TECHNICAL FIELD

The present disclosure pertains to a food product comprising fungi biomass and a food additive. This disclosure also relates to a method for producing the food product and the use of the food product for preparing a finished consumer food product.

BACKGROUND

In 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. In 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. In recent years, these issues have led to a heavily increased demand for meatresembling 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 sources.

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.

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 of dietary 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 “lacking 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

One or more of the above objects may be achieved with a food product in accordance with claim 1 , a method for producing the food product in accordance with claim 16, the food product according to claim 29 produced using the method disclosed herein, and/or use of a food product according to claim 30. Further embodiments are set out in the dependent claims and in the following description. A food product as disclosed herein comprises fungi biomass and at least one food additive. The fungi biomass comprises a filamentous mycelium network wherein the at least one food additive is present in an amount of 0.05% by weight or more of the total weight of the fungi biomass and the food additive, wherein the at least one food additive is integrated in the filamentous mycelium network. More than 50% of the filamentous mycelium network are aligned substantially in planes extending in a first direction, thus forming a lamellar structure in which the filamentous mycelium network comprising the at least one integrated food additive is substantially intact.

The fungi biomass as disclosed herein may be obtained from different species within Ascomycota and Zygomycota phyla, excluding yeasts. The fungi biomass is advantageously obtained from fungi of the genera selected from the list consisting of Rhizopus, Neurospora, Aspergillus, Trichoderma, Pleurotus, Ga noderma, Inonotus, Cordyceps, Ustilago, Tuber, Fusarium, Pennicillium, Xylaria, Trametes, or any combination thereof.

The fungi biomass is advantageously obtained from a fungus selected from the group consisting of the species Aspergillus oryzae, Rhizopus oryzae, Rhizopus oligosporus and Rhizopus microspores, Fusarium graminareum, Cordyceps militaris, Cordyceps sinensis, Tuber melanosporum, Tuber magnatum, Pennicillium camemberti, Neurospora intermedia, Neurospora sitophila, Xylaria hypoxion, or any combination thereof.

Preferably the fungi biomass is obtained from a fungus selected from the group consisting of the species Neurospora intermedia, Neurospora crassa, Aspergillus oryzae, Rhizopus oryzae, Rhizopus microsporus and Rhizopus oligosporus. More preferably the fungi biomass is obtained from a fungus selected from the group consisting of the species Rhizopus oryzae, Rhizopus microsporus and Rhizopus oligosporus.

A particular drawback with meat- and fish-replacement products currently available is that the texture is not satisfactory when being used as a fillet or large pieces of meat- or fishreplacement instead of minced meat- or fish-replacement. The present invention is based on the finding that fungi biomass comprising at least one food additive integrated in the filamentous mycelium network provides benefits in terms of texture and taste of a meat- or fish-replacement product, including fillets or steak replacements. To achieve the desired texture and taste of the food product, the at least one food additive should be present in an amount of 0.05% by weight or more of the total weight of the fungi biomass and the food additive. In conventional meat- and fish-replacement food products comprising fungi biomass, the fungi biomass may be mixed with the food additive in a separate step after production of the fungi biomass when the fungi biomass has its final water content. Such mixing implies destruction of the filamentous mycelium network structure.

Alternatively, in other conventional meat- and fish-replacement food products comprising fungi biomass, the fungi biomass is mixed at a high-water content with a heat-setting gel ingredient such as egg albumin and then heated to create a firm gel structure containing fungal biomass and other ingredients. Such process implies that the final product does not have an intact mycelium network, rather a dispersion of cells in a gel matrix. The food product disclosed herein is completely free from animal derived products.

However, when used herein, the terms “the filamentous mycelium network is substantially intact” or “a substantially intact filamentous mycelium network” are intended to mean that the filamentous mycelium network formed during the fermentation of the fungi culture and the subsequent process steps, which becomes aligned in planes extending in a first direction forming a laminar structure during the process steps subsequent to the fermentation, remains aligned substantially in planes extending in a first direction forming a laminar structure, also after the at least one food additive has been integrated in the filamentous mycelium network. The at least food additive is integrated into the filamentous mycelium network in such a manner that the network is not interrupted or destroyed but remains in planes extending in the same direction forming a laminar structure of filaments after integration.

Optionally, 50% or more, such as 70% or more, or 80% or more of the filamentous mycelium network are aligned substantially in planes extending in a first direction, thus forming a lamellar structure. The fact that the fibers of the filamentous mycelium network each extend in the first direction, i.e. , the same direction, provides an enhanced and meat- or fish-like texture to the food product according to the present disclosure.

The food product described herein comprises at least one integrated food additive, but it may optionally also comprise more than one integrated food additive, such as two or more integrated food additives, such as three or more integrated food additives.

The integrated food additive may be selected from the group consisting of food fibers, starches, proteins, fats, oils, food flours, hydrocolloids, and gelling agents. Food additives selected from this group, and which are integrated into the filamentous mycelium network structure according to the present disclosure, have been seen to provide a fungi biomass food product which has a pleasant softness and chewiness.

The integrated food additive may be selected from the group consisting of rice starch, potato starch, corn starch as well as other modified starches, potato fibers, bamboo fibers, pea fibers, oat fibers, canola oil, pea protein, soy protein, and hydrocolloids such as methylcellulose, carrageenan, and alginate.

The integrated food additive may be present in an amount of 0.05% by weight or more of the total weight of the fungi biomass and integrated food additive, optionally 0.1 % by weight or more, optionally 0.5% by weight or more, or 2% by weight or more of the total weight of the fungi biomass and integrated food additive. Optionally, integrated food additive is present in an amount within the range of from 0.05% to 20% by weight. Optionally, the integrated food additive is present in an amount within the range of from 0.01 % to 15%, such as from 0.5 to 12.5%, such as from 2% to 10% by weight of the food product.

A particular drawback with meat-replacement products currently available is that the texture is not satisfactory when being used as a fillet or large pieces of meat- or fish-replacement compared to the texture of minced meat- or fish-replacement. The present invention is based on the finding that fungi biomass comprising at least one food additive integrated in the filamentous mycelium network provides benefits in term of texture and taste of a meat-replacement product.

Due to the inventive process disclosed herein, the filamentous mycelium network comprising at least one integrated food additive may be substantially intact. This has been found by the present inventors to provide an enhanced meat- or fish-like texture of the fungi biomass food product.

The food product disclosed herein may be a final food product being in the form of a fillet, such as a replacement for whole-cut fish, chicken or whole-cut meat. The food product may be cut or sliced to form a final consumer food product.

The toughness of the food product may be within the range of from 2 000 g.s to 15 000 g.s., optionally within the range of from 3 500 g.s. to 10 000 g.s, as measured by the Knife blade test disclosed herein. It has been discovered that food products according to the present disclosure that have a toughness, as measured by the Knife blade test of from 2 000 g.s to 15 000 g.s., preferably within the range of from 3 500 g.s. to 10 000 g.s., have a chewiness resembling that of meat- and fish-products and are neither too soft and mushy nor too tough and chewy. The firmness of the food product may be within the range of from 2 000 g to 10 000 g., optionally within the range of from 4 000 g. to 6 000 g., as measured by the Knife blade test disclosed herein. It has been discovered that food products according to the present disclosure that have a firmness within the range of from 2 000 g to 10 000 g., preferably within the range of from 4 000 g. to 6 000 g., have a bite profile resembling that of meat- and fish-products and are neither too soft and mushy nor too hard and chewy.

The food product disclosed herein may also be an intermediate food product being in the form of bits each having the size of from 1 mm to 16 mm, as measured at the largest cross-section of each bit. For an intermediate food product according to the present disclosure, which is intended to be used for forming for example balls, patties, or a minced food product it has been found that the provision of at least a part of the meat- or fish-replacement products in bits of fungi biomass having a size within the range of 1 mm to 16 mm enhances the texture and reduces the gumminess of the final product.

Optionally, the food product comprises two groups of bits, a first group wherein the bits each has a size varying between 1 mm and 6 mm and a second group wherein the bits each has a size varying between 6 mm and 16 mm. For an intermediate food product according to the present disclosure, which is intended to be used for forming for example balls and patties, it has been found that if the sizes of the bits vary in the intermediate food product, the texture of the final product has a relatively high resemblance with meat products.

The food product disclosed herein may be a final consumer food product in the form of balls, patties, fillet, meat-like cuts, fish replacements, whole-cut meat replacements, sliceable products such as hams, bacon, or spreadable products such as pastes. The food product may alternatively be an intermediate food product used to form balls, patties, fillet, meat-like cuts, fish replacements, whole-cut meat replacements, sliceable products such as hams, bacon, or spreadable products such as pastes.

When the fungi biomass food product is an intermediate food product intended to form a final product by shaping and/or mixing with further ingredients, the food product may further comprise one or more ingredients selected from the following group: protein from soybean, pea, chickpea, wheat, rice, mung bean, potato, fava bean, lupin bean, egg or dairy; fat or oil from soybean, rapeseed oil, soybean oil, canola oil, coconut oil, sunflower oil or shea butter; binders and additives such as methylcellulose, xanthan gum, alginate, locust bean gum, agar-agar, gum arabic, egg white protein, sources of carbohydrates such as starch, wheat flour, potato flour, rice flour, oat flours, apple extract and sources of fiber such as pea, sugarcane, wheat, cellulose, oats and apple.

A method for manufacturing a food product according to the present disclosure comprises the steps of: a) cultivating fungi under aerobic submerged fermentation conditions using a closed fermentation vessel with liquid substrate media while stirring to obtain a fungi biomass comprising a filamentous mycelium network; b) processing the fungi biomass obtained from step a) by heating to a temperature within the range of from 50-85°C; c) separating the fungi biomass obtained from step b) from the liquid cultivation media, such as by filtration or decanting, such that the biomass has a water content within the range of from 85% to 98%; d) adding at least one food additive to the fungi biomass obtained in step c) to provide a food product comprising food additive in an amount of from 0.05% by weight or more relative to the total weight of fungi biomass and food additive in step f); e) mixing the fungi biomass and the at least one food additive, thereby integrating the at least one food additive into the filamentous mycelium network without disrupting the filamentous mycelium network; and f) dewatering, such as by pressing or centrifuging, the fungi biomass obtained from step e) to substantially orient the filamentous mycelium network in a single plane, such that a fungi biomass food product is obtained having a water content within the range of from 50 to 85 % by weight, as measured by weighing of the fungi biomass before and after an oven drying step, thus forming a lamellar structure and wherein the filamentous mycelium network comprising the at least one integrated food additive is substantially intact.

The fungi biomass is advantageously obtained from fungi of the genera selected from the list consisting of Rhizopus, Neurospora, Aspergillus, Trichoderma, Pleurotus, Ganoderma, Inonotus, Cordyceps, Ustilago, Tuber, Fusarium, Pennicillium, Xylaria, Trametes, or any combination thereof.

The fungi biomass is advantageously obtained from a fungus selected from the group consisting of the species Aspergillus oryzae, Rhizopus oryzae, Rhizopus oligosporus and Rhizopus microspores, Fusarium graminareum, Cordyceps militaris, Cordyceps sinensis, Tuber melanosporum, Tuber magnatum, Pennicillium camemberti, Neurospora intermedia, Neurospora sitophila, Xylaria hypoxion, or any combination thereof.

Preferably the fungi biomass is obtained from a fungus selected from the group consisting of the species Neurospora intermedia, Neurospora crassa, Aspergillus oryzae, Rhizopus oryzae, Rhizopus microsporus and Rhizopus oligosporus. More preferably the fungi biomass is obtained from a fungus selected from the group consisting of the species Rhizopus oryzae, Rhizopus microsporus and Rhizopus oligosporus.

After the dewatering step f), and after having formed the laminar structure wherein the filamentous mycelium network remains in planes extending in the same direction, the filamentous mycelium may not revert to the slurry state of step e).

The water content in a slurry of the fungi biomass and the liquid cultivation media in step c) may be within the range of from 85 to 95%, such as above 90%, and the at least one food additive may be added in amounts of 0.002% or more relative to the weight of the slurry of the fungi biomass, the at least one food additive and water. The amount food additive relative to the weight of the slurry depends on the amount of food additive desired for the final product.

Step c) may include washing, such as with water, to remove the cultivation media. Water may be added to provide the water content level of 85% to 98%.

The process that is considered a cultivation or aerobic fermentation process, i.e. , the process carried out in step a), may take place in a stirred-tank bioreactor, airlift reactor or bubble column reactor, where the liquid medium is agitated by aeration and/or stirring. The process is advantageous compared to a so-called solid-state fermentation process in that the produced fungi biomass is essentially free of any leftover substrate particles, which would otherwise prevent obtaining the improved texture as described in the present invention.

The process is a cultivation or aerobic fermentation process in a closed, sterile vessel with media containing a single or several different carbon sources originating from processed grain crops or glucose-, fructose- or lactose-containing substrates in a monomeric or oligomeric form.

The process related to the harvesting and post-fermentation treatment of the fungi biomass, includes a heat-treatment step, according to step b) for inactivation of the fungi biomass.

In step d) wherein the at least one food additive is added to the fungi biomass obtained in step c) to provide a food product comprising food additive in an amount of from 0.05% by weight or more relative to the total weight of fungi biomass and food additive in step f), the slurry, prior to pressing of the fungi biomass, the water content may be about from 85 % by weight to about 98 % by weight, optionally above 90 % by weight. The at least one food additive may be added in an amount of between 0.002 % by weight to 15 % by weight of the additive relative to the weight of the slurry. Optionally the at least one food additive may be added in an amount of between 0.02% to 12.5%, such as in an amount of 0.08% to 10% by weight of the additive relative to the weight of the slurry.

In step d) the at least one food additive may optionally be added to the fungi biomass obtained in step c) to provide a food product comprising food additive in an amount of 0.1 % by weight or more relative to the total weight of fungi biomass and food additive in step f), optionally the at least one food additive is added to the fungi biomass obtained in step c) to provide a food product comprising food additive in an amount of 0.5% by weight or more of the total weight of the fungi biomass and the food additive.

In step e) the fungi biomass is mixed with the at least one food additive to integrate the food additive into the filamentous mycelium network in a manner which does not disrupt the filamentous mycelium network. The at least one food additive is integrated into the filamentous mycelium network in such a manner that the network is not interrupted but remains substantially intact.

The heat treatment step is a process used for inactivating the fungi biomass after fermentation, whereby the fungi biomass together with the liquid fermentation substrate, a diluted version of it, or the biomass submerged in water, may be heated to a temperature within the range of 50° to 85°C for a period of time between 1 .5 min and 1 h. The biomass may furthermore be heat treated before or after pressing in step f) using exposure to steam for 5-20 minutes.

Step b) may include processing the fungi biomass obtained from step a) by heating to a temperature within the range of from 60-85°C.

The fungi biomass may be washed after heat treatment, the fungi biomass may be rinsed with tap water, distilled water or water containing 0-5% of salt (NaCI), with a water temperature of 6- 15°C, for 3-60 min.

The stirring in step a) may be performed with a rotation velocity within the range of from 150 to 300 RPM, corresponding to a tip speed of 0.63 m/s to 1 .26 m/s in a vessel with a Rushton turbine with a diameter of 8 cm, optionally within the range of from 150 to 250 RPM. This has been shown to provide enhanced Toughness and Firmness values of the fungi biomass food product. The heating of the biomass in step b) may be performed by submersion of the fungi biomass in heated water or heated culture media or by using steam. In particular heating of the fungi biomass by steaming was found to provide benefits to the food product provided in step f) in terms of beneficial toughness values.

The at least one food additive may be selected from the group consisting of food fibers, starches, proteins, hydrocolloids and gelling agents.

The at least one food additive may be selected from the group consisting of rice starch, potato starch, corn starch as well as other modified starches, potato fibers, bamboo fibers, pea fibers, oat fibers, canola oil, pea protein, soy protein, and hydrocolloids such as methylcellulose, carrageenan, and alginate.

The food product described herein comprises at least one integrated food additive, but it may optionally also comprise more than one integrated food additive, such as two or more integrated food additives, such as three or more integrated food additives.

The fermentation in step a) may be performed at a temperature within the range of from 25 to 42 °C, optionally within the range of from 30 to 35 °C. The temperature may be constant within this range.

The fermentation in step a) may be performed at a pH within the range of from 3.0 to 8.0, optionally within the range of from 4.0 to 5.5.

The method may comprise a mechanical treatment step g) of mechanically treating the food product by breaking the biomass into smaller pieces, optionally wherein the smaller pieces are bits having a size within the range of from 1 mm to 16 mm, as measured at the largest crosssection of each bit. For an intermediate food product according to the present disclosure, which is intended to be used for forming for example balls, patties or a minced food product, it has been found that the provision of at least a part of the meat- or fish-replacement products in bits of fungi biomass having a size within the range of 1 mm to 16 mm enhances the texture and reduces the gumminess of the final product.

Step g) may comprise mechanically treating the food product by breaking the biomass into two groups of bits, a first group wherein the bits each has a size varying between 1 mm and 6 mm, and a second group wherein the bits each has a size varying between 6 mm and 16 mm. For an intermediate food product according to the present disclosure, which is intended to be used for forming for example balls and patties, it has been found that if the sizes of the bits vary in the intermediate food product, the texture of the final product has a relatively high resemblance with meat products.

The method may further comprise a storing step h) comprising storing and freezing the food product, wherein the food product is sealed in an atmosphere free from oxygen and thereafter refrigerated or frozen. It has been seen that benefits may be obtained for the food product according to the present disclosure in terms of Toughness and Firmness values for samples being sealed in an atmosphere free from oxygen and thereafter refrigerated or frozen.

The present disclosure further relates to a food product manufactured by a process wherein a) a fungi is cultivated under aerobic submerged fermentation conditions using a closed fermentation vessel with liquid substrate media while stirring to obtain a fungi biomass comprising a filamentous mycelium network; b) the fungi biomass obtained from step a) is processed by heating to a temperature within the range of from 50-85°C; c) the fungi biomass obtained from step b) is separated from the liquid cultivation media, such as by filtration or decanting, such that the biomass has a water content within the range of from 85% to 98%; d) at least one food additive is added to the fungi biomass obtained in step c) to provide a food product comprising food additive in an amount of from 0.05% by weight or more relative to the total weight of fungi biomass and food additive in step f); e) the fungi biomass and the at least one food additive are mixed, thereby integrating the at least one food additive into the filamentous mycelium network without disrupting the filamentous mycelium network; and f) the fungi biomass obtained from step e) is dewatered, such as by pressing or centrifuging to substantially orient the filamentous mycelium network in a single plane, such that a fungi biomass food product is obtained having a water content within the range of from 50 to 85 % by weight, as measured by weighing of the fungi biomass before and after an oven drying step, thus forming a lamellar structure and wherein the filamentous mycelium network comprising the at least one integrated food additive is substantially intact.

The food product obtained by the above process may be mechanically treated breaking the biomass into two groups of bits; a first group wherein the bits each has a size varying between 1 mm and 6 mm, and a second group wherein the bits each has a size varying between 6 mm and 16 mm. For an intermediate food product according to the present disclosure, which is intended to be used for forming for example balls and patties, it has been found that if the sizes of the bits vary in the intermediate food product, the texture of the final product has a relatively high resemblance with meat products.

The food product may be stored by freezing the food product, wherein the food product is sealed in an atmosphere free from oxygen and thereafter refrigerated or frozen. It has been seen that benefits may be obtained for the food product according to the present disclosure in terms of Toughness and Firmness values for samples being sealed in an atmosphere free from oxygen and thereafter refrigerated or frozen.

The present disclosure furthermore relates to a use of the product according to the present disclosure for preparing a meat-replacement consumer product selected from the list consisting of balls, patties, fillet, meat-like cuts, fish replacements, whole-cut meat replacements, sliceable products such as hams, bacon, or spreadable products such as pastes.

DESCRIPTION OF FIGURES

Figure 1 schematically illustrates a method for preparing a fungi biomass food product according to the present disclosure.

Figure 2 shows a graph relating to the growth of fungi spores into fungi filaments, the variation in pH and the observed morphology of the mycelium in suspension.

Figure 3 shows a graph relating to the growth of fungi spores into fungi filaments with varying concentrations of yeast extract in different media, the morphology of the mycelium in suspension was observed.

Figure 4 shows a graph illustrating the correlation between propeller stirring speed of a stirred tank bioreactor of 30L, and the Toughness of the biomass obtained, measured through a Knife Blade test on a texture analyzer.

Fig. 5 shows a graph comparing the values of toughness and firmness of a Knife Blade test performed on fungi biomass samples which were treated with different concentrations of starch of fibers.

Fig. 6 shows a graph illustrating the results of a sensory analysis of fungi biomass samples which were treated with different concentrations of fibers and analyzed either fresh or frozen and thawed before analysis. Fig. 7 shows images obtained through stereomicroscopy indicating alignment of fungal mycelium according to a lamellar structure and comparing samples with and without embedded food additives.

Fig. 8 shows a graph illustrating the values of Toughness and Firmness of a Knife Blade test performed on fungi biomass samples which were subjected to different pressures during the dewatering step.

Fig. 9 illustrates schematically products made with fungi biomass containing different bits sizes for achieving different textures.

Fig. 10 shows a graph with values of Hardness from a texture profile analysis (TPA) test of balls formed with different protein sources.

Fig. 11 shows a graph with values of Toughness and Firmness of a Knife Blade test performed on fungi biomass samples which were treated differently during downstream processing.

Fig. 12 shows a graph of values Toughness and Firmness of a Knife Blade test performed on fungi biomass samples which were frozen either vacuum-packed or packed in a bag with air.

DETAILED DESCRIPTION

Fig. 1 schematically illustrates a method for preparing a fungi biomass food product according to the present disclosure including a first step of pre-culture preparation of the fungi cultures and a second step of cultivating fungi under aerobic submerged fermentation conditions processing the fungi biomass. In steps not shown, the fungi biomass is heated to inactivate the fungi biomass after fermentation and separation from the liquid cultivation media, such as by filtration. The resulting biomass has a water content within the range of from 85% to 98%.

In a texture enhancement step, at least one food additive is added to the fungi biomass and mixed with the fungi biomass, thereby integrating the at least one food additive into the filamentous mycelium network. In a subsequent dewatering step/pressing step, the product obtained in the texture enhancement step is pressed to reduce the water content, such that a fungi biomass food product is obtained having a water content within the range of 50-85 % by weight. By pressing the fungi biomass, the filamentous mycelium network may furthermore be oriented substantially in a single plane forming a laminar structure in which the filamentous mycelium network comprising the integrated food additive is substantially intact. To prepare an intermediate or a final fungal biomass food product, the fungi biomass obtained after dewatering by pressing is cut and shaped to a desired size and shape. If the final desired product is a ball or patty, the fungi biomass may first be cut into the smaller pieces and bits, each having a size within the range of from 1 mm to 16 mm, as measured at the largest crosssection of each bit. Thereafter the bits may be formulated to balls or patties by mixing the bits with further ingredients and shaping the resulting mince into the desired shape.

Experiment 1 : Impact of growth media on pre-culture morphology

Fungal spore suspensions of filamentous fungi species able to sporulate in solid agar plates were prepared by flooding a Potato dextrose Agar (PDA) plate culture with 10-20 mL of sterile water and spores scraped off the PDA plate surface with a disposable, sterile spreader. Spores were counted in a hemocytometer under a light microscope and used directly as inoculum for liquid cultivations.

Fungi pre-culture preparation

Precultures were prepared with fungi of different species within Ascomycota and Zygomycota, such as Neurospora intermedia, Neurospora crassa, Aspergillus oryzae, Rhizopus oryzae, Rhizopus microsporus and Rhizopus oligosporus filamentous fungi species, by adding 1 mL of fungal spore suspension (10 ⁷ spores/mL) obtained as described above, per 100 mL of growth media in Erlenmeyer flasks (volumes 100-2000 mL) with or without baffles. A defined culture media was composed of (NH ₄ ) ₂ SO ₄ at 7.5 g/L, KH ₂ PO ₄ at 3.0 g/L, MgSO ₄ .7H ₂ O at 0.6 g/L, CaCI ₂ .2H ₂ O at 1 .0 g/L, Glucose at 20 g/L and Yeast extract at 5g/L.

All precultures of these organisms resulted in filamentous mycelium structures in suspension. Precultures were sieved and pressed manually in a benchtop press, revealing an aggregated filamentous press-cake with a meat-like fibrous structure. This indicated a similar suitability for working with filamentous mycelium to modify said mycelium structure through embedding of food additives prior to pressing. For the remainder of experiments, the selected Rhizopus microsporus, oligosporus and oryzae species were used.

The flasks were filled with liquid growth media (as listed in Fig. 2) to a maximum of 20% of the total flask volume and the fungi cultures of the three Rhizopus species were incubated at 30- 35°C for 18-24h and shaken at 100-150 rpm). Germination of Rhizopus fungal spores into fungal mycelium in liquid media was compared between defined growth media and complex media as listed in Fig. 2. Defined growth media contained (NH ₄ ) ₂ SO ₄ at 7.5 g/L, KH ₂ PO ₄ at 3.0 g/L, MgSO ₄ .7H ₂ O at 0.6 g/L, CaCI ₂ .2H ₂ O at 1.0 g/L, Sucrose at 20 g/L and 5 ml/L of a trace metal solution as described in (Hill, T. W., and E. Kafer (2001) "Improved protocols for Aspergillus minimal medium: trace element and minimal medium salt stock solutions,” Fungal Genetics Reports: Vol. 48, Article 8.). Complex media was produced by replacing sucrose and trace metal solution by 20 g/L of the tested ingredients: corn flour, wheat breadcrumbs, dry gluten free bread, potato starch, standard dry white bread, wheat flour, fine milled oat husks or wheat bran in separate flasks. pH of all cultures was adjusted to 5.5 as initial pH. The different growth media were thereafter evaluated for biomass growth and morphology changes as described below.

Evaluation of growth of pre-cultures in different media

Rhizopus fungal cultures were cultivated for 24h, during which initial and final pH values, as well as the dry solid content of the culture were recorded. Growth of fungi culture was measured by weight of the dry solid content, which includes fungi and insoluble solids, together with measurement of the decrease in pH of the culture.

Solid dry content in fungi samples was calculated by weighing three sample replicates before and after an oven drying step and comparing the values. The oven drying step was carried out in a convection oven where samples were dried at 105°C overnight (10-16h). The measurement is stopped when the weight change is less than 1 mg during a 90-second time frame.

The mycelia macro-level morphology of the fungi was observed at the end of 24 hours of cultivation. It turned out that growth did not differ significantly between the three different Rhizopus precultures when cultivated under the conditions as disclosed above. Therefore, only results obtained for Rhizopus oligosporus are represented in Figures 2 and 3 and Table 1 below. In Fig. 2 black bars show the relative decrease in pH units from initial pH (pH 5.5) to pH at 24h, while white bars show the solid concentration in g/L at 24h. The column on the right shows the observed morphology of the mycelium in suspension. For example, when Rhizopus is grown in a complex medium comprising wheat bran for 24 hours, pH decreases about 1 .3 pH units from 5.5 to about 4.2, a solid content of about 9.5g/L and filaments of medium size are produced.

Precultures with filamentous morphology resulted in a better product with a more defined lamellar alignment after pressing, less crumbly behaviour and improved mouthfeel. The medias that combined filamentous morphologies with good fungal growth (indicated by pH drop and solid content) were then regarded the best for the process preculture.

Addition of yeast extract to growth media

Semi-defined rich complex media was also tested using the above-mentioned potato starch, wheat breadcrumbs or corn flour media, but instead of the trace metal addition, 4-8 g/L of yeast extract was added. Macro-level morphology was analysed visually through an Erlenmeyer flask, while micro-level morphology was observed under a light microscope. Three biological replicates were compared for all observations. Table 1 shows different morphologies of Rhizopus fungi in suspension in liquid media containing yeast extract at different concentrations. The left column specifies the substrate used in the media, while the different columns towards the right show the different morphologies formed at different amounts of yeast extract added to such media.

Table 1

Interestingly, addition of yeast extract was shown to be beneficial for formation of soft filamentous structures on the fungal culture when more pure carbon sources such as potato starch were used. The addition of yeast extract promoted growth of the fungi in a filamentous form in all media (see Table 1) while simultaneously maintaining or increasing biomass yield (Fig 3).

Fungi from different Rhizopus precultures with either pellet or filamentous morphologies were then inoculated in a defined growth media containing (NH ₄ ) ₂ SO ₄ at 7.5 g/L, KH ₂ PO ₄ at 3.0 g/L, MgSO ₄ .7H ₂ O at 0.6 g/L, CaCI ₂ .2H ₂ O at 1 .0 g/L, Sucrose at 20 g/L and 5 ml/L of a trace metal solution as described above. Cultures were grown in a 30L STR bioreactor in aerated conditions at 0.5vvm for 24h. The resulting biomass obtained from the fermented cultures was analyzed visually, and it was observed that a filamentous morphology already in the preculture is a prerequisite for growth of fungi into a filamentous mycelia structure during the fermentation. Since it was concluded that all three Rhizopus species resulted in substantially the same morphology when cultivated under the same conditions, all subsequent experiments were performed using the species Rhizopus oligosporus.

Experiment 2: Modulation of texture using bioreactor stirring speed variations

The effect on texture such as firmness and toughness of pressed fungi biomass fermentation settings such as stirring speed variations was tested on the filamentous fungi species Rhizopus oligosporus.

Liquid bioreactor fermentation conditions

A volume of 2L of fungus preculture grown for 16-24h on corn flour media with 8g/L yeast extract, was used to inoculate 20L of media in a 30L stirred-tank bioreactor using the defined growth media described in Experiment 1. The initial pH of the medium was adjusted to 4.0-5.5 with 5M NaOH. Fermentation conditions were thereafter kept constant at pH 4.0 using NH ₃ as a base for pH titration, an air flow of 12 L/min (0.6 vvm) and a temperature of 30-35°C for 24 hours. Sterilisation of the liquids in the bioreactor was done by heating the liquid with steam (via the bioreactor’s double jacket) to 121 °C and 1 bar overpressure for 20min.

A factorial experimental design was conceived to explore the effect of different fermentation conditions on fungi biomass texture. The fermentation was carried out multiple times with varying rotation speeds (150 rpm, 200 rpm and 250 rpm) and different fermentation times (18h, 24h and 30h) in a 30L STR bioreactor with Rushton turbine-type of propellers (i.e., a vessel with a diameter of 26 cm and a Rushton turbine propeller with a diameter of 8).

The fermented samples were harvested and pressed in a hydropress at a pressure of 2.5 bar to remove water, promote fiber alignment and create a solid material with less than 80% water content.

Texture analysis using a Knife Blade method

Samples of pressed 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 Firmness 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 Knife Blade method described herein eguals the method of shear measurement using the 45-degree chiselled blade mentioned in the following two references: Lyon, B. G., and C. E. Lyon. "Assessment of three devices used in shear tests of cooked breast meat." Poultry science 77.10 (1998): 1585-1590.

(https://www.sciencedirect.com/science/article/pii/S00325 79119412054) and in Eastridge, Janet S., and Morse B. Solomon. "A METHOD TO EVALUATE SHEAR FORCE MEASUREMENTS FROM VARIOUS INSTRUMENTS." International Congress of Meat Science and Technology Proceedings. 2005 (http://icomst-proceedings.helsinki.fi/papers/2005 04 18. pd

As seen in table 2, the toughness and the firmness of the fermented product obtained was influenced by the stirrer speed during the fermentation.

Table 2.

The results from the testing’s are illustrated in Fig. 4 showing a graph illustrating the correlation between propeller stirring speed of a stirred tank bioreactor of 30L size and the Toughness of the biomass obtained, measured through a Knife Blade test on a texture analyzer.

A correlation between higher stirrer speeds and lower toughness values was found with an R ² value, i.e., the coefficient of determination, being 0.748.

Experiment 3: Texture enhancement during downstream processing

Texture enhancement Fungi biomass was harvested from a 300 L bioreactor grown in the same conditions as described in Exp. 2 (defined sucrose media) at a speed of 150 RPM and subjected to a heat treatment procedure by incubating in water or culture media for 10-20 min at 65-72°C. After fermentation, the fungi biomass was concentrated by flowing medium with biomass through large sieve-like filters. Water content of the fungi biomass was controlled to retain more than 80% water content and to the point in which its behaviour was one of a viscous liquid and not a solid. Batches of biomass was mixed at a constant stirring rate for 5 minutes in the presence of each one of the food additives rice starch, potato starch, corn starch as well as other modified starches, potato fibers, bamboo fibers, pea fibers, oat fibers, canola oil, pea protein, soy protein, and hydrocolloids such as methylcellulose, carrageenan, and alginate in an amount of 0.5%, 2%, 5% and 10% food additive relative to the final wet product weight.

The batches of biomass with embedded ingredients were pressed in a hydropress system using a water pressure of 2.0-2.5 bar, in which force is applied in a single plane to simultaneously dewater the samples and promote fiber alignment. Water content in the samples was decreased from 85-99.9% in which the biomass has a liquid behaviour from low to very-high viscosity, to 63-85% where the biomass is in a wet solid form. Fungi biomass containing either rice starch or potato fibers as embedded ingredients provided a mouthfeel that most resembled meat of the different combinations tested.

Texture analysis of enhanced biomass

Samples of fungi biomass embedded with rice starch or potato fibers at a 2% and 5% weight of the final wet product were cooked in a convection oven for 15 min at 120 °C and allowed to stabilize back to a room temperature of about 15°C. Samples were then 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 the Knife Blade method described in Experiment 2.

The results from the Texture analysis are presented in Fig. 5 and show Toughness (black) and Firmness (white) values obtained when performing a Knife Blade test on fungi biomass samples which were treated with different concentrations of the food additives rice starch or potato fibers to give additive embedded fungi biomass products.

Samples with embedded rice starch or potato fibers at both 2% and 5% concentrations had significantly softer textures shown by lower Toughness and Firmness values compared to a control subjected to the same procedure without any embedding of the food additive in the filamentous mycelium network of the fungi. Biomass embedded with added potato fiber provided better chewiness and mouthfeel.

Sensorial analysis of biomass comprising potato fibers integrated in the filamentous mycelium network

A sensory panel of eleven trained individuals was subjected to blind and randomized testing of samples of fungi biomass enhanced with 1 wt.-%, 2.5 wt.-%, 4 wt.-% or 6 wt.-% potato fibers (as calculated based on the total weight of the food product) according to the present disclosure. The fungi biomass samples comprising potato fibers integrated in the filamentous mycelium network as described above were either analysed immediately or frozen at -20°C for 3 days before testing. All samples were thawed and allowed to reach a temperature of 20°C before analysis. The individuals of the sensory panel were asked to answer the following questions on a scale of 1-10: “Rate the chewiness of the sample”, “Rate the firmness of the sample”, ”How pleasant is the texture of the sample?”, “How pleasant is the taste of the sample?”.

Fig. 6 shows the Sensory analysis results of the fungi biomass samples treated with different concentrations of potato fibers and which were either analysed fresh or frozen and thawed before analysis. As may be seen in Fig. 6, samples comprising potato fibers integrated in the filamentous mycelium network were rated with lower chewiness, lower firmness, with a more pleasant texture and more pleasant taste.

Stereomicroscopy analysis of pressed biomass comprising fibers integrated in the filamentous mycelium network

Fungal biomass was cultivated and pressed as described above with either no additives added during production, or by adding food additive such that the fungal biomass product contained 4wt.-% potato fibres and 2wt.-% canola oil integrated in the filamentous mycelium network as calculated based on the total weight of the fungal biomass product. Samples were generated by cross-cutting surfaces with a razor blade while frozen (“Cross-Cut”) or tearing by hand after being defrosted (Tearing). These samples were then examined with Zeiss SteREO Discovery. V8 stereomicroscope equipped with Achromat S 0.5x objective (Carl Zeiss MicroImaging GmbH, Gottingen, Germany) and imaged using an Olympus DP-25 single chip colour CCD camera (Olympus Life Science Europa GmbH, Hamburg, Germany) and the Cell ^A P imaging software (Olympus). The obtained images in Fig. 7 show that the fungal mycelial network is substantially aligned forming a lamellar structure. Where additives are present, these are seen embedded in the mycelial network. When tearing or simulating a bite, the sample with embedded fibre and oil additives was shown to separate according to its lamellar structure and reveal aligned mycelial fibres.

Experiment 4: Effect of applied pressure during dewatering for toughness

Texture analysis of biomass pressed at different pressures

Samples of biomass containing 4wt.-% potato fibres integrated in the filamentous mycelium network (as calculated based on the total weight of the fungal biomass product) were pressed in a hydropress system using a water pressure of either 1 bar, 1.5 bar, 2 bar or 2.5 bar. Water content in the samples varied between 85% and 64% after pressing. The samples were pressed in a single direction causing the fungi mycelium to align according to planes perpendicular to the pressure applied, as illustrated in Fig. 7.

Samples were analysed using the Knife Blade method described in Experiment 2.

Fig. 8 illustrates the results of the analysis of a Knife Blade test performed on the fungi biomass samples comprising 4wt.-% potato fibres which were subjected to different pressures during the dewatering step, with the values of toughness being shown in black and the values of firmness being shown in white.

As may be seen in Fig. 8, increase in pressing pressure resulted in samples with lower water content and higher toughness in a way that both Toughness and Firmness directly correlate to pressure applied during dewatering.

Experiment 5: Preparation of heterogenous matrixes for final product formulation

Preparation of mixes with different sized bits

Fungi biomass was fermented and dewatered as disclosed in Experiment 2 above. The obtained fungi biomass was further prepared by embedding 4% potato fiber, 2% rice starch and 5% canola oil, and pressed in a belt press system at 6 bar so that the mycelium aligned in laminar structures. The obtained food product was ground in an enterprise-system grinder using hole sizes of 5mm, 8mm and 12mm forming bits of 5mm, 8mm and 12mm as measured at the largest cross-section of each bit. For each 1 kg of fungi biomass bits, 200 g oat flour, 50 g potato starch and 300 g water were added, and everything was mixed in a planetary mixer using a beater tool head for 3 minutes. Different mixes were formed using only 5mm, 8mm and 12mm particles, or using 50/50 ratios of 5 mm + 12 mm or 8 mm + 12 mm. Balls of 15g were formed and baked at 125°C for 20 min.

Fig. 9 illustrates a representation of products made with fungi biomass containing different bits sizes for achieving different textures. The samples which had larger bits sizes or a higher ratio of these showed coarser textures on low mixing rates, allowing the distinction of intact biomass bits. High mixing rates promoted the formation of a homogenous mass with a highly fibrous visual appearance and high gumminess mouthfeel. Coarser, heterogenous mixes obtained though low mixing rates are more favourable in the composition of formed meat-like products.

Preparation of hybrid products with textured plant proteins

Hybrid samples were prepared by mixing fungi biomass bit sizes of 5 mm as described above in this same experiment and bits of either hydrated textured soy protein or hydrated textured pea protein up to 8 mm size in an amount of 5 wt.-% as calculated based on the total weight of the hybrid product and shaping it in the form of balls with a diameter of 25mm. Texture properties were analyzed using the TPA method described below.

Texture Profile Analysis (TPA)

Binding properties of ingredients in the hybrid samples 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)

Cohesiveness = AreaFT4:61 AreaFT 1 :3 (area of second deformation relative to area of the first deformation)

Gumminess = Hardness x Cohesiveness

Springiness = Time-diff4:51 Time-diff41 :2 (percentage of product height that is regained after first deformation) Chewiness = Gumminess x Springiness

Resilience = AreaFT2:31 AreaFT 1 :2 (area of the second half of the first deformation relative to area of the first half)

Adhesion = AreaFT3:4 (pulling strength during retraction from sample) Fig. 10 illustrates the values of Hardness obtained from a texture profile analysis (TPA) test of balls formed with different protein sources. Either textured pea protein, textured soy protein, fungi biomass or fungi biomass mixed with textured pea or soy protein, or pea protein powder were analyzed.

Fungi biomass was shown to have a hardness between textured soy and pea protein in this application, and mixes of these sources shown to have intermediate values as shown in Fig. 10. The hybrid mixes were able to create a mixed structure of fibers and crumbles.

Table 3 shows values from the TPA test of these hybrid balls, as the average of 5 replicate samples. Balls which include fungi biomass show improved values of springiness, cohesiveness, gumminess and chewiness compared to using textured soy protein, and less than textured pea protein. Such results demonstrate applicability of this protein source to food product formulations that currently contain soy and pea protein.

Table 3.

Experiment s: Product storage preparation methods

Heat treatment methods

Fungi biomass was fermented as in Experiment 2 above in a 300 L stirred tank bioreactor. The fungi biomass was harvested and concentrated to a viscous liquid mass with a water content above 80%. A first set of samples was submerged in water at the defined temperatures 72°C and 85°C, and incubated for 1 .5 min or 10 min. After the heat treatment with hot water, samples were washed in cold water and pressed at 2.5 bar in a hydropress device down to a solid state with a water content of 75%.

A second set of samples was washed, pressed at 2.5 bar in a hydropress device, and thereafter subjected either to a steaming process using a steam-saturated vessel (high steam) or a container with steam mixed with hot air to about 50% steam saturation (low steam), both for 10 min.

One portion of each sample was stored under vacuum in a sealed bag, while another portion was sealed with air in a plastic bag. All samples were frozen at -20°C and tested after 4 days for texture and sensorial analysis.

The fungi biomass samples were thus treated differently during downstream processing. Samples were either heat treated at 72°C (HT-72), heat treated at 85°C (HT-85), treated with saturated steam atmosphere (high steam) or 50% steam-saturated atmosphere (low steam), and thereafter frozen either vacuum-packed or packed in a bag with air.

Samples were cut into a cuboidal shape of the size 12 mm x 10 mm x 2.5 mm (length x width x height) and analyzed using the texture analysis Knife Blade method defined in Experiment 2. Toughness and Firmness vales were obtained, and the results are shown in Fig. 11 , wherein values of toughness are illustrated in white and values of firmness are illustrated in black.

Clear effects were observed such as steam treatments providing lower toughness values than hot water treatments, and in some cases storage in air showed a decrease in toughness.

Freezing and vacuum storage Fungi biomass fermented as disclosed in Experiment 2 above was harvested from a 300 L stirred tank bioreactor and heat treated in water at a temperature between 65°C and 72°C. The biomass was pressed at 2.5 bar in a hydropress device down to a solid state with a water content of 75%. Samples were cut into cubes of 15 mm size and frozen at -20°C. One portion of the cubes was stored under vacuum in a sealed bag, while another portion of the cubes was sealed with air in a plastic bag. The cubes were defrosted after 1 day and 10 days for texture analysis. Samples with a cuboidal shape of the size 12 mm x 10 mm x 2.5 mm (length x width x height) were analyzed using the texture analysis Knife Blade method defined in Experiment 2 and Toughness and Firmness were obtained for these samples. The Toughness and Firmness values for the vacuum sample defrosted after 1 day was taken as 100% to enable comparative studies of all the samples and analyse % decrease in texture over time.

The results of the texture analyses are shown in Fig. 12 with the Toughness values shown in white and the Firmness values obtained shown in black.

The results show a decrease in Toughness and Firmness for the samples packed in contact with air after 10 days which is not observed after 1 day. Vacuum packing for 10 does not seem to affect the texture of the samples.

Experiment 7: Industrial patty forming using fungi biomass with enhanced texture

Fungi biomass was produced in a 600 L bioreactor using stirring speeds between 200-250 rpm. The biomass was harvested, heat treated at a temperature between 65°C and 75°C and its texture enhanced according to the invention by mixing at medium speed in a mixing tank with the addition of a final volume of 3% potato fibers, 2% rice starch and 5% canola oil. Biomass was pressed at 2 bar, cut into 10 cm x 5 cm pieces, blast frozen to -20°C and packaged in vacuum. Samples were then defrosted to 10°C and ground in an industrial meat grinder using 8 mm and 12 mm diameter holes. Ground fungi biomass was mixed with 15% to 40% of a mix containing potato starch, rice starch, oat flour, spices and flavours. Water and canola oil were added to the mix in a planetary mixer. The mix was processed and formed using an industrial patty former at a rate of up to 12 000 balls/hour, frying in canola oil at 180°C for 30 sec, baking at 130°C for 20 min, blast freezing and vacuum packaging.

The same process was repeated using fungi biomass obtained from a 300 L bioreactor fermentation using the following differences in the process: 150 rpm fermentation, no texture enhancing step, pressing at 2.5 bar. The balls resulting from this process were regarded in terms of taste, texture, form and processibility as suitable for commercial production of non-animal meatball-like products.