Food Product Comprising a Pure Fungi Biomass

Abstract

The present disclosure relates to a food product comprising a pure fungi biomass, the pure fungi biomass comprising fungi of a species belonging to the genus Rhizopus, wherein the dry weight of the food product comprises within the range of from 10% to 100% of dry weight of the fungi biomass the fungi biomass comprising fungi biomass fibers, wherein 50% or more, such as 70% or more, or 80% or more of the fungi biomass fibers are aligned substantially in planes extending in a first direction. The present disclosure also relates to a method for producing the food product and use of a pure fungi biomass for preparing a food product.

Claims

1. A food product comprising a pure fungi biomass, the pure fungi biomass comprising fungi of a species belonging to the genus Rhizopus, wherein the dry weight of the food product comprises within the range of from 10% to 100% of dry weight of the fungi biomass, the fungi biomass comprising fungi biomass fibers, wherein 50% or more, such as 70% or more, or 80% or more of the fungi biomass fibers are aligned in planes extending in a first direction.

2. The food product according to claim 1, wherein the dry weight of the food product comprises within the range of from 25% to 100% of dry weight of the pure fungi biomass.

3. The food product according to claim 1, wherein the dry weight of the food product comprises within the range of from 50% to 100% of dry weight of the pure fungi biomass.

4. The food product according to any one of claims 1 to 3, wherein the fungi are of the species Rhizopus oryzae, Rhizopus microsporus var. oligosporus, Rhizopus arrhizus, Rhizopus delemar, or Rhizopus stolonifer.

5. The food product according to any one of the preceding claim, wherein the amount of protein in the total pure fungi biomass (dry weight) is within the range of from 25% to 70%.

6. The food product according to any one of the preceding claim, wherein the amount of essential amino acids in the fungi biomass (dry weight) is within the range of from 30% to 70% of the total amino acid content.

7. The food product according to any one of claims 1 to 6, wherein the food product further comprises hydrocolloid, optionally xanthan gum, locust bean gum, carrageenan, sodium alginate, starch or methylcellulose.

8. The food product according to any one of the preceding claims, wherein the food product further comprises 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, oats, apple extract and sources of fiber such as pea, sugarcane, wheat, cellulose, oats and apple.

9. The food product according to any one of the preceding claims, wherein the food product is in the form of a patty, nugget, burger, sausage, paste, chunk, fillet, extrudate, granules, cake, meat substitute, meat extender, jerky, fish-like product, seafood-like product, snack, beverage, dessert or baked goods.

10. A method for producing a food product, the method comprising the steps of: a) cultivating fungi of a species belonging to the genus Rhizopus under aerobic submerged fermentation conditions using a closed fermentation vessel with liquid substrate media containing a carbon source originated from processed grain crops or glucose-, fructose- or lactose-containing substrates in a monomeric or oligomeric form to obtain a fungi biomass comprising fungi biomass fibers, b) processing the fungi biomass obtained from step a) by heating to a temperature of from 50° C. to 75° C. for a period of time of from 5 minutes to 1 h, c) separating the fungi biomass obtained from step b) from the liquid cultivation substrate by pressing the fungi biomass in a second direction thereby providing a pure fungi biomass having 50% or more, such as 70% or more, or 80% or more, of the fungi biomass fibers aligned in planes extending in a first direction, the first direction being perpendicular to the second direction, and d) incorporating the processed and pressed pure fungi biomass obtained in step c) in a food product.

11. The method according to claim 10, wherein the pure fungi biomass from step c) is mechanically treated to break the biomass into smaller pieces before incorporation of the fungi biomass into the food product in step d).

12. The method according to claim 10 or 11, wherein the food product is homogenized in the presence of any one or more of the 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, oats, apple extract and sources of fiber such as pea, sugarcane, wheat, cellulose, oats and apple.

13. The method according to any one of claims 10 to 12, wherein the pure fungi biomass is frozen at a temperature below −10 C.° before being incorporated in the food product or wherein the food product of step d) is frozen at a temperature below −10 C°.

14. The method according to any one of claims 10 to 13, wherein the cultivation in step a) takes place at 25 to 37° C.

15. The method according to any one of claims 10 to 14, wherein the cultivation in step a) includes stirring of the liquid substrate media and the fungi biomass at a rotation speed of from 100 rpm to 300 rpm.

16. The method according to any one of claims 10 to 15, wherein the pressure applied in step c) is within the range from 0.5 to 10 bar.

17. The method according to any one of claims 10 to 16, wherein the method prior to step c) and after step b) comprises a step b2) of incubating the heated fungi biomass in an aqueous solution having a salt concentration of from 0.5% to 10%, optionally the salt is NaCl.

18. The method according to any one of claims 10 to 16, wherein the method prior to step c) and after step b) comprises a step b2) of incubating the heated fungi biomass in water an aqueous solution having a pH within the range of from 6 and 10.

19. The method according to any one of claims 10 to 18, wherein the pure fungi biomass is mixed with a hydrocolloid solution, such as Xantham gum, optionally in an amount of from 0.1% to 5% of the total fungi biomass dry weight.

20. The method according to claim 19, where the hydrocolloid solution is added either during or after separation of the fungi biomass step in step c).

21. The method according to any one of claims 10 to 20, wherein in step a) a pH within the range of from 2.5 and 7.5 is maintained for the duration of the fermentation.

22. The method according to any one of claims 10 to 21, wherein the carbon source originates from a wheat flour-based substrate, optionally a wheat flour based substrate with minerals.

23. The method according to claim 22, wherein the amount of the wheat flour based substrate is within the range of from 5 g/L to 50 g/L dry weight based on the liquid substrate media.

24. The method according to any one of claims 10 to 23, wherein the pure fungi biomass subsequent to any one of steps c), d) is exposed to UV-B light, such as during a period of time of about 1 minute to 60 minutes.

25. Use of a pure fungi biomass comprising fermented fungi of a species belonging to the genus Rhizopus for preparing a food product in the form of a patty, nugget, burger, sausage, paste, chunk, fillet, extrudate, granules, cake, meat substitute, meat extender, jerky, fish-like product, seafood-like product, snack, beverage, dessert or baked goods.

Description

DESCRIPTION OF FIGURES

[0068] FIGS. 1A and 1B show images obtained through stereomicroscopy indicating alignment of fungal mycelium according to a structure having aligned fungi biomass fibers compared to commercial mycoprotein which does not present this structure.

[0069] FIG. 2 shows a graph illustrating values of Hardness from a texture profile analysis (TPA) test of balls formed with fungi biomass mixed with different classes of ingredients.

[0070] FIG. 3 shows a graph illustrating Compression Work from a texture profile analysis (TPA) test of balls formed with fungi biomass mixed with different classes of ingredients.

[0071] FIG. 4 illustrates the results of a comparison with a meat-ball style food product according to the present disclosure and six other non-animal based meatball-style products and one animal-based meatball product.

[0072] FIG. 5 illustrates the results of a comparison between a pure fungi biomass according to the present disclosure and other protein sources with respect to their respective water holding capacity (WHC).

[0073] FIG. 6 illustrates the results of a comparison between a pure fungi biomass according to the present disclosure and other protein sources with respect to their respective water solubility index (WSI).

[0074] FIG. 7 illustrates the results of a comparison between a pure fungi biomass according to the present disclosure and other protein sources with respect to their respective oil holding capacity (OHC).

[0075] FIG. 8 illustrates the correlation between propeller stirring speed of a stirred tank bioreactor of 30 L size and the Toughness of the biomass obtained, measured through a Knife Blade test on a texture analyzer.

[0076] FIGS. 9A-9C shows three graphs illustrating parameters obtained from a Knife Blade test performed on fungi biomass samples which were grown in liquid bioreactors with two different type of blades: a high-shear mixing and a low-shear mixing blades.

[0077] FIG. 10 shows a graph illustrating the values of toughness of a Knife Blade test performed on fungi biomass samples which were subjected to different concentrations of NaCl and then pressed, and compares them to meat samples and commercial mycoprotein.

[0078] FIG. 11 shows a graph illustrating the values of toughness of a Knife Blade test performed on fungi biomass samples which were subjected to different pH values and then pressed, and compares them to meat samples and commercial mycoprotein.

[0079] FIG. 12 shows the water content of different fungi biomass samples that were incubated with different concentrations of NaCl, at different pH values, or pressed at different pressures.

DETAILED DESCRIPTION

[0080] The present disclosure relates to the process for producing edible, food-grade fungi biomass/mycoprotein through a fermentation process on carbohydrate substrates, and the application of the resulting pure fungi biomass in a food product for human consumption. The filamentous fungi used in the invention are cultivated in liquid media under aerobic conditions and belong to the genus Rhizopus. The food products containing this fungi biomass are in the area of meat replacements, meat extenders, protein-enriched food products, fiber-enriched food products, or novel mycoprotein-containing food products.

[0081] To achieve the texture and taste profile acceptable and desired for these types of products, the biomass can be processed using the following described methods. After harvesting from the fermentation, the biomass needs to be heat treated at 50-70° C. for 5-60 min, and after that a washing step may be applied to remove off-tastes. A pressing step may also be applied for dewatering after this treatment. For this pressing step, to create a good meat-like structure, the fungal mycelia should be pressed from one single direction, and allowed to expand in the direction perpendicular to the pressing direction. This creates a flow field for fibers that promotes alignment and creation of a lamellar structure, which has resemblances to meat when cut. In combination to this, the equipment may allow for the creation of large press cakes, which are beneficial for the creation of larger meat-like chunks, which have added value as opposed to small mince-like pieces. To create the combination of the pressing effect and obtaining large press cakes, the following industrial equipments can be used as examples: an hydropress, a piston press, a belt press, a cold-press or similar systems. Other common dewatering equipments such as conventional filter presses and membrane filter presses will create forces in multiple directions while the fungi biomass is concentrated and dewatered, and therefore not create the desired alignment, while other equipments like decanters and centrifuges will not allow the obtention of large filter cakes and might not also exert a pressing force in a single direction.

[0082] The pressing step affects significantly the biomass texture so that biomass conditions before and during pressing step influence the outcome. Salts and other solutes causing osmotic pressure such as but not limited to NaCl can be added to the biomass-containing solution before pressing in concentrations of, for example, 0.1% to 10%, which has an effect of decreasing the biomass toughness values when measured with a knife blade shear test in a texture analyzer. Toughness of the pressed biomass, as well as dry matter content of the press cake is also influenced by adjusted pH of the biomass-containing solution before pressing. Increase in pH is correlated to decrease in toughness, and pressing should occur between pH 6 and 10 for the better effects of creating a product with meat-like toughness values.

[0083] Fungi Biomass

[0084] The present disclosure relates to pure fungi biomass produced by a fermentation/cultivation process from a starter-culture/pre-culture. The strain of filamentous fungi belong to the genus of Rhizopus, such as Rhizopus oryzae, Rhizopus microsporus var. oligosporus (also called Rhizopus oligosporus), Rhizopus arrhizus, Rhizopus delemar, or Rhizopus stolonifer.

[0085] The pure fungi biomass is an edible product for use in a human food product, either alone or in combination with other ingredients.

[0086] The pure fungi biomass is rich in protein with an unusually favourable amino acid composition, low carbohydrate content and low fat content. The percentage of protein is in the range of 25 to 70%. The percentage of essential amino acids is 30-70% of the total amino acid content. The percentage of dietary fiber is in the range of 1 to 10%. The values for fatty acids in dry weight are: 0.1-10 g/100 g for total fat, 0-4 g/100 g saturated fatty acids. 0-5 g/100 g monounsaturated fatty acids and 0-8 g/100 g polyunsaturated fatty acids.

[0087] Experimental Section

[0088] 1.1 Microorganism

[0089] The zygomycete fungi Rhizopus oryzae, Rhizopus oligosporus, Rhizopus arrhizus, Rhizopus delemar, and Rhizopus stolonifer were used throughout these tests.

[0090] The strains were maintained on potato dextrose agar (PDA) plates containing (in g/L): agar 15, dextrose 20, potato extract 4. New plates were prepared by incubating for 4-5 days at 35° C. and then storing them in the fridge (4° C.). The plates were renewed every 2 months from a 25% glycerol stock kept at −80° C.

[0091] 1.2 Inoculum Preparation

[0092] For preparation of spore suspension, a plate was flooded with 10-20 mL of sterile water and spores scraped off the surface with a disposable, sterile spreader. Spores were counted in a hemocytometer under a light microscope, and used directly as inoculum for plates and liquid cultivations.

[0093] 1.3 Cultivation in Shake Flasks

[0094] R. oryzae, R. oligosporus, R. arrhizus, R. delemar, and R. stolonifer were cultivated in Erlenmeyer flasks (volumes 100-2000 mL) with or without baffles, filled with liquid growth medium to a maximum of 20% of the total flask volume. Growth media contained either glucose or sucrose as the carbon source in the range of 10-40 g/L.

[0095] Additional media components: nitrogen source in the form of ammonium, as well as potassium, magnesium and calcium in the form of phosphates, sulphates or chlorides.

[0096] 1 mL of spore suspension (10{circumflex over ( )}7 spores/mL) per 100 mL of growth media was added to each flask, followed by incubation at 30-35° C. for 18-24 h under shaking (100-150 rpm).

[0097] 1.4 Cultivation in Bioreactor

[0098] To 20 L growth media in a 30 L stirred-tank bioreactor, 1-3 L of preculture from shake flasks were added, as well as 10-50 mL of spore suspension. The pH was adjusted to 5-6 with 5M NaOH. Sterilisation of the liquid in the bioreactor was done by heating up the liquid with steam (via the bioreactor's double jacket) to 121° C. and 1 bar overpressure for 20 min.

[0099] Batch cultivation was carried out for 24 h, at 30-35° C., stirring at 150 rpm, under aeration, and the pH was kept between 5.0 and 6.0. Semi-continuous cultivation was carried out by harvesting part of the biomass and growth media after 18-24 h, and filling up to 20 L again with fresh, sterile growth media.

[0100] A fungal biomass yield of between 0.2 and 0.3 g pure biomass/g substrate could be reached at the end of the fermentation (24 h).

[0101] 1.5 Protein Content of Dry Fungal Biomass

[0102] Fungal biomass harvested from shake flask and bioreactor cultivations was dried at 65-105° C. for 24-48 h until no change in mass occurred and all water had been removed. The dried biomass was milled in a TissueLyser ballmill for 30 sec to obtain a fine powder. The elemental composition of the powder was analysed in an Elemental Analyser to obtain the percentages of nitrogen, hydrogen, sulphur and oxygen. To estimate the protein content of the fungal biomass, the nitrogen content of the dried biomass was multiplied with the factor 6.25.

[0103] 1.6 Nutrient Content of Rhizopus Fungal Biomass

[0104] The nutritional composition of the Rhizopus biomass was analysed in detail by an external accredited laboratory (ALS Scandinavia AB). One representative example of the nutritional composition of the dry biomass showed the following values (per 100 g): 340 kcal (or 1400 kJ), 60.29 g protein, 3.74 g carbohydrates, 5.97 g fat, 12.30 g fiber. One representative example of the nutritional composition of the wet biomass showed the following values (per 100 g): 85 kcal (or 350 kJ), 15.07 g protein, 0.94 g carbohydrates, 1.49 g fat (of which 0.34 g saturated fat, 0.40 g monounsaturated, 0.68 g polyunsaturated fat), 3.08 g fiber.

[0105] One representative example of the amino acid content (% of total protein) of the fungal biomass of Rhizopus is summarised in Table 1 below, with essential amino acids being marked with an asterisk.

TABLE-US-00001 TABLE 1 Content of amino acids (in % of total protein) in Rhizopus biomass. Alanine 10.16% Arginine 3.39% Asp. Acid 5.98% Cystine 1.39% Glut. Acid 8.37% Glycine 7.17% Histidine* 3.59% Isoleucine* 7.97% Leucine* 9.36% Lysine* 8.96% Methionine* 2.99% Phenlyalanine* 5.78% Proline 2.39% Serine 2.59% Threonine* 6.18% Tryptophan* 0.80% Tyrosine 3.78% Valine* 9.16% Essential amino acids 54.78% BCAA 26.49%

[0106] Regarding micronutrients, the following values were obtained for one representative measurement of the dry Rhizopus biomass (per 100 g): 509.1 mg calcium, 1004.5 mg potassium, 116.5 mg magnesium, 148.6 mg sodium, 238.5 mg sulfur, 2550.2 mg phosphorus, 10.8 mg iron, 15.5 mg zinc, 5.2 mg copper, <0.2 ug vitamin B12, 1.0 ug vitamin B6, 1.5 ug vitamin D2 and <12.5 ug vitamin D3.

[0107] Experiment 2: Cultivation on Synthetic Media in 300 L Bioreactor

[0108] 2.1 Process Parameters

[0109] Cultivations on sugar substrate were scaled up to a 300 L bioreactor (manufactured by Belach Bioteknik) with a working volume of 250 L. The process parameters were the following: 25-45 L/min constant aeration through an arrangement of porous air spargers, 30-35° C., stirring with a single Rushton impeller, pH 5-6 (adjusted with 5M NaOH). The cultivation media had the same composition as described in 1.3 with sucrose as the main carbon source.

[0110] For batch cultivation, all biomass was harvested after 24 h. For semi-continuous cultivation, an initial batch cultivation was carried out for 24 h, followed by harvesting of 50-80% of the fermentation liquid and filling up with fresh, sterile growth media to 250 L for another round of 18-24 h fermentation.

[0111] 2.2 Heat Treatment Regimes

[0112] Heat treatment of biomass to inactivate the fungus was conducted at 50-70° C. for 5-60 min. The best results were achieved by heat-treating the biomass at 55-65° C. for 10-20 min. Several different methods were tried, including heating up the biomass together with the fermentation liquid to 60° C. in the bioreactor, or immersing the harvested biomass in 60-65° C. hot water. The success of heat inactivation was checked by placing heat-treated biomass on PDA plates and incubating at 35° C. for 24 h. Sporulation showed an insufficient heat treatment.

[0113] The best results were achieved with immersing the harvested and filtered biomass into 65° C. water for 10 min while stirring. No sporulation was detected after this treatment, and the biomass had a pleasant texture and appearance without appearing too cooked.

[0114] 2.3 Fed-Batch/Semi-Continuous Fermentation Mode

[0115] The concentration of dry biomass obtained during the semi-continuous process was 3.12 g/L, with a resulting productivity (g/L*h) of 0.134 dry biomass.

[0116] Experiment 3: Production of a Meat/Chicken-Like Product Containing Rhizopus Biomass

[0117] 3.1 Fermentation Process Parameters

[0118] The process parameters were the same as described in Experiment 2.

[0119] 3.2 Treatment of Biomass after Fermentation

[0120] Upon harvesting, the biomass was subjected to a heat treatment step. The biomass was washed and pressed in a single direction in order to remove water and orient the fibres in a single plane. For minimally treated samples, the pressed biomass with a water content of 70-80° C., was broken into small pieces. The biomass had a fibrous texture, no off-flavour or off-taste and a beige colour.

[0121] Prior to pressing, one sample contained hydrocolloids integrated into the biomass network by mixing the biomass with a hydrocolloid solution of 1 to 5%. The resulting press cake with or without integrated hydrocolloids was sliced into chunks ranging from 1 cm to 10 cm in size. Said chunks were then further reduced in size by processing in a blender or hacking machine by themselves or in the presence of a hydrocolloid solution added during the size reduction step.

[0122] The fungal biomass treated with hydrocolloid solution, when grinded to small particles, forms a mince-like form that has binding properties among the biomass particles, which was not a strong trait of the raw biomass without addition of said hydrocolloid. The presence of hydrocolloid solution with the biomass also resulted in an increase of water retention capacity when the mix was subjected to a freeze-thaw cycle. Within 24 h at 4° C. after thawing the samples, the biomass sample alone would lose up to 24% of its weight as water released. The addition of xanthan gum after the biomass pressing step decreased this to an 8% weight loss, while addition to the biomass before the pressing step decreased it to a 5% loss.

[0123] 3.3 Preparation of Food Product Functional Formulations

[0124] The biomass that was mixed with different solutions of hydrocolloids was successfully used in the forming of balls and patties such as burgers. The structure and cohesiveness of the ball/patty were improved when solutions of the hydrocolloids xanthan gum, locust bean gum, sodium alginate, potato starch or methylcellulose were used, compared to the same structure formed with the biomass without use of any additions.

[0125] The mixing of biomass at 60-85% water content with sodium alginate and starch at 2% (w/w) each, and subsequent treatment with heat in a wet environment (steaming or boiling), led to the gelation of the obtained structure, making it suitable for use in sliceable meat alternative food products.

[0126] 3.4. Shelf Life Analysis

[0127] The microbiological activity of the biomass and preparations of such was analyzed after treatment and after conservation at −18° C. for 6 months. No activity was detected either in the fresh biomass post-treatment of conserved in the freezer for 6 months.

[0128] In another experiment, formed balls using the fungi biomass in the presence of hydrocolloids were conserved in vacuum at 4° C. for 8 days. After reopening the vacuum packages, samples with and without hydrocolloids were equally well preserved. The taste, flavour, texture and smell profiles were similar to recently defrosted or freshly produced samples.

[0129] 3.5 Comparison of Fiber Texture by Stereomicroscopy

[0130] Samples of the pressed biomass or of a commercial mycoprotein chicken-like product were generated by cross-cutting surfaces with a razor blade while frozen. These samples were then examined with a Zeiss SteREO Discovery.V8 stereomicroscope equipped with Achromat S 0.5× objective (Carl Zeiss Microlmaging GmbH, Göttingen, Germany) and imaged using an Olympus DP-25 single chip colour CCD camera (Olympus Life Science Europa GmbH, Hamburg, Germany) and the Cell{circumflex over ( )}P imaging software (Olympus).

[0131] The obtained images in FIGS. 1A and 1B show that the fungal mycelium from pressed samples is substantially aligned forming a lamellar structure, which is not present in the existing commercial mycoprotein product. This structure creates a much more appealing product as a meat replacement compared to the existing market alternatives.

[0132] Samples were pressed in a hydropress system with radial pressure, in a belt press system, and in a hydraulic piston-based system that apply the same concepts of single direction force applied with room for perpendicular expansion. The same product effect was observed as in FIGS. 1A and 1B.

[0133] Experiment 4: Production of a Final Product (“Meatball”) Containing Rhizopus Biomass

[0134] 4.1 Preparation of Product from Fungi Biomass

[0135] Upon harvesting, the biomass was subjected to a heat treatment step. The biomass was washed and pressed in a single direction in order to remove water and orient the fibres in a single plane. The biomass was then frozen under vacuum conditions and thawed after a week. The biomass was cut down to pieces from 0.5 to 2 cm and blended together with selected culinary ingredients from a list of ingredients with synergistic effects for the product texture, such as oat flour, potato starch, almond flour, rice flour, wheat flour, corn starch and bread crumbs. To this was also added flavour ingredients such as spices, salt, sugar and flavour enhancers. To this it was also added hydrocolloids such as methylcellulose, sodium alginate or carrageenan. The mix was blended for up to 15 minutes and balls were formed with the resulting mass. The balls were heat treated in an oven at 140° C. for 40 min and deep fried at 180° C. for 2 min.

[0136] 4.2 Sample Preparation of Mycoprotein Mixed with Different Ingredients for Texture Profile Analysis

[0137] Samples of pure frozen biomass with water content between 70%-75% were defrosted and ground with a single-blade enterprise cutting system using a hole diameter of 8 mm. The ground biomass was then mixed with different ingredients composing of different starches, gelling agents, fibers, flours, fats and proteins in a concentration of 5% for a sample in the shape of a ball with 15 g weight. In the case of gelling agents, a 5% solution was prepared according to manufacturer's indications and 5% was added as an ingredient. Ingredients were also heat-treated by incubating in an oven for 125° C. for 15 min or cold-treated by placing at −4° C. for 2 h.

[0138] 4.3 Texture Profile Analysis (TPA)

[0139] Binding properties of these ingredients 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=AreaFT4:6/AreaFT1:3 (area of second deformation relative to area of the first deformation)

Gumminess=Hardness×Cohesiveness

Springiness=Time-diff4:5/Time-diff41:2 (percentage of product height that is regained after first deformation)

Chewiness=Gumminess×Springiness

Resilience=AreaFT2:3/AreaFT1:2 (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)

[0140] Mixing with the different ingredients changed all parameters measured by the TPA in the fungi biomass samples in significant ways, from −95% to up to 500% as seen in FIG. 2 and FIG. 3.

[0141] FIG. 2 shows a graph illustrating values of Hardness from a texture profile analysis (TPA) test of balls formed with fungi biomass mixed with different classes of ingredients. Each box represents the variation of values within a single class of ingredients. For some classes, different boxes were created for each temperature treatment (HT: High-temperature, RT/C: Room temperature/cold).

[0142] FIG. 3 shows a graph illustrating values of the Compression Work of the first peak from a texture profile analysis (TPA) test of balls formed with fungi biomass mixed with different classes of ingredients. Each box represents the variation of values within a single class of ingredients. For some classes, different boxes were created for each temperature treatment (HT: High-temperature, RT/C: Room temperature/cold).

[0143] This shows the compatibility of the fungi biomass with many ingredients used in the food industry to achieve a variety of textures.

[0144] 4.2 Sensory Analysis

[0145] A small sensory test panel evaluated 7 competitor plant-based ball products, as well as a meat-based ball product, regarding visual appearance, smell, texture and taste. The testers classified the balls made from Rhizopus fungi biomass to be of identical (non-statistically significant difference) quality to 4 of the existing plant-based commercial alternatives and superior to 3 others (FIGS. 1A and 1B). The result of the sensory test is illustrated in FIG. 4.

[0146] 4.3 Storage Conditions

[0147] The products created with the fungi biomass were frozen and conserved at −18° C. Packaging the products in vacuum conditions eliminated any gain of off-flavours and unpleasant taste over time and preserves the product longer due to elimination of oxygen.

[0148] Experiment 5: Production of a Dry Biomass-Containing Food Ingredient

[0149] 5.1 Preparation of Dried Fungi Biomass

[0150] Biomass obtained from the fermentation process was harvested, filtered and pressed to remove water. The pressed biomass was cut into small pieces and dried at 85-105° C. over the course of 16-24 h to remove water. The resulting dry mass was grinded in a ball mill to obtain a fine powder.

[0151] 5.2 Water Holding Capacity and Water Solubility Index

[0152] The method used for measuring water holding capacity (WHC) and water solubility index (WSI) was adapted from Miedziandka et al, with modifications (Miedzianka et al., 2014; https://doi.org/10.1016/j.foodchem.2014.03.054). 0.1 g of dry fungal biomass powder (dried at 45° C. until all water was removed and milled in a ball mill) was added to each tube and the exact biomass weight was noted down. 10 mL of MilliQ-water was added, creating a 1% suspension (w/v). The samples were then stirred for 20 s every 10 min for one hour using a laboratory vortex mixer on full speed. The samples were centrifuged (4000 g, 25 min, room temperature).

[0153] The supernatant was discarded and added to pre-weighed beakers and left to dry overnight at 85° C. The pellets left in the centrifuge tubes were incubated at 50° C. for 25 min before weighted. The values of the dried pellets, supernatants and volumes were used for calculating the WHC and WSI according to the formulae used in Miedziandka et al. 2014: [0154] Initial weight of sample=A [0155] Final dried weight of remaining pellet=B [0156] Final dried weight of supernatant=C

[00001]$WHC=\frac{B-A}{A}$$\mathrm{WSI}=\frac{A-C}{A}$

[0157] Comparisons were made with commonly used protein sources such as pea and soy protein, Rhizopus biomass had a comparable WHC as illustrated in FIG. 5.

[0158] As to the WSI and as illustrated in FIG. 6, Rhizopus biomass has a comparable WSI to the commonly used protein from soybean and chickpea.

[0159] 5.3 Oil Holding Capacity

[0160] The method used for measuring oil holding capacity (OHC) was adapted from Miedziandka et al., with modifications (Miedzianka et al., 2014; https://doi.orq/10.1016/j.foodchem.2014.03.054). 500 mg of dry fungal biomass powder (dried at 45° C. until all water was removed and milled in a ball mill) was added to 15 mL centrifuge tubes (the exact weight was noted down), followed by addition of 5 mL corn oil. The samples were mixed for 30 s using a laboratory vortex mixer on full speed. The samples were then centrifuged (2000 g, 30 min, RT), and the volume of oil separated after centrifugation was determined using a measure cylinder. These values were used to determine the OHC according to the formula used in Miedziandka et al. 2014: [0161] D=Initial volume of oil [0162] E=Final volume of oil separated [0163] F=Weight of sample added

[00002]$OHC=\frac{D-E}{F}$

[0164] As illustrated in FIG. 7 and compared to commonly used protein, Rhizopus fungi biomass has a fairly low OHC, similar to potato and pea protein.

[0165] Experiment 6: Production of Edible Biomass with Increased Vitamin D Content

[0166] 6.1 Preparation of Fungal Biomass

[0167] Upon harvesting, the biomass was subjected to a heat treatment step. The biomass was washed and pressed to remove water. The pressed biomass was cut into small pieces, vacuum-packaged and frozen.

[0168] 6.2 UV Treatment

[0169] For the UV treatment, half-thawed Rhizopus biomass was spread in a 2 cm layer onto the surface of a UVP Transilluminator PLUS (Jena Analytik) and exposed to UV-B light (302 nm, 25 W) at setting “mild”. The biomass pieces were covered with aluminium foil and exposed for 10 min and mixed to ensure even exposure. This was repeated every 10 min for 45 min in total. As control, an unexposed sample was included in subsequent analysis. Samples were frozen again and sent to an external lab for analysis of vitamin D2 and D3 content.

[0170] 6.3 Vitamin D Content Results

[0171] The UV-exposed samples and reference (non-exposed samples) were analysed for Vitamin D2 and Vitamin D3 content. The UV-treated samples reported a 1878-fold improvement in Vitamin D2 levels compared to the non-exposed samples. (Table 2) The protein content of the biomass was not affected by the UV treatment.

TABLE-US-00002 TABLE 2 Vitamin D2 and D3 contents of UV-exposed Rhizopus biomass. Sample Vitamin D.sub.2 (μg/kg) Vitamin D.sub.3(μg/kg) Reference 32.3 <12.5 UV-treated samples 60 100 <12.5

[0172] Experiment 7: Varying Shear Rate and Profile During Liquid Fermentation

[0173] Evaluating Effect of Different Shear Rates in Bioreactor Mixing

[0174] Aerated bioreactor fermentations were carried out as described in Experiment 1, section 1.4. Fermentation experiments were performed at varying stirrer rotation speeds (150 rpm, 200 rpm and 250 rpm), in which increased stirring speeds created higher shear rates in the fermentation broth. The fermentation was carried out multiple times with varying rotation speeds.

[0175] Samples were then 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.

[0176] Texture Analysis Using a Knife Blade Method

[0177] Samples of solid fungi biomass were prepared as a cuboid shape of 20 mm×10 mm×5 mm (length×width×height) for texture analysis. Texture analysis was carried using a Stable Microsystems TA.TX Plus-C equipped with a Knife Blade (70 mm width×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 1 mm/s until trigger force of 20 g is sensed, which in turn the plot was acquired as the blade moved for 20 mm at 2 mm/s. A curve plot was obtained showing measured Force×Time, and the parameter of Toughness was defined as the total area below the curve in g.Math.s.

[0178] Toughness values varied between 25000 g.Math.s and 10000 g.Math.s and a correlation between higher stirrer speeds (higher shear rate) and lower toughness values was found with an R2=0.748. Results can be seen in FIG. 8.

[0179] Evaluating Effect of Different Mixing Blade Types

[0180] Fungi biomass was grown as described in experiment 2 in a 300 L vessel with a 3-blade propeller stirring system. A second system was also setup with the same conditions but using a Rushton turbine as stirring system. 3-blade propellers provide a low-shear radial mixing while Rushton turbines provide high shear radial mixing. Samples were harvested from the two bioreactors and processed in the same conditions as described in experiments 2 and 3. Samples from the high shear fermentation (Rushton turbine) had a profile observed by a panel of 4 people described as more “grainy”, “crumbly” and having a “shorter bite”, and less meaty than the low shear (3-blade propeller) fermentation samples.

[0181] Texture analysis using the Knife Blade method described above was performed on samples derived from the two different bioreactor systems. The plots of force×blade distance were analyzed and the profile from the two samples were significantly different. The force profile of high shear samples was often a single peak, indicating a brittle material with one single breaking point, while the low-shear samples had often multiple peaks, indicating several pressure points over the blade penetration. The following parameters were measured: “Break force” indicates the force value of the first peak observed in the plot, “Max force” indicated the force value of the highest peak in the plot, “Distance to max force” indicates the blade travel distance from trigger point (trigger of 20 g force) at which Max force is measured, “Toughness” indicates the total positive area of the plot. Results are shown in FIGS. 9A-9C. FIG. 9A shows that for many points in the low shear sample the break force is lower than the max force, while in the high shear these are equal, and the force values are much lower for the high shear samples. This indicated a brittleness and easier breaking in the high shear samples and a longer bite with several pressure peaks in the low shear samples. FIG. 9B shows how the max force distance is much lower for the high hear samples, meaning that high shear samples not only break easier, they break sooner. The toughness values in FIG. 9C are also much higher or lower-shear samples, meaning there is a much higher chewiness when biting for low shear samples. These results indicate lower shear fermentation such as by using propeller blades is more suitable to produce meat-like products since the bite profile is much more favourable for these products.

[0182] Experiment 8: Use of Different Pressing Conditions with Salinity and pH

[0183] Fungi biomass was produced as explained in experiment 2. Prior to the pressing step and after the washing step, the biomass was incubated for 30 minutes in just water or water 1% or 5% NaCl concentrations, or pH values of 3.0, 6.0, 9.0 or 11.7. The pH was adjusted by addition of NaOH or HCl. The incubated biomass was then placed in a hydropress system which applied a directional radial pressure so that the samples were pressed in a single plane and could flow/expand in a perpendicular direction to the force applied. The press was allowed to reach 0.5, 1.0 or 2.0 bar and held at the intended pressure for 5 minutes.

[0184] In parallel, to compare with the texture of meat, pieces of chicken breast (chicken) and pork chop (pork) were boiled in boiling water for 25 minutes and then allowed to cool down to room temperature. To compare with the texture of another mycoprotein “chicken-like bites” available in the market, a sample of “Quorn bites” (Marlow foods) was bought from a retailer and thawed to room temperature. Samples of fungi biomass, meat and commercial mycoprotein samples were then prepared and analysed by texture analysis using the Knife Blade method as described in Experiment 7.

[0185] FIG. 10 illustrates toughness values of three different fungi biomass samples according to the present invention, one being incubated in water, one being incubated in 1% NaCl and one being incubated in 5% NaCl prior to a respective pressing step at 2.0 bar. The results shown in FIG. 10 show that addition of 1% NaCl reduces the toughness of the sample significantly, while 5% NaCl produces a sample that has toughness values close to boiled chicken breast. All these samples were tougher than the commercial mycoprotein product. The effect of salt is thought to be related to variations of osmotic pressure in the cells that results in a different level of turgency pressure, therefore affecting product toughness.

[0186] FIG. 11 illustrates toughness values of three different fungi biomass samples according to the present invention pressed at 2.0 bar and with respective pH value of 3.0, 6.0 and 9.0 prior to pressing. The samples at different pH values also shown significant differences, in which high pH values is associated with lower toughness. Samples at pH 9.0 had a toughness closest to the meat samples.

[0187] The water content of all samples above was measured, which is shown in FIG. 12. Samples at pH 9 and 11.7 showed much higher water content, and were also perceived as softer. This indicates changes in pH affect the water holding capacity of the mycelium and this can be used to retain more water, resulting in juicier products, but also having a softer texture. Increases in pH is therefore beneficial for meat-like characteristics such as increased juiciness and adequate toughness.