A food composition product and manufacturing method

Abstract

A food composition comprising protein, dietary fiber, starch, and filamentous fungi, as well as a method for manufacturing the food composition by dry extrusion of protein, dietary fiber, and starch, and followed by solid state fermentation by filamentous fungi, are disclosed

Description

[0001] A FOOD COMPOSITION PRODUCT AND MANUFACTURING METHOD

[0002] TECHNICAL FIELD

[0003] The present disclosure generally relates to food compositions and products comprising them. The disclosure relates particularly, though not exclusively, to a food composition which is manufactured by fermenting in solid state fermentation a dry extrudate obtained from a mixture comprising protein, dietary fiber, and starch.

[0004] BACKGROUND

[0005] This section illustrates useful background information without admission of any technique described herein representative of the state of the art.

[0006] Currently, there is a need to expand sustainable food production. Alternative protein products, particularly plant-based meats, have seen rapid growth in recent years. Plantbased foods are often designed to mimic ground meats. There remains a need to develop new methods for producing plant-based biomass with textures suitable for meat substitutes, enabling efficient and sustainable production of foods that meet sensory, nutritional, and environmental objectives.

[0007] SUMMARY

[0008] It is an aim to solve or alleviate at least some of the problems related to prior art. An aim is to provide an ingredient suitable for use as a meat substitute. Furthermore, an aim is to provide a more climate friendly food product. Further, an aim is to provide a mycelium-based plant-based food composition which has good nutritional characteristics and appealing texture profile.

[0009] The appended claims define the scope of protection. Any example and technical description of an apparatus, system, product and / or process not covered by a claim is presented as an example useful for understanding the invention.

[0010] According to a first aspect is provided a method for manufacturing a food composition comprising: preparing a mixture comprising a protein, dietary fibre, and starch; carrying out dry extrusion on the mixture to provide a porous extrudate comprising fibrillated protein; and fermenting the extrudate by solid state fermentation with filamentous fungi. In an embodiment, the present mixture contains 35-80 wt-%, preferably 40-75 wt-%, more preferably 40-70 wt-%, most preferably 40-60 wt-% protein, or about 45wt-%, based on dry weight.

[0011] In an embodiment, in the present method the mixture contains dietary fiber 5-30wt-%, preferably 10-30wt-%, more preferably 20-30wt-%, most preferably about 25wt-%, based on dry weight.

[0012] In an embodiment the present mixture comprises protein, 5-30wt-% dietary fibre, and starch.

[0013] In an embodiment, in the present method the mixture contains starch 10-35wt-%, preferably 15-30wt-%, more preferably 20-25wt-%, most preferably about 20wt-%, based on dry weight.

[0014] In an embodiment, in the present method the mixture is prepared by mixing at least one protein concentrate with at least one separate source of dietary fiber and starch; or mixing at least one protein concentrate, at least one separate source of dietary fiber, and at least one separate source of starch.

[0015] In an embodiment the mixture is prepared by mixing to achieve 35-80wt-% protein, 10-35wt- % dietary fiber, and 5-30wt-% of starch.

[0016] In an embodiment, in the present method: the protein is at least one of plant protein, legume protein, faba protein, pea protein, soy protein, cereal protein, oat protein, wheat protein, pseudocereal protein, oil seed protein, oil seed press cake, rapeseed protein, sunflower protein, cellular agriculture protein (e.g. single cell protein, microalgae protein, whole biomass protein, precision fermentation made structural proteins, cultured animal cell protein); and / or the dietary fiber is at least one of plant fiber, cereal bran, pseudocereal bran, wheat bran, dietary fiber sourced from legume, or dietary fiber from cellular agriculture (e.g. whole biomass of cellular agriculture); and / or the starch is at least one of plant starch, cereal flour, legume flour, and pseudocereal flour.

[0017] In an embodiment the plant protein is an isolated plant protein.

[0018] In an embodiment the plant fiber comprises fruit peel, citrus fruit peel, and / or apple peel. Optionally, the plant fiber is isolated dietary fiber from any or each of these sources.

[0019] In an embodiment the plant starch comprises at least one of potato starch, maize starch, wheat starch, rice starch, tapioca starch, and cassava starch. Optionally, the starch is isolated starch from any or each of these sources. In an embodiment, in the present method the extrudate is hydrated before fermenting to a moisture content of 40-75wt-%, preferably 55-70wt-%, more preferably 60-65wt-%.

[0020] In an embodiment, in the present method the extrudate is hydrated before fermenting to a moisture content of 50-75wt-%.

[0021] In an embodiment, in the present method the dry extrusion is carried out in operating conditions to obtain an extrudate having a density in the range 100-1000 g / l, preferably 200- 900 g / l, more preferably 300-800 g / l, more preferably 300-700 g / l, more preferably 300-600 g / l, most preferably 300-500 g / l, or about 400g / l. An advantage of carrying out dry extrusion to achieve a porous extrudate with these densities is that solid state fermentation provides a food structure and composition with advantageous properties.

[0022] The density can be measured according to Example 10.

[0023] In an embodiment dry extrusion is carried out using a twin-screw extruder.

[0024] An exemplary embodiment of suitable dry extrusion parameters for a twin-screw extruder is specified in Table 1.

[0025] In an embodiment, the extrudate has a moisture content of 5-20wt-%, preferably 5-15wt-%, more preferably 5-10wt-%, more preferably 6-9wt-%, more preferably 6-8wt-%.

[0026] In an embodiment, in the method the protein content of the fermented food composition obtained by the method is not significantly decreased during fermenting; and / or the vitamin content (e.g. niacin content) of a fermented food composition obtained by the method is increased during fermenting; and / or essential amino acid profile is preserved (e.g. the tryptophan content of the fermented food composition obtained by the method is not significantly changed during fermenting).

[0027] In an embodiment, in the present method the solid state fermentation is carried out at 20- 40°C for 12-168h, preferably 20-40°C for 12-72h, preferably 20-37°C for 12-48h, more preferably at about 30°C for about 20-40h.

[0028] In an embodiment, the present method further comprises at least one step of drying, sterilization, mechanical homogenization.

[0029] The present inventors have surprisingly found that with the present method it is possible to obtain a product which can be used as a meat alternative, or as an ingredient in further food products. The obtained product has good nutritional characteristics. Additionally, the obtained product has good texture properties. In an embodiment the mixture, and / or the extrudate, contains protein in the range of 35-80 wt-% based on dry weight preferably, preferably 35-75 wt-%, 35-70 wt-%, 35-65 wt-%, 35- 60 wt-%, 35-55 wt-%, 35-50 wt-%, 35-45 wt-%, or 35-40 wt-%, based on dry weight.

[0030] In an embodiment, the mixture and / or the extrudate contains protein in the range of 40-80 wt-% based on dry weight, preferably 40-75 wt-%, 40-70 wt-%, 40-65 wt-%, 40-60 wt-%, 40-55 wt-%, 40-50 wt-%, or 40-45 wt-%, based on dry weight.

[0031] In an embodiment, the mixture and / or the extrudate contains protein in the range of 45-80 wt-% based on dry weight, preferably 45-75 wt-%, 45-70 wt-%, 45-65 wt-%, 45-60 wt-%, 45-55 wt-%, or 45-50 wt-%, based on dry weight.

[0032] In an embodiment, the mixture contains dietary fiber in the range of 5-30 wt-% based on dry weight, preferably 10-30 wt-%, 15-30 wt-%, 20-30 wt-%, or 25-30 wt-% based on dry weight.

[0033] In an embodiment, the mixture contains dietary fiber in the range of 5-25 wt-% based on dry weight, preferably 10-25 wt-%, 15-25 wt-%, 20-25 wt-%, or 25-25 wt-%, based on dry weight.

[0034] In an embodiment the mixture contains about 25wt-% dietary fiber based on dry weight.

[0035] According to a second aspect is provided a food composition manufactured by the present method and comprising 30-60 wt-% of protein, 20-30 wt-% of dietary fibre, 10-50 wt-% starch, and filamentous fungi.

[0036] In an embodiment the present food composition has at least one of: an average hardness of 60-90N; and / or an average springiness of 0.7-0.8; and / or an average cohesiveness of 0.6-0.7; and / or an average chewiness of about 25-45; and / or an average resilience of 0.2- 0.4.

[0037] According to a third aspect is provided a meat alternative comprising the present food composition.

[0038] In an embodiment, the present food composition has at least one of the following characteristics high in protein, high in fiber, low in fat, low in sugars, a source of iron / contains iron, as specified in Annex of Regulation (EC) No 1924 / 2006, lastly amended by Regulation (EU) No 1047 / 2012.

[0039] According to a fourth aspect is provide a method for manufacturing a food product, comprising providing the present food composition, optionally mixing with other food product ingredients, and carrying out extrusion to obtain the food product. In an embodiment, in the present method for manufacturing a food product the food product is at least one of minced meat, meat ball, sausage, steak, minced-meat patties, such as hamburger patties, nuggets, or hot dogs.

[0040] According to a seventh aspect is provided a food product manufactured according to present method.

[0041] The present method may provide a more environmentally sustainable and / or climate friendly food composition and / or food product.

[0042] According to a further aspect, there is provided a use of the present food composition as a meat substitute, preferably a substitute for minced meat, meat balls, sausages, and / or minced-meat patties, such as hamburger patties.

[0043] BRIEF DESCRIPTION OF THE FIGURES

[0044] Some example embodiments will be described with reference to the accompanying figures, in which:

[0045] Fig. 1 shows a flow chart of the present method.

[0046] Fig. 2 shows development of solid state fermentation.

[0047] Fig. 3 shows water binding capacity results of the obtained product.

[0048] Fig. 4 shows oil binding capacity results of the obtained product.

[0049] Fig. 5 shows hardness and springiness results of the obtained product.

[0050] Fig. 6 shows cohesiveness, chewiness, and resilience results of the obtained product.

[0051] Fig. 7 shows microscopy (reflection images, 5x objective) sample structure without staining after fermenting for 18h, 24h, and 30h.

[0052] DETAILED DESCRIPTION

[0053] Food-grade or food refers herein to compositions, mixtures, products, ingredients and / or compounds that are edible and suitable for human consumption. As used herein, suitable for human consumption does not (necessarily) imply regulatory approval.

[0054] The fungal mycelia or mycelium has typically a network structure which contributes to the texture of the present mixture when fermented in the present porous extrudate in solid state fermentation. Further, fungal mycelium is rich in protein and dietary fiber and contributes to the nutritional value of the mixture. In an embodiment the dietary fiber is at least one of cereal bran, pseudocereal bran, wheat bran, dietary fiber sourced from legume, or dietary fiber from cellular agriculture (e.g. whole biomass of cellular agriculture).

[0055] The present method may comprise including further components or ingredients and combining or mixing the further components or ingredients with the mixture. For example, the present method may comprise adding water to obtain a desired water content.

[0056] The present method may comprise further treatment or processing of the mixture. For example, the method may comprise freezing, heating, drying, and / or shaping or forming the mixture. The shaping may for example be or comprise moulding and / or extruding.

[0057] The present mixture may be used as such. Optionally, the present mixture is seasoned. The spices in the optional seasoning are not particularly limited, and may for example be spices conventionally used for seasoning food, for example minced meat products.

[0058] In an embodiment the protein is at least one of legume protein, faba protein, pea protein, soy protein, cereal protein, oat protein, wheat protein, pseudocereal protein, oil seed protein, oil seed press cake, rapeseed protein, sunflower protein, cellular agriculture protein (e.g. single cell protein, microalgae protein, whole biomass protein, precision fermentation made structural proteins, cultured animal cell protein).

[0059] In the present application, the term fibrillated protein means material produced by dry extrusion. During dry extrusion proteinaceous material is fed into an extruder and subjected to controlled thermal, shear, and pressure conditions, causing denaturation and partial unfolding of the protein molecules. As the material flows through the extruder, the proteins reorganize and form fibrils. Parameters including temperature, moisture, shear rate, and extrusion speed are adjusted to achieve fibrillation and specific extrudate characteristics such as porosity and density. The resulting extrudate comprising fibrillated protein is suitable for use as a medium for the subsequent solid state fermentation.

[0060] The present food product may optionally comprise other components in addition to the present mixture. For example, the food product may comprise further plant protein or plant protein component, ingredient, or product. In such embodiments, the present mixture may complement said plant protein component(s) or ingredient(s) for example in a meat substitute. The present mixture may for example be used with (other) plant proteins or plant protein components to improve the colour and / or texture of the food product.

[0061] The use of the present food composition is not limited to vegetarian products. For example, the present food composition may replace a portion of the meat in a meat-containing food product. For example, the present food composition may be a hamburger patty comprising the present food composition and minced meat, such as beef. Such hybrid products may be considered more climate or environmentally friendly and / or provide health benefits compared to their conventional counterparts. Such hybrid products may comprise components or ingredients used in their conventional counterparts, such as breadcrumbs and eggs in meatballs or the like.

[0062] In an embodiment fermenting the extrudate by solid state fermentation with filamentous fungi comprises fermenting with at least one strain of filamentous fungi.

[0063] In an embodiment solid state fermentation is carried out at least one strain of filamentous fungi selected from Ascomycota or Basidiomycota. In an embodiment the strain of filamentous fungi is selected from at least one of Rhizopus microsporus var. oligosporus, Neurospora crassa, Neurospora intermedia, Aspergillus oryzae, Fusarium venenatum, Monascus purpureus, Agaricus bisporus, Lentinus edodes, and Pleurotus ostreatus.

[0064] In an embodiment, solid state fermentation is carried out at a temperature in a range from 20°C to 40°C, preferably from 25°C to 37°C, more preferably from 28°C to 32°C.

[0065] In an embodiment, solid state fermentation is carried out for about 12-168h, preferably for about 16-40h, more preferably for about 20-35h.

[0066] In an embodiment, solid state fermentation is carried out at a temperature in a range from 20°C to 40°C and for about 12-168h, preferably for about 16-40h, more preferably for about 20-35h.

[0067] In an embodiment, solid state fermentation is carried out at a temperature in a range from 25°C to 35°C and for about 12-168h, preferably for about 16-40h, more preferably for about 20-35h, more preferably for about 17-31h, or about 18h, about 24h, or about 30h.

[0068] In an embodiment, solid state fermentation is carried out at a temperature in a range from 28°C to 32°C and for about 12-168h, preferably for about 16-40h, more preferably for about 20-35h, more preferably for about 17-31h, or about 18h, about 24h, or about 30h.

[0069] In an embodiment the present food composition has an average hardness of 60-90N, preferably 60-70N, or about 65N. In another embodiment the food composition has a hardness of 80-90N, preferably about 85N or about 90N. A higher hardness is typically obtained with a longer fermentation time, such as with a fermentation time of about 30h. Hardness can be analysed as described in Example 7. In an embodiment the present food composition has an average springiness of 0.6-0.8, preferably 0.65-0.8, more preferably 0.7-0.8. Springiness can be analysed as described in Example 7.

[0070] In an embodiment the present food composition has an average cohesiveness of about 0.5- 0.8, preferably 0.55-0.75, more preferably 0.6-0.7.

[0071] Cohesiveness can be analysed as described in Example 7.

[0072] In an embodiment the present food composition has an average chewiness of about 25-45, preferably 25-40, more preferably 30-40.

[0073] Chewiness can be analysed as described in Example 7.

[0074] In an embodiment the present food composition has an average resilience of about 0.2-0.4, preferably 0.25-0.35, more preferably 0.25-30.

[0075] Resilience can be analysed as described in Example 7.

[0076] Density can be analysed as described in Example 10. The skilled person is able to set the operating conditions of the extruder to achieve desired characteristics, such as a desired density, of the extrudate. As is seen in the micrographs in Fig. 7, typically in the present method, during fermentation the amount of mycelia is increased while no changes are observed in the extrudate.

[0077] In an embodiment, the present food composition has an average density of about 100-1000 g / l, 200-800 g / l, preferably 300-600 g / l, more preferably 350-450 g / l, most preferably about 400 g / l.

[0078] In an embodiment, the present porous extrudate, or the present food composition, has an average density of about 100-1000 g / l, preferably 150-1000 g / l, more preferably 200-1000 g / l, more preferably 250-1000 g / l, more preferably 300-1000 g / l, more preferably 350-1000 g / l, more preferably 400-1000 g / l.

[0079] In an embodiment, the present porous extrudate, or the present food composition, has an average density of about 100-900 g / l, preferably 150-900 g / l, more preferably 200-900 g / l, more preferably 250-900 g / l, more preferably 300-900 g / l, more preferably 350-900 g / l, more preferably 400-900 g / l.

[0080] In an embodiment, the present porous extrudate, or the present food composition, has an average density of about 100-800 g / l, preferably 150-800 g / l, more preferably 200-800 g / l, more preferably 250-800 g / l, more preferably 300-800 g / l, more preferably 350-800 g / l, more preferably 400-800 g / l.

[0081] In an embodiment, the present porous extrudate, or the present food composition, has an average density of about 100-700 g / l, preferably 150-700 g / l, more preferably 200-700 g / l, more preferably 250-700 g / l, more preferably 300-700 g / l, more preferably 350-700 g / l, more preferably 400-700 g / l.

[0082] In an embodiment, the present porous extrudate, or the present food composition, has an average density of about 100-600 g / l, preferably 150-600 g / l, more preferably 200-600 g / l, more preferably 250-600 g / l, more preferably 300-600 g / l, more preferably 350-600 g / l, more preferably 400-600 g / l.

[0083] In an embodiment, the present porous extrudate, or the present food composition, has an average density of about 100-500 g / l, preferably 150-500 g / l, more preferably 200-500 g / l, more preferably 250-500 g / l, more preferably 300-500 g / l, more preferably 350-500 g / l, more preferably 400-500 g / l.

[0084] In an embodiment, the present porous extrudate, or the present food composition, has an average density of about 300-500 g / l, preferably 310-490 g / l, more preferably 320-480 g / l, more preferably 330-470 g / l, more preferably 340-460 g / l, more preferably 350-450 g / l, more preferably 360-440 g / l, more preferably 370-430 g / l, more preferably 380-420 g / l, more preferably 390-410 g / l, most preferably about 400 g / l.

[0085] In an embodiment dry extrusion is carried out at a pressure selected from the range 10-100 bar, preferably 15-50 bar, preferably 20-40 bar, preferably 20-30 bar, more preferably 23- 26 bar.

[0086] A density below 10OOg / l is advantageous, because it provides a good texture of the food product after fermentation. Additionally, it improves water absorption during fermentation, thereby enhancing fermentation.

[0087] In an embodiment any or all chemical analyses and amounts of analyzed entities are measured as described in the Examples below, in particular as described in Example 8.

[0088] Preferably, in the present method the insoluble dietary fibre present in the mixture may reinforce the porous structure of the extrudate comprising fibrous protein and starch, and surprisingly maintains the porous structure throughout the solid state fermentation, thereby enabling production of a meat-like structure by a simple two-step process.

[0089] The present invention if further illustrated by the following numbered embodiments: Embodiment 1 : A method for manufacturing a food composition comprising: preparing a mixture comprising a protein, dietary fibre, and starch; carrying out dry extrusion on the mixture to provide a porous extrudate comprising fibrillated protein; and fermenting the extrudate by solid state fermentation with filamentous fungi.

[0090] Embodiment 2: The method according to Embodiment 1 , wherein the mixture contains 35- 80 wt-%, preferably 40-75 wt-%, more preferably 40-70 wt-%, most preferably 40-60 wt-% protein, based on dry weight.

[0091] Embodiment 3: The method according to Embodiment 1 or 2, wherein the mixture contains dietary fiber 5-30tw-%, preferably 10-30wt-%, more preferably 20-30wt-%, most preferably about 25wt-%, based on dry weight.

[0092] Embodiment 4: The method according to any one of Embodiments 1-3, wherein the mixture contains starch 10-35wt-%, preferably 15-30wt-%, more preferably 20-25wt-%, most preferably about 20wt-%, based on dry weight.

[0093] Embodiment 5: The method according to any one of Embodiments 1-4, wherein the mixture is prepared by: mixing at least one protein concentrate with at least one separate source of dietary fiber and starch; or mixing at least one protein concentrate, at least one separate source of dietary fiber, and at least one separate source of starch.

[0094] Embodiment 6: The method according to any one of Embodiments 1-5, wherein: the protein is at least one of legume protein, faba protein, pea protein, soy protein, cereal protein, oat protein, wheat protein, pseudocereal protein, oil seed protein, oil seed press cake, rapeseed protein, sunflower protein, and cellular agriculture protein such as single cell protein, microalgae protein, whole biomass protein, precision fermentation made structural proteins, or cultured animal cell protein; and / or the dietary fiber is at least one of cereal bran, pseudocereal bran, wheat bran, dietary fiber sourced from legume, or dietary fiber from cellular agriculture such as whole biomass of cellular agriculture; and / or the starch is at least one of cereal flour, legume flour, and pseudocereal flour. Embodiment 7: The method according to any one of Embodiments 1-6, wherein the extrudate is hydrated before fermenting to a moisture content of 50-75wt-%, preferably 55- 70wt-%, more preferably 60-65wt-%.

[0095] Embodiment 8: The method according to any one of Embodiments 1-7, wherein the dry extrusion is carried out in operating conditions to obtain an extrudate having a density in the range 100-1000 g / l, preferably 200-900 g / l, more preferably 300-800 g / l, more preferably 300-700 g / l, more preferably 300-600 g / l, most preferably 300-500 g / l, or about 400g / l.

[0096] Embodiment 9: The method according to any one of embodiments 1-8, wherein the protein content of the fermented food composition obtained by the method is not significantly decreased during fermenting; and / or the vitamin content, such as niacin content, of a fermented food composition obtained by the method is increased during fermenting; and / or essential amino acid profile is preserved.

[0097] Embodiment 10: The method according to any one of Embodiments 1-9, wherein solid state fermentation is carried out at 20-40°C for 12-168h, preferably 20-40°C for 12-48h, more preferably at about 30°C for about 20-40h.

[0098] Embodiment 11 : A food composition manufactured by the method according to any one of Embodiments 1-10, and comprising 30-60 wt-% of protein, 20-30 wt-% of dietary fibre, IQ- 50 wt-% starch, and filamentous fungi.

[0099] Embodiment 12: The food composition of Embodiment 11 having at least one of: an average hardness of 60-90N; and / or an average springiness of 0.7-0.8; and / or an average cohesiveness of 0.6-0.7; and / or an average chewiness of about 25-45; and / or an average resilience of 0.2-0.4.

[0100] Embodiment 13: A meat alternative comprising the food composition of Embodiment 11 or 12.

[0101] Embodiment 14: A method for manufacturing a food product, comprising providing a food composition according to any one of Embodiments 11 or 12, optionally mixing with other food product ingredients, and carrying out extrusion to obtain the food product.

[0102] Embodiment 15: A food product manufactured according to Embodiment 14. EXAMPLES

[0103] The following examples are provided to better illustrate the claimed invention and they are not to be interpreted as limiting the scope of the invention, which is defined by the claims. To the extent that specific materials or apparatus are mentioned, it is merely for purposes of illustration and is not intended to limit the invention, either.

[0104] Materials

[0105] The following materials were used to manufacture food compositions according to the present invention.

[0106] Protein concentrate: Faba protein concentrate (AGT Foods, 60% protein)

[0107] Source of starch: Whole grain oat flake flour (O 2000 FF, Fazer)

[0108] Source of dietary fiber: Coarse wheat bran (V 4000, Fazer)

[0109] Strains

[0110] Filamentous fungi (Ascomycota, Basidiomycota) such as Rhizopus microsporus var. oligosporus, Neurospora crassa, Neurospora intermedia, Aspergillus oryzae, Fusarium venenatum, Monascus purpureus, Agaricus bisporus, Lentinus edodes, and Pleurotus ostreatus.

[0111] Example 1 - dry extrusion

[0112] Dry extrusion of the mixture was carried out in the following operating conditions:

[0113] Twin-screw extruder APV MPF 19 / 25, Baker Perkins Group Ltd.

[0114] 4 mm die

[0115] 100-220°C, water between 5-30wt-%.

[0116] Conditions were selected to provide porous extrudate structure which does not turn into mush or collapse when rehydrated before fermentation

[0117] Table 1. Dry extrusion parameters. Example 2 - fermentation

[0118] The following fermentation conditions were used:

[0119] 187.5 g of extruded raw material I SacO2 box with HEPA filter

[0120] Moisture content: 62-64 %

[0121] Inoculation: 105spores / g

[0122] Incubation at 30°C, in dark

[0123] Preferably 25% air space of the container volume is used.

[0124] In an additional example, the following fermentation conditions were used. These conditions enabled growth of fungi and mycelium development:

[0125] Inoculation: 102-107, preferably 105spores / g

[0126] Porous structure and filling degree of raw material to facilitate adequate air flow,

[0127] Raw material moisture content: 30-95%, preferably 50-65%

[0128] Water activity: 0.6-0.99, preferably 0.9-0.99

[0129] Temperature: 0-55°C, preferably 20-37°C

[0130] Preferably in dark to avoid sporulation

[0131] Fermentation time: at least 10h, preferably 24-30h pH (2-10, preferably 5.5-7.5)

[0132] The experimental setup and development of fermentation is shown in Fig. 2.

[0133] For analysis of the fermented material in blocks, sides were cut off, and from the middle 8- 10x1 cm thick pieces were cut for analysis regarding thickness, colour, cooking, TPA, moisture, microscopy, microbial analysis, protein digestibility, freeze drying, and chemical analysis, functional properties.

[0134] Example 3 - freeze drying

[0135] For the freeze drying experiments, 8 blocks from each time point were freeze dried (Alpha 1-4, Martin Christ) and afterwards milled with Fritsch mill, 0.5mm sieve.

[0136] Example 4 - cooking

[0137] For the cooking experiments, pieces were cut, weighed, and placed on a tray. Samples were baked in an oven for 6min (200°C). The cooled samples were weighed again after oven to calculate the moisture loss Example 5 - water binding capacity (WOB) and oil binding capacity (OBC)

[0138] Protocol WBC / OBC: 1g of raw material or freeze-dried and milled SSF sample was mixed to 10 mL of distilled water / rapeseed oil and shaken in 10 min intervals for 30 min. Suspension was centrifuged 2000 x g / 10 min, supernatant poured to a new tube and sediment, weighed (+ for WBC: sediment dried at 105 C oven o / n and weighed). The method is adapted from Silvennoinen et al. 2021 ,

[0139] Figures 3 and 4 show the results of WBC and OBC, respectively. I MB SPSS version 28 was used for the statistical analyses. One-way ANOVA with Tukey’s post hoc test.

[0140] Example 6 - protein solubility

[0141] Protocol: 5% suspension was made to milliQ and stirred for 15 min. Suspension was centrifuged at 10000 x g / 15 min in room temperature, as adapted from Silvennoinen et al 2019, with modifications to stirring time, doi: 10.1007 / s11947-019-02307-w, supernatant was collected for dumas protein analysis. Dumas combustion method AACC 46-30.01.

[0142] Protein solubility was the highest for the raw material mix. Dry extrusion decreased protein solubility. Protein solubility increased as a function of fermentation time.

[0143] Example 7 - textural properties

[0144] Shrinkage

[0145] Protocol: Digital caliper was used to measure the thickness from the middle of the sample. Results are average of 20 measurements. High deviation was due to the cutting of the sample from the block was uneven (thickness between 10-13 mm).

[0146] Average change after cooking

[0147] 24h: 1.3 mm shrinkage

[0148] 30h: 1.6 mm shrinkage

[0149] 30 h samples were slightly thicker compared to 24 h samples, which might affect the shrinkage result.

[0150] Moisture loss after cooking

[0151] Protocol: Sample was weighed before (w1) and after oven (w2) and moisture loss was calculated as (w1-w2) / w1.

[0152] Results were calculated as average of 20 measurements.

[0153] Average change after cooking

[0154] 24h: 5.6 g weight loss 30h: 5.5 g weight loss

[0155] Fresh sample weight was between 15-21g. 30 h samples were slightly heavier on average.

[0156] Colour after cooking

[0157] Protocol: 1 slice analysed from each of the 5 blocks as 10 replicate measurements before and after oven with Minolta CR-400 chroma meter. Results are average of 50 measurements.

[0158] Delta E:

[0159] 24h before vs after: 8.3

[0160] 30h before vs after: 8.9

[0161] 24h vs 30h before: 0.8

[0162] 24h vs 30h after: 0.9

[0163] Samples were slightly darker after the oven, with higher yellowness and redness values

[0164] Moisture content of fresh sample

[0165] Protocol: moisture content of each box was measured by mixing together samples from texture profile analysis (TPA). For 18 h sample which wasn’t texture analysed, samples from the middle of a box were mixed together (4 boxes instead of 5).

[0166] 6 g of sample was measured to pre-weighed cup and placed in 105 °C oven for overnight and weight recorded again in the morning. Moisture was calculated as difference between sample weight before and after the oven.

[0167] Results are average of 4 measurements for 18 h sample and 5 measurements for 24 and 30 h samples.

[0168] Moisture content increased as a function of fermentation time, Anova 0.016 (<0.05):

[0169] 18h -> 62.5% ± 0.7 0.6 0.9

[0170] Texture profile analysis

[0171] Protocol: Texture profile analysis (TPA) was measured with Texture analyser TA.Xtplus (Stable Micro Systems). Rectangular pasta probe (38x50.5 mm) was used to press the samples with test speed of 5 mm / sec, 50% strain, and 5 sec between the compressions. Samples were cut with a mould (38x50.5 mm) to the same size as the probe. Results are average of 20 measurements for 24h sample and 19 measurements for 30h sample, and shown in figures 5 and 6, and in the Table 2 below. T-test: <0.001 , samples are significantly different from each other, except in springiness >0.05.

[0172] Textural parameters associated with meat like features, such as hardness and chewiness, were improved.

[0173] Table 2. TPA results.

[0174] Example 8 - Chemical analysis

[0175] Moisture was analysed gravimetrically at VTT.

[0176] Protein analyzed at VTT - Dumas method using nitrogen analyser (rapid MAX N exceed, Elementar Analysensysteme GmbH, Langenselbold, Germany) according to AACC method 46-30.01 (AACC, Crude Protein - Combustion Method, Approved Methods of Analysis, 11th Ed. Method 46-30.01. Cereals & Grains Association, St. Paul, MN, U.S.A. 1995, 8. htp: / / dx.doi.Org / 10.1094 / AACCIntMethod-46~30.01). A conversion factor of 6.25 was used to calculate the protein content from the nitrogen content.

[0177] Dietary fiber analyzed at VTT - AOAC 2011.25 (McCleary et al. 2012). McCleary B V., DeVries J, Rader J, Cohen G, Prosky L, Mugford D, Champ M, Okuma K. 2012.

[0178] Determination of Insoluble, Soluble, and Total Dietary Fiber (CODEX Definition) by Enzymatic-Gravimetric

[0179] Method and Liquid Chromatography: Collaborative Study. J AOAC Int. 95(4):933-936.

[0180] Fat - At Eurofins according to NMKL 160 standard.

[0181] Sugars - At Eurofins according to AOAC 982.14 by HPAEC-PAD (high-performance anion- exchange chromatography with pulsed amperometric detection).

[0182] Iron - At Eurofins according to internal method by ICP-OES instrument (Inductively coupled plasma-optical emission spectroscopy).

[0183] Thiamin - At Eurofins according to EN 14122:2014 by LC-FLD (liquid chromatography fluorescence detector).

[0184] Riboflavin - At Eurofins according to EN 14152:2014 by LC-FLD. Niacin - At Eurofins according to EN 15652:2009 by HPLC (high performance liquid chromatography).

[0185] GOS - At Eurofins according to internal method by HPAEC-PAD detector.

[0186] The results of the chemical analysis are shown in Tables 3, 4, and 5. Protein content increased further as a result of fungal biomass accumulation. Shorter fermentation time avoided the protein hydrolysis. No change in essential amino acids was observed. In situ production of vitamins (eg. Niacin). Niacin content increased during fermentation (24h). Table 3. Proximate and other nutritional composition, per dry matter of sample.

[0187] Table 4. Proximate and other nutritional composition, fresh samples.

[0188] *sugars=glucose, fructose, galactose, sucrose, lactose, maltose **GOS; galacto-oligosaccharides = raffinose, stachyose, verbascose The fermented food samples were found to be high in protein, high in fiber, low in fat, low in sugars, and a source of iron, as specified in Ell food nutritional claims.

[0189] Amino acids were analyzed at Eurofins according to the method ISO 13903:2005 by IC-LIV (ion chromatography UV) detector, for tryptophan Ell 152 / 2009 by LC-FLD detector.

[0190] When the score is <1 , there is too little amino acids as compared to the requirement (for older child, adolescent, adult).

[0191] Thus, the quality of protein, when evaluated only from the amino acid composition, is incomplete.

[0192] Reference for calculating the amino acid score: FAO 2013. Dietary protein quality evaluation in human nutrition. Report of an FAO expert consultation. FAO food and nutrition paper 92, 2013.

[0193] Table 5. Amino acid scores

[0194] *SAA=sulfur amino acids=methione + cysteine+cystine

[0195] *AAA=aromatic amino acids=phenylalanine + tyrosine + tryptophan

[0196] Fermentation increased free amino acid content in the samples.

[0197] Free amino acids analyzed at VTT using UHPLC-MS / MS method (high-throughput ultra- high performance liquid chromatography-tandem mass spectrometry).

[0198] Example 9 - microscopy

[0199] The following analyses were carried out:

[0200] Stereomicroscopy

[0201] Cross sectional surfaces were imaged as such without staining.

[0202] Objectives used: 1x, 2x, 4x and 6.3x The quantity of mycelia was increased along the fermentation being hardly visible at the time point of 18h and very dense at the time point of 30h.

[0203] Confocal laser scanning microscopy a) without staining using reflection-based imaging mode (Figure 7):

[0204] Pieces fitting in a silicone well (diameter 20mm, depth 2.6mm) attached on an objective slide were cut from each sample.

[0205] Imaging using confocal laser scanning microscope with resolution of 1024x1024 using two objectives: 5x objective (depth 371-1787 pm, z-step 19.3 pm) and 10x objective (depth 223-692 pm, z-step 5.5 pm)

[0206] Increase of mycelia was clearly visible. No clear changes in extrudate structures were observed.

[0207] Confocal laser scanning microscopy b) with staining for localisation of cell walls and protein Pieces fitting in a silicone well (diameter 20mm, depth 2.6mm) attached on an objective slide were cut from each sample.

[0208] Mixture of stains was applied on top of the sample: 0.01 % (w / v) Calcofluor White (fungal cell walls, cereal beta-glucan, cellulose), ex. 405nm, em. 424-476nm; and 0.02 % (w / v) Fast Green (protein), ex. 633nm, em. 644-723nm

[0209] Staining in +4°C for 4 min and sealing with a cover slip

[0210] Imaging using confocal laser scanning microscope with resolution of 1024x1024 using two objectives: 5x objective (depth 389-956 pm, z-step 17.7pm); and 10x objective (depth 89-293 pm, z-step 5.0 pm).

[0211] Increase of mycelia was clearly visible.

[0212] Stainability of plant cell wall particles decreased along the fermentation time probably indicating hydrolysis of these structures.

[0213] Organisation of protein seemed to be decreasing along the fermentation time when using 10x objective, indicative of protein hydrolysis.

[0214] Example 10 - density

[0215] A method modified from AAACC 10-05.01 was used to measure densities of the porous extrudate. A 500 ml measuring glass was filled with dry rapeseeds. Then, a small amount of the rapeseeds were poured into another measuring glass, and a small amount of sample (about 2-8 g) was added and their weight was recorded. Then, more rapeseeds were poured from the first measuring glass so that the sample in the second measuring glass was fully covered and surrounded by the rapeseeds. Then this was repeated until the measuring glass was full of sample and rapeseeds. The volume of the rapeseeds left in the first measuring glass was recorded. Sample density was calculated as the volume of the rapeseeds left corresponding of the volume of the sample in the second measuring glass divided by the weight of the sample. Results are shown in Table 6.

[0216] Table 6. Density of the porous extrudate.

[0217] The scope of protection sought for various embodiments of the invention is set out by the independent claims. Any example or embodiment not falling within the scope of the independent claims is presented herein as an additional example or additional embodiment useful for understanding the claimed invention.

Claims

CLAIMS1 . A method for manufacturing a food composition comprising: preparing a mixture comprising a protein, dietary fibre, and starch; carrying out dry extrusion on the mixture to provide a porous extrudate comprising fibrillated protein; and fermenting the extrudate by solid state fermentation with filamentous fungi.

2. The method according to claim 1 , wherein the mixture contains 35-80 wt-%, preferably 40-75 wt-%, more preferably 40-70 wt-%, most preferably 40-60 wt-% protein, based on dry weight.

3. The method according to claim 1 or 2, wherein the mixture contains dietary fiber 5- 30tw-%, preferably 10-30wt-%, more preferably 20-30wt-%, most preferably about 25wt-%, based on dry weight.

4. The method according to any one of claims 1-3, wherein the mixture contains starch 10-35wt-%, preferably 15-30wt-%, more preferably 20-25wt-%, most preferably about 20wt-%, based on dry weight.

5. The method according to any one of claims 1-4, wherein the mixture is prepared by: mixing at least one protein concentrate with at least one separate source of dietary fiber and starch; or mixing at least one protein concentrate, at least one separate source of dietary fiber, and at least one separate source of starch.

6. The method according to any one of claims 1-5, wherein: the protein is at least one of legume protein, faba protein, pea protein, soy protein, cereal protein, oat protein, wheat protein, pseudocereal protein, oil seed protein, oil seed press cake, rapeseed protein, sunflower protein, and cellular agriculture protein such as single cell protein, microalgae protein, whole biomass protein, precision fermentation made structural proteins, or cultured animal cell protein; and / or the dietary fiber is at least one of cereal bran, pseudocereal bran, wheat bran, dietary fiber sourced from legume, or dietary fiber from cellular agriculture such as whole biomass of cellular agriculture; and / or the starch is at least one of cereal flour, legume flour, and pseudocereal flour.

7. The method according to any one of claims 1-6, wherein the extrudate is hydrated before fermenting to a moisture content of 50-75wt-%, preferably 55-70wt-%, more preferably 60-65wt-%.

8. The method according to any one of claims 1-7, wherein the dry extrusion is carried out in operating conditions to obtain an extrudate having a density in the range 100- 1000 g / l, preferably 200-900 g / l, more preferably 300-800 g / l, more preferably 300- 700 g / l, more preferably 300-600 g / l, most preferably 300-500 g / l, or about 400g / l.

9. The method according to any one of claims 1-8, wherein the protein content of the fermented food composition obtained by the method is not significantly decreased during fermenting; and / or the vitamin content, such as niacin content, of a fermented food composition obtained by the method is increased during fermenting; and / or essential amino acid profile is preserved.

10. The method according to any one of claims 1-9, wherein solid state fermentation is carried out at 20-40°C for 12-168h, preferably 20-40°C for 12-48h, more preferably at about 30°C for about 20-40h.

11. A food composition manufactured by the method according to any one of claims 1- 10, and comprising 30-60 wt-% of protein, 20-30 wt-% of dietary fibre, 10-50 wt-% starch, and filamentous fungi.

12. The food composition of claim 11 having at least one of: an average hardness of 60-90N; and / or an average springiness of 0.7-0.8; and / or an average cohesiveness of 0.6-0.7; and / or an average chewiness of about 25-45; and / or an average resilience of 0.2-0.4.

13. A meat alternative comprising the food composition of claim 11 or 12.

14. A method for manufacturing a food product, comprising providing a food composition according to any one of claim 11 or 12, optionally mixing with other food product ingredients, and carrying out extrusion to obtain the food product.

15. A food product manufactured according to claim 14.

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