and oats) for comparison (Table 2).

To calculate the percentage of mycoprotein and of side stream in the final product, the carbohydrate content was compared. Regular mycoprotein contains 0.53 g of carbohydrates while bread contains 48 to 56 g. The resulting product of fermentation of bread contains 6.98 to 10.5 g of carbohydrates, which means the fungi has consumed 80 to 87% of the original substrate, remaining 13 to 20% of bread or dough in the final product (Table 2). Fragments of the dough could be observed microscopically in the final product as shown in Figure 1, B, and compared with the mycoprotein grown in synthetic medium (Figure 1, A).

Fibers contain was doubled in biomass grown in oats (Table 2). A good sign of fungi production is the protein content of the final product. The protein content was higher in mycoprotein grown in finely grinded oats compared to the one grown in larger oat particles (>1 mm), indicating a better growth and hence a higher mycoprotein content (Table 2). lf it's assumed that no proteins are consumed from the substrate, the percentage of mycoprotein in the final product ranges from 70 to 78% in mycoprotein grown with bread or dough, and 40 to 48% in mycoprotein grown with oats (Table 2).

Table 2. Nutritional composition of mycoprotein, bread and mycoprotein produced with bread or dough.

Mycoprotei Bread Mycoprotei Mycoprotei Oats (gl Mycoprotei Mycoprotei n (gl 100 g) n with n with 100 g) n with oats n with oats (gl 100 g) Dough (gl Bread (gl (particles (particles 100 g) 100 g) <1mm) (gl >1mm) (gl 100 g) 100 g) Water content 65-73 g 20.3 - 27.4 68.7 g 74.8 g 66.7 g 79.4 g / 9 side-stream Ash / / 1.57g 1.11 g 2.3g / Crude protein (in 14.4 g 8.3 - 9.1 g 13.2 g 14.2 g 10.4 g 9.31 g 8.37 g wet weight) Crude protein (in 41.0 - 53.3 12.4 - 12.8 42.2 g 56.4 g 31.2 g 59.7 g 52.3 g dry weight) g g Crude fat 2.48 g 2.2 - 6.4 g 6.05 g 2.91 g 4.22 g 3.3 g / Carbohydrates 0.53 g 48 - 56 g 10.5 g 6.98 g 4.79 0.33 g / Energy value 415 kJ/ 99 / 627 kJ/ 150 468 kJ/ 112 507 kJ/ 121 335 kJ/ 80 / (kJ) kcal kcal kcal kcal kcal Fructose / / <0.04 g 0.18 g <0.04 g <0.04 g / Glucose / / 0.55 g 0.77 g <0.04 g <0.04 g / Lactose / / <0.04 g <0.04 g / <0.04 g / Maltose / / <0.04 g <0.04 g 3.54 g <0.04 g / Saccharose / / <0.04 g <0.04 g 0.14 g <0.04 g / Galactose / / <0.04 g <0.04 g / <0.04 g / Total sugar 0.39 g 5.4 - 7.9 g 0.55 g 0.95 g / / content Total omega 6 1.3 g / 1.46 g 0.74 g 1.8 g 1.33 g / fatty acids Total omega 3 0.008 g / 0.23 g 0.06 g 0.08 g 0.05 g / fatty acids Ratio 167.7 / 6.52 11.45 21.6 28.7 / omega6/omega3 Total fatty acids / / 5.66 g 2.59 g / / Sodium 0.51 g 0.65 - 0.95 6.9 mg <5 mg <5 mg 0.06 g / NaCl 9 0.017 g <0.012 g <0.012 g Gluten nd / nd 57 mg/ kg / / Dietary fiber 3.1 g / / 10.7 g 6.1 g / Percentage of 100% 0% 80% 87% 0% / mycoprotein (based on carbohydrate consumption) Percentage of 100% 0% 70.1% 77.7% 0% 47.7% 40.3% mycoprotein (based on protein content) Percentage of 0% 100% 20% 13% 100% / Microscopy pictures from the biomass generated by growing fungi in dough or sucrose (Figure 1) showed dough fragments remaining in the final product (Figure 1, B) compared to the sucrose grown mycoprotein (Figure 1, A).

Texture of mycoprotein was very soft, and this texture could be modulated by the addition of more vegetable substrate which forms part of the end product (Figure 2). 16 Texture Profile Analysis (TPA) Texture properties were analyzed through a standard Texture Profile Analysis using a Stable Microsystems TA.TX Plus-C equipped with a P/100 Stainless Steel Compression Platten with a diameter of 100 mm and an acquisition rate of 500 PPS. The plate was set to compress the samples by 60% of the sample size using a trigger force of 20 g in a 2-cycle analysis at a test speed of 1 mm/sec. The deformation curve of the sample was obtained, from which the parameters Force 1, Force2, Area FT1:2, Time-diff 1:2, AreaFT1:3, AreaFT2:3, AreaFT3:4 (negative), AreaFT4:6, and Time-diff4:5, according to the manufacturer's protocol.

From these parameters, the following parameters were calculated: Hardness = Force2 (peak force of the first compression of the product) Springiness = Time-diff4:5 /Time-diff41:2 (percentage of product height that is regained after first deformation) Chewiness = Gumminess x Springiness Adhesiveness = AreaFT3:4 (pulling strength during retraction from sample Example 2: Evaluating growth of fungi in different side-streams Mycoprotein was grown in presence of several different side-streams as described in the previous example, briefly as follows. 20 g/ L of bread, dough, oats, wheat flour, corn starch, rapeseed filter cake, potato peel, potato fiber, brewer's spent grain (BSG) or corn flour were blended with a commercial blender and autoclaved in bottles at 120°C for 20 min. Then, the media was added to shake flasks (0.5 L) and inoculated with spore suspensions made from Rhizopus plates (0.7% of total volume) and allowed to ferment for 22 - 96 h at 35°C while shaking (120 rpm). The resulting biomass was dewatered, washed and heat treated at 70°C for 10 min. The biomass was sieved and dried to calculate growth yields (g /L). The protein content was calculated by NMKL 6 method by Eurofins Food and Feed Testing Sweden AB, Lidköping. 17 The yields varied greatly depending on the incubation times and substrates, ranging from 0.175 to 11.5 g/ L (Table 3). Protein contents of 27.1 to 54.3% proved good fungal growth in most substrates. Accessibility of the fungi to the substrates also proved to be important for good growth yields. For example, corn and wheat flours showed better growth than corn starch, which precipitated in the medium making it less available to the fungi during growth.

The fungi grown with wheat flour showed fiber structures microscopically (Figure 3).

Table 3. Cultivation time (h) final yield (g/ L) and protein content (%) of mycoprotein grown with different side-streams as substrate. substrate Yield (g/i.) cuitivetien Protein time (h) content (%) Breed i Deugh 3.07 - 7.1 i 22 - 25 31.9 - 44.9 iiäkeieid iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii iifláiliíjá iiiiiiiiiiiiiii iiiiiiiiiiiiiiiii 1154531 iiiiiiiiiiiiiiiiiiiiiii i oat i oet reeiduee 6.58 i 0.004 i 48 Na others i wheat flour 2.13 - 11.5 i 24 - 96 27.1 Corn starch 0.175 - 5.33 24 - 96 29.5 Repeeeed filter 16.13i2.2s 48 08.9 cake Åtlïàotaàttottpetel AAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA AAAA A418 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAA (i 0.91 ...pètá.t.ó..fi.bé} ............... .š..g...ó..;...1...í..à ............... -24 .............................. .Na ............................ .. (Ulšreweritsttstpetnt AAAAAAA LLLLLLLLLLLLLLLLLLL N24 LLLLLLLLLLLLLLLLLLLLLLLLLL LLLLLLLLLLLLLLLLLLLLLLLLLLL *i grain (BSG) 18 To determine the capacity of the fungi to digest different particle sizes of substrate, eight types of substrates were selected (Figure 4). Each substrate was added to 2 L shake flasks (400 ml of sample volume, 20 g/L solid material). The experiment was performed in duplicates. The shake flasks were incubated for 25 h at 35°C in a shaking incubator (120 rpm). The solid material was sieved out after the incubation period and pictures were taken of the final product.

Figure 3 shows the substrate before and after fermentation. As illustrated, the fungi were not able to digest all of the substrates completely, resulting in indigestible particles ending up in the final product. When comparing the different oat substrates for example, it is clear that reduction in particle size can decrease the amount of substrate ending up in the final product. Example 3. Growth of biomass in different sources of starch and fiber Media was prepared with eleven different side-stream substrates (20 g/ L) together with chemicals (NH4)2SO4 (10 g/ L) and KH2PO4 (5.25 g/ L). As a control, synthetic media was also prepared with 35 g/ L sugar; 10 g/ L (NH4)2SO4; 5.25 g/ L KH2PO4; 1.06 g/ L MgSO4; 1.75 g/ L CaClz and 0.25% trace solution. pH of the different media was measured and set to 5.5 using HCl or NaOH. The media were then autoclaved at 120°C for 20 min, and distributed in different shake flasks (0.5L/ flask). The shake flasks were then inoculated with Rhizopus spore suspension (0.7% of total flask volume). pH was measured again, then the shake flasks were incubated for 24h at 35°C while shaking (120 rpm). pH was measured at the end of the fermentation and pH drop was calculated to determine growth. The biomass was then sieved, evaluated for filamentous morphology and color (quality 1-4) and finally dried for growth yield calculation (g/ L) (Figure 5).

Example 4. Using different side-stream particle sizes to change the sensorial properties of the final product Oat residues were obtained from the process that extracts oat protein to a liquid fraction. The oat residues remining consisted of wet granules rich in undigestible fiber and carbohydrates. The granules had a size between 500 and 3000 um. Part of the granules was also crushed with a ball mill until a fine powder with particle size below 500 um was formed. Fungal spores from Rhizopus were used to inoculate a pre- 19 culture of 20L containing pre-culture media composed of 20 g/L Glucose, 7.5 g/L ammonium sulfate, 3 g/L potassium phosphate and 3 g/L yeast extract. This preculture was used to inoculate a bioreactor of 250L containing 18 g/L dry weight of oat substrate, 2 g/L glucose, 0.75 g/L ammonium sulfate and 0.3 g/L potassium phosphate. The bioreactor was kept at a constant aeration of 0.5 vvm using regular air, kept at a pH between 4.0 and 5.5 and a temperature between 30 and 35C. After 48h, the biomass was harvested by sieving and heat treated between 65 and 72C for 20 min. The biomass was then washed with cold water and pressed in a hydropress system at a pressure of 2 bar. The resulting biomass had considerable amounts of oat residue fiber observed at naked eye from the presence of darker structures in sizes same or smaller than the initial particle size. The same process was repeated using the same media as the preculture for the fermentation, yielding pure mycelium biomass with a white colour without oat residues.

Texture analysis using a Knife Blade method Samples of solid fungi biomass were prepared as a cuboid shape of 20 mm x 10 mm x 5 mm (length x width x height) for texture analysis. Texture analysis was carried using a Stable Microsystems TA.TX Plus-C equipped with a Knife Blade (70 mm width x 3 mm thick, 45°-chisel end) and guillotine block. The sample was placed in the centre of the guillotine block and cut with the knife blade starting at a position of 20 mm and a descending speed of 2 mm/s for 30 mm. A curve plot was obtained showing measured Force x Time, and the parameters of Peak Force was defined as the maximum Force value of the curve in g, while Toughness was defined as the total area below the curve in g-s.

The results from the testing's are illustrated in Fig. 6 showing that increasing particle size reduces the chewiness mouthfeel, which is represented by toughness and peak force values.

Example 6. Creating a chunk of meat-like food product from mycoprotein grown in bread waste Fungal cells were grown in a 250 L bioreactor as explained in example 4, with the exception that bread waste sidestreams were used instead of oat residues as carbon source. The bread samples included rye bread with insoluble particles. The biomass from the fermentation was harvested and remains of undigested bread were observed. The biomass was heat treated and pressed in a hydropress system, resulting in a press cake with a length between 10 and 20 cm. The press cake was then sliced into 3mm thick slices and marinated in broth.

The biomass slices were identified with a taste and mouthfeel as bacon, kebab or in general meaty thin slices.

Example 7. Creating mycoprotein using potato streams and then using potato fiber in the end for an improved texture Fungai cells were grown in a 250 L bioreactor using a mix of potato wash water and potato peel. The potato peel was dry and milled and added in amounts of 30 g/L to the wash water to create the fermentation media. The fungal biomass was harvested and processed as described in example 6. The resulting product had a less compact and more moist mouthfeel compared to samples of pure fungi biomass. To the samples was also added potato fiber and an effect of moist, more tender and less compact mouthfeel could be felt.

Confocal microscopy Samples with integrated fiber materials was analyzed by fluorescent microscopy. For this, the frozen samples were thawed overnight at +4°C prior to staining and imaging with confocal laser scanning microscopy (CLSM) equipment consisting of a Zeiss LSM 710 (Zeiss, Jena,Germany) attached to a Zeiss Axio lmager.Z microscope. Small pieces (dimensions 1cm x 1cm x 0.3 mm) were cut from the middle of thawed samples. The components present in the samples were visualised with two staining combinations. The first staining combination included staining of fungal cell walls (cellulose and chitin) and protein by adding 40 pl of 0.01% (w/v) Calcofluor White (Fluorescent brightener 28,Aldrich, Germany) and 40 pl of 0.1% (w/v) Rhodamine B (Merck, Darmstadt, Germany) on top of the sample surface at 1 min intervals without rinsing between, respectively (Fulcher et al.1989, Auty 2013).The second staining combination included staining of fungal cell walls (cellulose and chitin) and lipids by 0.01% (w/v) Calcofluor White (Fluorescent brightener 28, Aldrich, Germany) and 0.01 % (w/v) Nile Red (Molecular Probes, 21 Eugene, OR, USA). Stained samples were examined on a microscope slide as sealed preparates covered with a cover slip. Diode laser line of 405 nm was used for excitation of Calcofluor and emission was collected at 425-480 nm. HeNe laser line 543 nm was used for excitation of Rhodamine B and Nile Red, and emissions were collected at 571-620 nm and 650-710 nm, respectively. Images were assembled of the optical sections taken using a 40x objective (Zeiss EC Plan-Neofluar,numerical aperture of 0.75) to the depth of 13-55 um with 0.76 um z step using ZEN software(Zeiss). The results shown in Figure 7 of this staining shows large plant structures interacting with fungal biomass and interrupting the compact structure, indicating that the mouthfeel benefits of fibers come partially from the disruption of the fungal mycelia compact structure. lt is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Unless expressly described to the contrary, each of the preferred features described herein can be used in combination with any and all of the other herein described preferred features.

Claims (1)

1.Claims A method for modifying the texture, such as the hardness, adhesiveness, springiness and/or chewiness, and/or nutritional value of a fungal biomass food product, said method comprising growing food-safe filamentous fungi on a vegetable substrate, wherein said vegetable substrate comprises, for said food-safe filamentous fungi, indigestible substance(s), such as fibers, and admitting at least part of said indigestible substances to remain in said fungal biomass food product. _ The method according to c|aim 1, wherein said vegetable substrate is a plant fiber substrate, bread, and/or dough, such as oat, potato, corn, wheat flour, rye flour, seaweed, wheat bran or any combination thereof. The method according to c|aim 1 or 2, wherein said vegetable substrate is a food waste substrate and/or a side stream substrate. _ The method according to any one of the preceding claims, wherein the vegetable substrate has a particle size of from about 100 to about 4000 um, such as from about 100 to about 3000 um, such as from about 100 to about 500 um or from about 500 to about 3000 um. The method according to any one of the preceding claims, wherein said method comprises the steps of: i) growing said food-safe filamentous fungi on said plant substrate to produce a fungal biomass; ii) harvesting, such as by dewatering, the fungal biomass of step i); and iii) optionally further dewatering, such as by pressing, the harvested fungal biomass of step ii), to obtain a fungal biomass. _ The method according any one of the preceding claims, wherein said food- safe filamentous fungi are grown in step i) using liquid aerobic fermentation. The method according to any one of the preceding claims, wherein said vegetable substrate is supplemented with nutrients, such as a nitrogen and/or phosphate source, trace metal(s), vitamin(s) and/or sulphate. _ The method according to any one of claims 1-6, wherein said vegetable substrate is not supplemented with nutrients.The method according to any one of the preceding claims, wherein said vegetable substrate is pre-treated, such as by reducing the size of the particles constituting the vegetable substrate, hydrolysing the vegetable substrate with enzyme(s) and/or microorganisms(s). 10.The method according to any one of the preceding claims, wherein said food-safe filamentous fungi are of the Zygomycota and/or Ascomycota phylum, exciuding yeasts, such as fungi of the genera Rhizopus, Neurospora, Aspergillus, Trichoderma, Pleurotus, Ganoderma, lnonotus, Cordyceps, Ustilago, Tuber, Fusarium, Pennicillium, Xylaria, Trametes, or any combination thereof. .The method according to any one of the preceding claims, wherein said fungi are of the species Aspergillus oryzae, Rhizopus oryzae, Fusarium graminareum, Cordyceps militaris, Cordyceps sinensis, Tuber melanosporum, Tuber magnatum, Pennicillium camemberti, Neurospora intermedia, Neurospora sitophila, Xylaria hypoxion, or any combination thereof. 12.The method according to any one of the preceding claims, wherein the amount of fungi in funga| biomass after fermentation and optionai dewatering is from about 40 to about 78 wt%. 13.A funga| biomass food product obtained or obtainable by the method of any one of the preceding cIaims. 14.The funga| biomass food product of claim 13, wherein said funga| biomass food product is a meat-replacement product, such as minced meat, meat siices, meat cubes, whole cut meat, shredded fish or spreads 15. Use of a vegetable substrate comprising, for filamentous food-safe fungi, indigestib|e substances for modifying the texture, such as the hardness, adhesiveness, springiness and/or chewiness, and/or nutritiona| value of a funga| biomass food product by admitting at least part of said indigestib|e substances to remain in said funga| biomass food product.