SE2151489A1

SE2151489A1 - A food product or food ingredient comprising fungi biomass with an increased intracellular fat content

Info

Publication number: SE2151489A1
Application number: SE2151489A
Authority: SE
Inventors: Otero Carlos Nunez; Teixeira Paulo Gonçalves; Nair Ramkumar Balachandran
Original assignee: Mycorena Ab
Priority date: 2021-12-07
Filing date: 2021-12-07
Publication date: 2023-06-08
Also published as: EP4444103A1; WO2023104906A1

Abstract

The present disclosure pertains to a food product or food ingredient comprising fungi biomass, the fungi biomass comprising fungal cells which comprises intracellular fat and/or oil in the form of droplets or accumulations such that within the range from 20% to 90% of the total volume of the fungal cells constitutes of the intracellular fat or oil. The present disclosure furthermore pertains to methods of preparing the food product or the food ingredient.

Description

A FOOD PRODUCT OR FOOD INGREDIENT COMPRISING FUNGI B|OMASS WITH AN INCREASED INTRACELLULAR FAT CONTENT TECHNICAL FIELD The present disclosure relates to a food product or a food ingredient comprising fungi biomass with an improved fat content and mouthfeel. The present disclosure also relates to methods for preparing the food product or the food ingredient.

BACKGROUND OF THE INVENTION ln recent years, the excessive use of meat as a dietary protein source has come under close scrutiny and received significant negative criticism. Several factors are at play, but the root cause of this movement can be narrowed down to tvvo key components. First, it is apparent that production, distribution and consumption of meat leads to substantial negative climate impact. Livestock rearing not only emits massive quantities of greenhouse gases due to its excessive use of land, water and resources, but also contributes to deforestation, biodiversity loss, eutrophication, and a range of other climate- related issues. Second, excessive consumption of animal-based protein is associated with a range of detriments to health and wellbeing that include but are not limited to higher prevalence of obesity, and elevated risks of cancer and cardiovascular disease. ln addition, the unsustainable practices that prevail in many parts of meat and dairy manufacturing contribute to increased risks of zoonosis as well as antibiotic resistance. ln recent years, these issues have led to a heavily increased demand for meat-resembling food products ('meat replacements') comprised of protein sources of non-animal origin ('alternative protein'). These forces have spilled over into the segment for fish as well. Consumers are increasingly looking for fish replacements based on alternative protein, despite the fact that production and consumption of fish is arguably not as harmful for the climate or for individual health as meat. The food manufacturing industry has responded by innovating heavily within the area, outputting large quantities of products that are perceived as capable of meeting the emerging needs of the market. Typically, these products are made using plant-based protein sources.

While it is apparent that plant-based protein sources have the potential to perform 2 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. Sources of fat in the alternative protein industry are scarce compared to traditional meat industry. Money and effort are invested in the development of new textures that resemble the texture of meat. The process of extrusion is common within the alternative protein food industry and contributes to the formation of meat analogue structures. However, during extrusion, the protein is subjected to high temperatures and pressure, and if fats are included pre- extrusion the process will degrade fats leaving the food product with an undesired aftertaste and degraded unsaturated fat content.

Most plant fats that have beneficial mouthfeel properties for the creation of meat analogues are composed mostly of saturated fatty acids. These fatty acids are unhealthy, as opposed to unsaturated, omega3-rich fatty acids, which have health benefits. Sources of omega3 are scarce, and this trend will only be further decreased in the future. This is particularly important for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), only found in marine environments. These essential omega3 fatty acids can only be consumed by diet supplements and/ or fish. Availability of fish in the future is uncertain due to overfishing and bad practise, making sources of these oils very valuable. ln addition to this, omega3 fatty acids are easily degraded upon heating, up to 60% loss in cooked fish. Plant-based proteins can only be coated by these fats, and since the cooking process generates high temperatures, they are degraded.

There is thus still need for a food product with a non-animal based, high-nutritious and environmentally friendly protein source which has a taste and texture resembling a wide variety of meat- and fish-based food product types, with improved fatty mouthfeels and nutritional contents. ln view of the above, the object of the present disclosure to provide an improved food product or food ingredient and methods for producing the same.

SUMMARY OF THE INVENTION One or more of the above objects may be achieved with a food product or food ingredient in accordance with claim 1, a method for producing the food product or food ingredient in 3 accordance with claim 15 or 16. Further embodiments are set out in the dependent claims and in the following description.

A food product or food ingredient as disclosed herein comprises fungi biomass. The fungi biomass comprises fungal cells comprising intracellular fat and/or oil in the form of droplets or accumulations such that within the range from 20% to 90% of the total volume of the fungal cells constitutes of the intracellular fat or oil.

The present invention is based on the discovery that mycoprotein enriched with fats that accumulate intracellularly provides a meat and/or fish like texture which fat can be preserved from the heat generated in process like extrusion or cooking of the food product. This also may also guarantee the delivery of the healthy omega3 fatty acids.

The total cell volume is, as described herein, calculated by image analysis. At least 10 cells are randomly selected, and the total area of each fungal cell is measured, the area occupied by fat/oil droplets is also measured, and the percentage occupied by the fat/oil droplets is calculated.

Optionally, the fungi biomass comprises fungal cells comprising intracellular fat and/or oil in the form of droplets or accumulations such that within the range from 20% to 70% of the total volume of the fungal cells constitutes of the intracellular fat or oil, such as 25% to 68% of the total volume of the fungal cells.

Omega3 fatty acids are essential nutrients which the human body cannot produce and can only be consumed by diet supplements and/ or fish. Sources of omega3 are scarce, and this trend Will only be further decreased in the future. This is particularly important for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), only found in marine environments. Availability of fish in the future is uncertain due to overfishing and bad practise, making sources of these oils very valuable. ln addition to this, omega3 fatty acids are easily degraded upon heating, up to 60% loss in cooked fish. Plant-based proteins can only be coated by these fats, and since the cooking process generates high temperatures, they are degraded. A fish replacement capable of retaining and protect the omega3 fatty acids is a great step towards better accessibility to essential nutrients. This may be provided with a food product or food ingredient according to the present 4 disclosure where the fat and/or oil is incorporated into the fungal cells and thus protected during the cooking process.

The fungi biomass may comprise fungal cells comprising intracellular fat and/or oil in the form of droplets or accumulations such that within the range from 25% to 90% of the total volume of the fungal cells constitutes of the intracellular fat or oil, optionally within the range from 30% to 90% of the total volume of the fungal cells constitutes of the intracellular fat or oil, such as within the range from 35% to 90% of the total volume of the fungal cells constitutes of the intracellular fat or oil. The fungi biomass may comprise fungal cells comprising intracellular fat and/or oil in the form of droplets or accumulations such that within the range from 25% to 80% of the total volume of the fungal cells constitutes of the intracellular fat or oil The fungal cells may have an intracellular fat content within the range of from 4 wt.% to 60 wt.%, based on the weight of the total fungi biomass. The fungal cells may have an intracellular fat content within the range of from 5 wt.% to 60 wt.%, based on the Weight of the total fungi biomass. The fungal cells may have an intracellular fat content within the range of from 7 wt.% to 60 wt.%, based on the weight of the total fungi biomass. The fungal cells may have an intracellular fat content within the range of from 9 wt.% to 25 wt.%, based on the total fungi biomass. The fungal cells may have an intracellular fat content within the range of from 10 wt.% to 60 wt.%, based on the weight of the total fungi biomass.

The fungi biomass may be obtained by submerged fermentation where the oil/fat is dispersed in the liquid media or by incubating the fungi biomass, obtained by submerged fermentation, in liquid media with the oil or fat dispersed therein. This provides a more evenly distributed fat and/or the oil in the fungi biomass and the excess fat/oil Which have not penetrated the fungal cells may be washed away, thereby ensuring that extracellular fat/oil is not left within the structure providing an undesired aftertaste upon cooking of the fungi biomass food product or food ingredient.

The droplets or accumulations of fat and/or oil may comprise lipophilic sensory-enhancing molecules, such as lipophilic flavours and lipophilic taste enhancers. As the fungal cells comprises oil/fat accumulated therein, lipophilic molecules which have previous been difficult to integrate with the fungal biomass structure may now advantageously be incorporated therein together with the oil/fat and be protected by the cell Walls of the fungal cells. Examples of such lipophilic flavours are compounds that resemble smoked taste, butter mint, cherry, mango, ginger, peanut, praline, smoked coconut, chicken, beef, pork, fish, animal fat, and overall, any flavour enhancers soluble in lipids.

The fat and/or oil may comprise or consist of one or a combination of the following oils/fat; canola oil, waste canola oil, linseed oil, sunflower oil, olive oil, waste olive oil, coconut fat, shea butter, peanut oil, corn oil, soybean oil, sesame oil, palm oil, echium oil, evening primrose oil, borage oil, mustard oil and walnut oil.

The fat and/or oil may have a fish origin, an algal origin and or is of animal origin or has a chemical composition and structure analogue to animal fat.

Optionally, 0.1 wt.% or more of the intracellular fat content is from Eicosapentaenoic Acid (EPA). Optionally, 0.2 wt.% or more of the intracellular fat content is from Eicosapentaenoic Acid (EPA). Optionally, 0.3 wt.% or more of the intracellular fat content is from Eicosapentaenoic Acid (EPA). Optionally, 0.5 wt.% or more of the intracellular fat content is from Eicosapentaenoic Acid (EPA). Optionally, within the range of from 0.1 wt.% to 5 wt.% of the intracellular fat content is from Eicosapentaenoic Acid (EPA).

Eicosapentaenoic Acid (EPA) is an omega-3 fatty acid obtained in the human diet by eating oily fish or fish oil and various types of edible algae. EPA may for example reduce symptoms of depression and help to prevent and reduce inflammation in the body.

Optionally, 0.1 wt.% or more of the intracellular fat content is from Docosahexaenoic Acid (DHA). Optionally, 0.2 wt.% or more of the intracellular fat content is from Docosahexaenoic Acid (DHA). Optionally, 0.3 wt.% or more of the intracellular fat content is from Docosahexaenoic Acid (DHA). Optionally, 0.5 wt.% or more of the intracellular fat content is from Docosahexaenoic Acid (DHA). Optionally, within the range of from 0.1 wt.% to 5 wt.% of the intracellular fat content is from Docosahexaenoic Acid (DHA).

Omega-3 [(n-3)] fatty acids have been linked to healthy aging throughout life. Fish-derived omega-3 fatty acids EPA and DHA have been associated with fetal development, cardiovascular function, and Alzheimer's disease. However, because our bodies do not efficiently produce some omega-3 fatty acids from marine sources, it is necessary to 6 obtain adequate amounts through alimentation. Studies have shown that EPA and DHA are important for proper fetal development, including neuronal, retinal, and immune function. EPA and DHA may affect many aspects of cardiovascular function including inflammation, peripheral artery disease, major coronary events, and anticoagulation. lt is therefore a desire to be able to prepare fish replacement products having corresponding health benefits as. With the present invention with the fat/oil being inside the fungal cells, a fish replacement product or ingredient may be obtained which is capable of retaining and protect the omega-3 fatty acid during cooking.

The fat and/or oil may be a triacylglycerol, diacylglycerol, monoglycerol and/or fatty acid. Triacylglycerol is the major form of dietary lipid in fats and oil, whether derived from plants or animals An omega-3 content of the fungal cells may be within the range of from 6 wt. % to wt. 21 % of the total volume of the fungal cells.

The intracellularfat and/or oil may have a melting point of 10°C or less, optionally 5°C or less or optionally 0°C or less.

The fungi biomass may be from a genus selected from one or more of the following; Rhizopus, Neurospora, Aspergi/lus, Trichoderma, Pleurotus, Ganoderma, Inonotus, Cordyceps, Usti/ago, Tuber, Fusarium, Pennici/lium, Xylaria, Trametes, or any combination thereof.

The fungi biomass may be from the species Aspergi/lus oryzae, Rhizopus oryzae, Fusarium graminareum, Cordyceps mi/itaris, Cordyceps sinensis, Tuber melanosporum, Tuber magnatum, Pennicillium camemberti, Neurospora intermedia, Neurospora sitophi/a, Xylaria hypoxion, or any combination thereof.

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, 7 oat flours, apple extract and sources of fiber such as pea, sugarcane, wheat, cellulose, oats and apple.

The food product may be in the form of a patty, nugget, burger, sausage, paste, chunk, fi||et, extrudate, granules, cake, meat substitute, meat extender, jerky, fish-like product, seafood-like product, snack, beverage, dessert or baked good, or the like. The fungi biomass comprising fungi of a species belonging to the genus Rhizopus has been found by the present inventors to provide a non-animal based food product with a taste, smell and consistence with a high resemblance of meat-based food products but without the negative impact on the climate provided by meat-based products.

A method for preparing a food product or food ingredient 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 while stirring, the liquid substrate media comprising oil and/or fat to an amount within the range of from 1 wt.% to 10 wt. %, optionally within the range of from 1 wt. % to 8 wt.%, or within the range of from 1 wt.% to 6 wt.% or within the range of from 1 wt.% to 5 wt.%, based on the total liquid substrate media, to obtain a fungi biomass comprising fungal cells comprising intracellular fat and/or oil in the form of droplets or accumulations such that within the range from 20% to 90% of the total volume of the fungi biomass constitutes of the intracellular fat or oil; b) separating the fungi biomass obtained from step a) from the liquid substrate media, optionally washing the fungi biomass with water; c) optionally heat treating the fungi biomass; and d) dewatering the fungi biomass obtained from step b) or c), such as by pressing or centrifuging.

An alternative method for preparing a food product or food ingredient according to the present disclosure, comprising the steps of a) cultivating fungi under aerobic submerged fermentation conditions using a closed fermentation vessel with liquid substrate while stirring to obtain a fungi biomass comprising fungal cells; b) separating the fungi biomass obtained from step a) from the liquid substrate media to obtain a fungi biomass, and optionally washing the fungi biomass with water; 8 c) incubating the fungi biomass obtained from step b) with a liquid media, the liquid media comprising oil and/or a fat to an amount within the range of from 1 wt. % to 12 wt.%, optionally within the range of from 1 wt. % to 10 wt.%, such as within the range of from 1 wt. % to 8 wt.%, or within the range of from 1 wt. % to 6 wt.%, based on the total liquid media, during a time period of from 5 hours or more at a temperature within the range of from 5°C to 45°C, optionally within the range of from 5°C to 35°C, to obtain a fungi biomass comprising fungal cells comprising intracellular fat and/or oil in the form of droplets or accumulations such that within the range from 20% to 90% of the total volume of the fungi biomass constitutes of the intracellular fat or oil; d) separating the fungi biomass obtained from step c) from the liquid media, and optionally washing the fungi biomass with water; optionally heat treating the fungi biomass; and f) dewatering the fungi biomass obtained from step c) or d), such as by pressing or centrifuging. lt has surprisingly been found by the present inventors that the amount of oil present in the liquid medium in the respective method is crucial to obtain a product according to the present invention. A too low amount of oil will be consumed by the fungi biomass as a substrate and too much oil may surprisingly lead to the fungi death, this is believed to be the result of too low oxygen levels for the fungi. Additionally, too much oil in the substrate medium has been seen to reduce the distribution of the oil/fat in the liquid while shaking, resulting in lower accessibility of the fungi to the oil and lower uptake into the fungi cells. Even more surprisingly it was found that a lower percentage of fungi biomass were obtained when using too high amounts of oil.

The fungi are cultivated in an aerobic submerged fermentation process, i.e., the process carried out in step a), may take place in a stirred-tank reactor bioreactor, airlift reactor or bubble column reactor, where the liquid medium is agitated by aeration and/or stirring. ln the alternative method according to the present disclosure, the fungi biomass may be incubated in step c) during a period of from 15h or more, optionally during a period of time 20h or more, optionally within the range of from 15h to 30h. 9 ln the alternative method according to the present disclosure, the fungi biomass may be incubated in step c) at a temperature within the range of from 5°C to 15°C. ln the alternative method according to the present disclosure, the incubation step c) may be provided under low level oxygen conditions in a presence of less than 30% of oxygen, optionally less than 20% of oxygen, as measured in the liquid media. ln the alternative method according to the present disclosure, the incubation step c) may carried out in an anaerobic environment and in the absence of light.

The fungi biomass may be from a genus selected from one or more of the following; Rhizopus, Neurospora, Aspergi/lus, Trichoderma, Pleurotus, Ganoderma, Inonotus, Cordyceps, Usti/ago, Tuber, Fusarium, Pennici//ium, Xylaria, Trametes, or any combination thereof.

The fungi biomass is from the species Aspergi/lus oryzae, Rhizopus oryzae, Fusarium graminareum, Cordyceps militaris, Cordyceps sinensis, Tuber me/anosporum, Tuber magnatum, Pennici//ium camemberti, Neurospora intermedia, Neurospora sitophi/a, Xylaria hypoxion, or any combination thereof.

The food product may be mixed or 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, oat flour, apple extract and sources of fibers, such as pea, sugarcane, wheat, cellulose, oats and apple.

The process may include a blending step with binders, protein powders, textured proteins, fresh proteins, flours, fats, gluten, egg.

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 methods may comprise a step of preparing a final food product or a final food ingredient comprising the fungal biomass.

The oil or fat added into the liquid substrate media in step a) in the first method or wherein the oil or fat added into the liquid media in step c) in the alternative method may comprise lipophilic sensory-enhancing molecules, such as lipophilic flavours and lipophilic taste enhancers. The oil or fat added into the liquid substrate media in step a) in the first method or wherein the oil or fat added into the liquid media in step c) in the alternative method may have a melting point of 10°C or less, optionally 5°C or less, or optionally 0°C or less.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graph illustrating the results from a textural analysis comparing toughness of fungi biomass sample with or without canola oil; Fig. 2 is a graph illustrating the results from a textural analysis comparing hardness of fungi biomass sample with or without canola oil; Fig. 3 illustrates photos of fungi biomass Where the floatability of the fungi biomass is compared; Fig. 4 illustrates a heat map showing the quantification of the floatability of the fungi biomass; Figs. 5A/5B show confocal pictures of intracellular fat deposits obtained from a light microscope of oil droplets in emulsions according to the present disclosure; 11 Fig. 6 shows confocal imaging taken from fungi not incubated with oil (negative control), fungi incubated with canola oil for Oh or 6h, fungi grown with canola oil for 24h and the same fungi grown for 24h heat treated; Fig. 7 shows a graph illustrating the quantification of the confocal images from Fig. 6; Fig. 8 shows confocal imaging taken from fungi incubated with coconut or sunflower oil; and Fig. 9 shows a graph illustrating the quantification of the confocal images from fungi incubated with different types of oils: canola, canola and olive, coconut, sunflower, linseed, cod liver, algae or lard DETAILED DESCRIPTION The present disclosure relates to a fungal biomass enriched with fats that are accumulated inside the fungal cells. The accumulation of the fat/oil within the fungal cells is achieved by incubating fungal biomass with different types of oils with incubation conditions as disclosed herein. The fungal biomass generated after the oil incubation was 2 to 10-fold fattier than the original fungal biomass and the fatty acid profile was similar to the fatty acid profile of the oil that the fungal biomass was incubated with (Example 1). Hence, the fatty acid profile was maintained after incubation, thus indicated that the fungi did not degrade the fat and that the fat remained within the fungal structure. The fatty fungal biomass obtained also showed lower toughness and a softer texture, which distinguished it from the original fungal biomass, as illustrated in Example 1 below. These interesting textural features could result in food products with an improved resemblance with fish or meat. lncubation of the fungal biomass at low temperatures is preferred, i.e., 10°C or lower, as high temperatures may create oxidation of oils. This can be prevented by adjusting the conditions at which the oils were incubated with the biomass, reducing the incubation time (from 24h to 6h), temperature (from 35°C to 10 °C) and oxygen levels (below 30%), as illustrated in Example 2. lt was seen that during incubation, the fungal cells took up the fat with which they were incubated when the conditions were according to the methods as described herein. This resulted in new fat deposits that were stored in the fungal cells characterized by globular structures, corresponding to lipid bodies, see Example 3. Treatment of fungal biomass under these conditions results in a product 12 with very favourable levels of healthy omega3 fatty acids (up to 7.7% EPA and 9.3% DHA) as shown in Example 4, and very low ratio of omega3/omega6 (0.33 to 2.85).

The general procedure of producing this biomass in the Examples disclosed herein was the following: o Plate cells of fungi from genus Rhizopus onto PDA plates and incubate at 35°C for 3 days. o Resuspend the spores in 10 mL sterile water per plate. o Prepare pre-cultures in rich corn media (Table 1) by adding spore suspension (0.6% of total volume). o lncubate at 35°C while shaking (120 rpm) for 24h. o lnoculate of a 20L bioreactor with 10% fungal preculture. Synthetic medium composition is shown in table 1. lf no oil is added throughout the fermentation, more sugar is added. o Addition of 4% canola oil throughout the fermentation (optional). o lncubate for 24h, with stirring speed of 200 rpm, air inlet at 14 L/min and addition of ammonia to regulate the pH to 4.5. o Harvest of the resulting biomass and wash with water. o lf the biomass was not incubated with oil during fermentation, incubate with canola oil for 6h at 10 - 35°C. Optionally, other vegetable oils can be used instead of canola: waste canola oil, linseed oil, sunflower oil, olive oil, waste olive oil, coconut fat, shea butter, peanut oil, corn oil, soybean oil, sesame oil, palm oil, echium oil, evening primrose oil, borage oil, mustard oil, walnut oil. o Harvest again and wash with water. o Heat treatment for 10 min at 70°C (optional). o Press at 2 bar for 5 min to dewater the biomass prior to fat analysis (optional).

However, the fungi biomass may of course be produced by any method as disclosed herein.

The fungal biomass produced by this procedure was rich in healthy fatty acids (unsaturated fatty acids 74.7 - 90.1%) and these fatty acids can be protected from the external environment by the fungal cell walls. 13 The described invention can be used to include beneficial fatty acids such as omega-3, in particular EPA and DHA in a vegan or vegetarian product and confer heat protection capacities to these fatty acids preventing heat-enabled degradation. ln addition to healthy fatty acids, incorporation of other fat-soluble molecules of interest can be achieved. These can be compounds with unique properties, antioxidants, pigments, or molecules that provide unique nutritional properties which are unstable or have low bioavailability on their own, but that can be incorporated by the fungi. Some examples are curcumin, CBD or CBD alternatives, lutein, resveratrol or astaxanthin. Moreover, sensory-enhancing molecules can be incorporated in the same way such as lipophilic flavours and lipophilic taste enhancer compounds. These can be added to the fats which serve as carriers of these lipophilic compounds to the intracellular environment.

Such flavour integration allows for a release of the flavours over time during cooking and chewing as opposed to a quick release if included in the outside of the fungi by simple addition.

Example 1: Macrostructural description of fat mvcoprotein Fungal biomass enriched with fat was produced according to the general description in the previous section, briefly as follows. Spore suspensions were created from PDA plates of Rhizopus sp, and used to inoculate (0.6% of total volume) shake flasks previously filled with corn pre-culture media (Table 1). ln a sample 1, incubation proceeded for 24h at 35°C while shaking (120 rpm), then it was used to inoculate (10% of total volume) a 20L working volume bioreactor (Techfors lnfors) filled with sterile synthetic media (Table 1, middle) and 4% canola oil. lncubation proceeded for 24h with stirring speed of 200 rpm, air inlet at 14 L/min and addition of ammonia to regulate the pH to 4.5. The fungal biomass was then harvested and washed thoroughly with water. The fungal biomass was finally pressed at 2 bar for 5 min and stored frozen at -20°C. ln parallel, the same experiment was performed on a sample 2, using linseed oil instead of canola oil. A third sample was obtained by growing the fungal biomass without oil using the synthetic media composition from table 1 at the right. The fungal biomass was left to grow for 18h, then 4% of canola oil was added to the reactor and incubation proceeded for 6h prior harvesting and washing. The fungal biomass was also pressed at 2 barfor 5 min and stored at -20°C until further use.

Table 1. Different growth media composition. 14 com pre-culture Synthetic medium Synthetic medium (fermentation with oil) (fermentation without oil) (NH4)2SO4 1 g/L (NH4)2SO4 10 g/L (NH4)2SO4 10 g/L KH2PO4 0.4 g/L KH2PO4 5.25 g/L KH2PO4 5.25 g/L Corn flour 20 g/L lVlgSO4-7H2O 1.06 g/L lVlgSO4-7H2O 1.06 g/L Yeast extract 3 g/L CaCl2 1.75 g/L CaCl2 1.75 g/L Sucrose 20 g/L Sucrose 35 g/L The fat content and fatty acid profile of the fungal biomass incubated with oil was evaluated, see Table 2 below. To determine it, 100g of biomass was sent to Eurofins Environment Testing AB, Lidköping, Sweden, Which measured the fat content by NMKL 160 method. An increase of almost 10-fold (2.25g to 23.2g) was observed when incubating the biomass with oil throughout the fermentation, while the fat content was lower if the biomass Was incubated forjust 6h (14g). The fatty acid profile of the biomass was also evaluated by the Eurofins Environment Testing AB using GC-FID. The profile of the fungal biomass was adapted to the one of the oils incubated with, and the omega3 levels in the biomass were highly increased, reducing the omega6/ omega3 ratio from 167.67 to 2-3 in canola or 0.33 in linseed oil. These results indicate that our process to produce fatty mycoprotein increase the fat content 5 to 10-fold and adapts the fatty acid profile of the fungi to the oil used during the incubation.

Table 2. Fat content and omega 6/3 levels of biomass incubated with canola, linseed or no oil. Fatty acid Omega 3 Omega 6 Ratio Saturated Unsaturated content (% of total (% of total omega 6/ fatty acids fatty acids (gl 100 g) fat) fat) omega 3 No oil 2.52 g 0.3% 56% 167.67 23.8% 74.9% Sample 1 23.2 g 9% 192% 2.13 8% 90.1% Canola oil (24h) Sample 2 22.5 g 47.8% 15.8% 0.33 10.3% 81 .8% Linseed oil (24h) Sample 3 14 g 7.7% 22% 2.85 11% 88.1% Canola oil (6h) A texture analyzer (TA.XTp|us from Stable Micro Systems (U K)) was used to evaluate properties of fungal biomass samples with or without canola oil (24h). Two different tests, knife blade (single compression/cutting) and TPA (double compression test using plate probe) were performed in order to evaluate the textural properties of the selected samples.

For the knife-blade test fungal biomass samples were taken from the freezer and left to defrost for about 30 minutes until half defrosted. The samples were cut into stripes with an average height and thickness of 5 and 10 mm respectively and were left to defrost completely before testing. For the texture profile analysis (TPA) measurements the mycoprotein samples were taken from the freezer and left to defrost for about 30 minutes until half defrosted. The samples were cut into cubes with a length and height of 20 and 5 mm respectively and were left to defrost completely before testing.

For the knife blade test the Promyc strip was placed centrally on the slit of the base plate. The blade was cutting through the sample at a rate of 2 mm/s and the maximum peak force (g) giving information about the hardness of the sample and the area under graph (g*s) being the work of compression and giving information about the toughness of the sample was measured. The test was repeated at least 5 times per sample. For the TPA test a flat base and a flat probe was used to measure the texture properties of the sample. The sample was placed at the centre of the base and was compressed twice with a compression speed of 5 mm/s. lt was used in strain-mode and the compression strain was set to 60 %. The hardness (g) of the samples were recorded and the test was repeated at least 5 times per sample.

The first observable difference was that the fungi biomass enriched with fat showed lower positive area and lower abs. positive force, which indicates lower toughness as illustrated in Fig. 1. The fungi biomass samples enriched with fat were also much softer than regular fungi biomass, as evidenced by measuring the hardness in g, as illustrated in Fig. 2 This texture enables improved preparation of different food products that require a fattier texture, such as fish and meat replacements.

Example 2: Reduction of the oil oxidation levels When preparing fungi biomass enriched with fat there may be an increased risk of oxidation if fat/oil remains extracellularly or if the fat/oil is oxidized prior to absorption of 16 oil/fat into the fungal cells. Oxidation of the oils may result in off flavours, which is not desired in the final product. To overcome this problem, the process of incubation of the fungi biomass with the oil was improved. The fungi biomass was harvested after the fermentation process was complete, then transferred to 6X shake flasks and incubated with canola oil at 10°C while shaking (120 rpm) in absence of light and with hypoxic conditions. Oxygen reduction was achieved by introducing an anaerobic environment generation bag (AnaerocultTM P, 1323820001, SigmaAldrich) and each shake flask was isolated with two layers of parafilm during the oil incubation process. Oxygen reduction was observed by colour change in AnaerotestW' Strips (1 .32371, SigmaAldrich). Oxygen reduction was also monitored using a DOT sensor and evaluating the dissolved oxygen levels every 2h for 6 hours, as illustrated in Table 4 below.

The reduction of the oil oxidation was proved by the total oxidation method (Totox), as may be seen in Table 3. Totox analysis were performed by an external company by titrimetry (peroxide value) and spectrophotometry (p-anisidine value). The oil showed some oxidation when it was added throughout the fermentation, such as in sample 1 and sample 2. The level of oxidation was drastically reduced by lowering the temperature of the oil incubation from 35°C to 10°C, according to sample 3. The sample 3 still presented an aftertaste when evaluated by a panel. This aftertaste disappeared when the oil was incubated at hypoxic conditions according to sample 4, likely caused by the reduction of the anisidine levels to less than 0.5.

Table 3. Different conditions to reduce the total oxidation (Totox) of canola oil.

Conditions for canola oil Anisidine Peroxide Totox Total fat incubation Sample 1 Bioreactor for 24h at 35°C 1.6 0.7 3 22.8% with air (14 L/ min) Sample 2 Bioreactor for 6h at 35°C, no 4 2 8 14% air Sample 3 Shake flasks for 6h at 10°C, 2.1 0.8 3.7 nd no air Sample 4 Shake flasks for 6h at 10°C, <0.5 0.9 1.8 11.3% no air, hypoxic environment 17 Table 4. Levels of dissolved oxygen with time using an anaerobic environment generation bag, in a shake flask, i.e., each shake flask was isolated with tvvo layers of parafilm during the oil incubation process.

Time Dissolved oxygen (h) (%) 0 20-21 2 4-4.8 4 3.1 6 3.1 Example 3: Lipid droplet accumulation within funqal cells Fungi biomass was grown as described in example 1, with the exception that only canola oil was used and samples were taken right after adding the oil (Oh) or at the end of the incubation with oil (6h). Fungi biomass was also grown in presence of oil for 24h as shown in example 1. The fungi biomass generated was evaluated to determine the fat concentration in the fungal filaments. By taking samples at different times during the oil incubation, an increase in the fat levels of the fungi was observed by two methods; floatability and microscopy. ln the floatability experiments, 100 g of fungi biomass grown by the example 1 procedure in presence of canola oil was selected (24h). As a control, 4% of canola oil was added to 100 g of fungi biomass grown without oil (Oh). The fungi biomass from the two different conditions were added to a respective 2L vessel filled with water up until 1.5L and left for 5 minutes before taking pictures illustrated in Fig. 3. Quantification of the intensity of the signal every 100 mL was achieved using the software imageJ and plotted into a heatmap, illustrated in Fig. 4, showing the percentage of the intensity of the signal relative to the different fractions of a 1.5L glass test tube. Quantification of the percentage of signal on the top and bottom fractions was performed.

The fungi biomass incubated for 24h with canola oil showed high floatability, with 61% of the biomass located in the top fraction of the vessel as may be seen in Fig. 4. The sample to which oil was added prior harvesting (Oh) did not show floatability (just 36% in the top fraction). These results indicate that oil uptake increases with longer incubation times. 18 To determine the location and quantity of the fat deposits, the samples were investigated microscopically using a LSM 700 inverted confocal microscope (Carl Zeiss). The same fungi biomass used for floatability experiments was stained with Nile red (72485, SigmaAldrich), a fluorophore commonly used to stain neutral lipids in cells, and counterstained with calcofluor white (18909, SigmaAldrich) which binds to fungal cell walls (rich in chitin). Staining with Nile red proceeded for 1h at 0.5 ug/ mL while shaking at room temperature. The remaining sample was washed with water twice, then transferred to a coverslip, where the samples were covered with 40 ul of 0.01% calcofluor white. Then, a drop of Neo-Mount (1 .09016.0500, Merk) was placed on top of the samples, and they were covered with a coverslip prior observation. Confocal pictures illustrated that the fat deposits were localized inside the fungal cells, see Figs. 5A and 5B, with Fig. 5B being a zoom from Fig. 5A. At the left image, calcofluor white (CW) is used for staining of the fungal cell walls, while at the right there is Nile red (NR) stained lipid droplets. White arrows in 5B point at lipid deposits in the cells.

The percentage of occupancy of lipids within the fungal cell was determined by calculating the area of the droplets and normalize to the total filament area in at least 10 fungal cells randomly selected. The results showed that 33% to 67% of the volume of the fungal cell are comprised of lipid droplets, which proved that the fat accumulates inside the fungal cells.

Fig. 6 shows confocal images taken on fungi biomass incubated with canola oil according to the present disclosure with the images taken from samples incubated at Oh, at 6h, and after 24h. After collection of the samples at the respective period of time, the samples were stained with calcofluor white (CW), showing the fungal cell walls (left images), while at the right the Nile red (NR) stained lipid droplets could be observed inside the cells. The images were then analyzed using imageJ by quantifying the fluorescent signal correspondent to the lipids stained with Nile red in at least 20 different filaments per condition, as shown in Fig. 7, which provides reliable and representative measurements. Z-stacks were transformed into a single picture per condition. Statistical significance was assessed by Mann-Whitney U test, * indicates p<0.05, ** p<0.01 and *** p<0.001. There was a higher lipid concentration in samples in which oil was added (Oh) compared to the negative control (no oil), indicating that direct addition of the lipid, in the amounts as disclosed herein, was enough to cause a small degree of lipid absorption. lt should 19 however be noted that the staining procedure involves 1 hour incubation with the Nile Red staining, hence, this may have an impact in the result.

A significant difference in the lipid accumulation was seen when the samples were incubated for 6h compared to the Oh sample. This difference was even higher between 6h and 24h incubation times. The Oh sample was significantly lower than the sample that was grown for 24h in presence of oil. These results indicate that a longer incubation with oils leads to higher uptake levels of lipids. ln addition, the 24h sample was heat treated for 10 minutes at 70°C and the lipid accumulation was quantified after. There was no difference between the samples before or after heat treatment, indicating that the lipids could be protected from the heat treatment within the fungal cells.

Example 4: Characterization of funqal biomass incubated with different oils Fungal biomass was produced by the method described in examples 1 and 2, with the following exceptions. Different types of oils (4%) were selected for the incubation with the biomass: canola (75%) with olive (25%) oils, coconut oil, sunflower oil, linseed oil, cod liver oil, EPA-rich algae oil (2.3 g diluted in sunflower oil) or animal fat (lard). The incubation conditions were adapted to each oil type, incubating at higher temperatures in oils with high melting point (lard) or lower temperatures to reduce oxidation in oils with high percentage of omega3 (algae, cod liver) (Table 5). lncubation proceeded for 6h in almost all oils, with the exception of canola and linseed oils (24h).

The results showed that a fungi biomass comprising oil/fat accumulated in the fungal cells could be achieved in all cases, although different fat contents resulted from different oil types and incubation conditions, see table 4. Oils with a high melting point (coconut oil, lard) did not result in very fat biomass, and incubation at 35°C in the bioreactor achieved the fattiest mycoprotein, see table 5. The fatty acid profile of the fungi biomass incubated with cod liver oil or EPA-rich algae showed high levels of marine omega3 DHA and EPA compared to other vegetable oils like canola or linseed, see table 6.

Table 5. lncubation conditions and fat content of mycoprotein incubated with different types of oils. Oil type Linseed Coconut Sunflower Canola Cod EPA- Porklard Canola + Liver rich Olive algae Fat content 22.5 5.2 25.2 25.1 9.3 4.9 3.75 23 (gl 100 g) lncubation 24 6 6 6 6 6 6 24 time (h) Percentage 4% 4% 4% 4% 4% 4% 3.5% 4% oil Temperature 35°C 35°C 35°C 35°C 10°C 10°C 42°C 35°C Vessel Reactor Reactor Reactor Reactor Shake Shake Shake Reactor flasks flasks flasks Comments Added Hypoxic Hypoxic Fat obtainedAdded throughout from pork throughout fermentation skin fermentation Lipid droplet accumulation within the cells occurred in all cases, coconut and sunflower oils are shown as examples in Fig. 7. Quantification of the fluorescent signal showed that lipids accumulated within the fungal filaments in a statistically significant way, see Fig. 8. 21 Table 6. Omega3 fatty acid profile and fat content of mycoprotein incubated with different oils. Fat content (gl EPA DHA Ratio omega3l 100g) omega6 Canola oil 23.2 <0.1% <0.1% 2.13 Linseed oil 22.5 <0.1% <0.1% 0.33 Cod liver oil 9.3 7.7% 9.3% 0.35 EPA-rich algae oil 4.9 1.1% <0.1% 34.8 Figure 4. Confocal imaging and quantification of the lipid signal in different samples. A) Confocal images were taken from each condition, and Z-stacks were transformed into a single picture per condition. Nile red (NR) stained lipid droplets. B) Quantification of the signal from confocal imaging of different oils. Two axis were used to show smaller differences between other oil samples.

Example 5: Fat accumulation at different time points To determine the increasing in fat content when incubating for 6h in comparison to 18h, fungi biomass was grown and harvested as stated in example 1 and 2, briefly as follows.

After 24h of fermentation, the fungi biomass was washed and transferred to shake flasks. Cod liver oil at 4% was added to the shake flasks and incubation proceeded in the dark for 6 to 18h at 10°C, in absence of air and shaking (130 rpm) to improve oil dispersion. After the stated hours of incubation, the samples were washed thoroughly with water and evaluated for total fat content by NMKL 160 method. The fatty acid profile was analysed by GC-FID.

The results showed an increase in the fat content from 6h to 18h incubation times of 7.2% (Table 7). The fatty acid profiles were similar at both time-points, indicating that the fat absorbed is not consumed or transformed to other fatty acids. These results also prove the time dependant absorption of the oil by the fungal biomass. 22 Table 7. Fat content and fatty acid profile of biomass Without oil or incubated with cod liver oil for 6h or 18h.

Example 6: Fat saturation of the funqal biomass Fatty Omega Omega Ratio EPA (% DHA (% SaturatedUnsaturated acid 3 (% of 6 (% of omega of total of total fatty fatty acids content total total 6/ fat) fat) acids (gl 100 fat) fat) omega 9) 3 No oil 2.52 g 0.3% 56% 167.67 <0.1 <0.1 23.8% 74.9% Cod 9.3 g 20.8% 7.3% 0.35 7.7 9.3 17.8% 74.7% liver oil (Sh) Cod 16.5 g 20.7% 5.9% 0.29 7.9 9.3 18.3% 74.6% liver oil (18h) The fungal biomass was incubated with canola oil at different percentages to determine the maximum capacity to retain fat. Three different samples of fungal biomass were incubated in liquid media for 24h in the presence of Canola oil. For the first sample the amount of Canola oil in the liquid media was 4 wt. %, for the second sample the amount of oil was 8 wt. % and in the third sample the amount of Canola oil in the liquid media was 12 wt. %.

The samples of fungal biomass (a total of 500 g in duplicates per condition) were incubated at 10°C in water (total volume of 2L) and absence of air (closed systems) with the % of oil stated. Table 8 Fat content (g/100g) Dry matter content (%) Canola 4% 8,4 25,3 Canola 8% 5,84 24,4 Canola 12% 4,11 31,5 23 As illustrated in table 8, it was surprisingly found that the amount of oil accumulated in the fungal cells reduced With an increasing amount of oil in the liquid media at a certain level and that the fat content in the fungal cells were significantly higher for the first sample, at 4 wt.% oil in the liquid media, compared to both the second and third samples.

Claims

1.Claims A food product or food ingredient comprising fungi biomass, the fungi biomass comprising fungal cells characterized in that fungal cells comprises intracellular fat and/or oil in the form of droplets or accumulations such that within the range from 20% to 90% of the total volume of the fungal cells constitutes of the intracellular fat or oil. The food product or food ingredient according to claim 1, wherein the fungal cells have an intracellular fat content within the range of from 4 wt.% to 60 wt.%, based on the weight of the total fungi biomass. The food product or food ingredient according to claim 1, wherein the fungal cells have an intracellular fat content within the range of from 9 wt.% to 25 wt.%, based on the total fungi biomass. The food product or food ingredient according to any one of claims 1 to 3, wherein the fungi biomass is obtained by submerged fermentation. The food product or food ingredient according to any one of the preceding claims, wherein the fat and/or oil comprises or consists of one or a combination of the following oils/fat; canola oil, waste canola oil, linseed oil, sunflower oil, olive oil, waste olive oil, coconut fat, shea butter, peanut oil, corn oil, soybean oil, sesame oil, palm oil, echium oil, evening primrose oil, borage oil, mustard oil and walnut oil. The food product or food ingredient according to any one of claim 1 to 5, wherein the fat and/or oil has a fish origin, an algal origin and or is of animal origin or has a chemical composition and structure analogue to animal fat. The food product or food ingredient according to any one of the preceding claims, wherein 0.1 wt.% or more of the intracellular fat content is from Eicosapentaenoic Acid (EPA). The food product or food ingredient according to any one of the preceding claims, wherein 0.1 wt.% or more of the intracellular fat content is from Docosahexaenoic Acid (DHA). The food product or food ingredient according to any one of the preceding claims, wherein the fat and/or oil is a triacylglycerol, diacylglycerol, monoglycerol and/or fatty acid. The food product or food ingredient according to any one of the preceding claims, wherein an omega3 content of the funga| cells is within the range of from 6 wt.% to 21 wt.%. The food product or food ingredient according to any one of the preceding claims, wherein the fungi biomass is from a genus selected from one or more of the following; Rhizopus, Neurospora, Aspergi/lus, Trichoderma, Pleurotus, Ganoderma, Inonotus, Cordyceps, Usti/ago, Tuber, Fusarium, Pennici/lium, Xylaria, Trametes, or any combination thereof. The food product or food ingredient according to any one of the preceding claims, wherein the fungi biomass is from the species Aspergi/lus oryzae, Rhizopus oryzae, Fusarium graminareum, Cordyceps militaris, Cordyceps sinensis, Tuber melanosporum, Tuber magnatum, Pennici/lium camemberti, Neurospora intermedia, Neurospora sitophila, Xylaria hypoxion, or any combination thereof. The food product or food ingredient according to any one of the preceding claims, wherein the droplets or accumulations of fat and/or oil comprises lipophilic sensory- enhancing molecules, such as lipophilic flavours and lipophilic taste enhancers. The food product or food ingredient according to any one of the preceding claims, wherein the intracellular fat and/or oil has/have a melting point of 10°C or less, optionally 5°C or less. A method for preparing a food product or food ingredient according to any one of claims 1 to 8, comprising the steps of cultivating fungi under aerobic submerged fermentation conditions using a closed fermentation vessel with liquid substrate while stirring, the liquid substrate media comprising oil and/or fat to an amount within the range of from 1 wt.% to 10 wt. %, optionally within the range of from 1 wt. % to 8 wt.%, or within the range of from 1 wt.% to 6wt.% or within the range of from 1 wt.% to 5 wt.%, optionally within the range of from 2 wt.% to 5 wt.% ,based on the total liquid substrate media, to obtain a fungi biomass comprising fungal cells comprising intracellular fat and/or oil in the form of droplets or accumulations such that within the range from 20% to 90% of the total volume of the fungi biomass constitutes of the intracellular fat or oil; separating the fungi biomass obtained from step a) from the liquid substrate media and thereby obtaining a fungi biomass, optionally washing the fungi biomass with water; optionally heat treating the fungi biomass dewatering the fungi biomass obtained from step b) or c), such as by pressing or centrifuging. A method for preparing a food product or food ingredient according to any one of claims 1 to 8, comprising the steps of a. cultivating fungi under aerobic submerged fermentation conditions using a closed fermentation vessel with liquid substrate while stirring to obtain a fungi biomass comprising fungal cells; separating the fungi biomass obtained from step a) from the liquid substrate media to obtain a fungi biomass, and optionally washing the fungi biomass with water; incubating the fungi biomass obtained from step b) With a liquid media, the liquid media comprising oil and/or a fat to an amount within the range of from 1 wt. % to 12 wt.%, optionally within the range of from 1 wt. % to 10 wt.%, such as within the range of from 1 wt. % to 8 wt.%, or within the range of from 1 wt. % to 6 wt.%, such as within the range of from 2 wt.% to 5 wt.% based on the total liquid media, during a time period of from 5 hours or more at a temperature within the range of from 5°C to 45°C, optionally within the range of from 5°C to 35°C, to obtain a fungi biomass comprising fungal cells comprising intracellular fat and/or oil in the form of droplets or accumulations such that within the range from 20% to 90% of the total volume of the fungi biomass constitutes of the intracellular fat or oil; separating the fungi biomass obtained from step c) from the liquid media to obtain a fungi biomass, and optionally washing the fungi biomass with water; optionally heat treating the fungi biomass dewatering the fungi biomass obtained from step c) or d), such as by pressing or centrifuging. The method according to claim 16, wherein the fungi biomass is incubated in step c) during a period of time from 15h or more, optionally during a period of time 20h or more, optionally within the range of from 15h to 30h. The method according to claim 16 or 17, wherein the fungi biomass is incubated in step c) at a temperature within the range of from 5°C to 15°C. The method according to any one of claims 16 to 18, wherein the incubation step c) is provided under low level oxygen conditions in a presence of less than 30% of oxygen, optionally less than 20% of oxygen, as measured in the liquid media. The method according to any one of claims 15 to 19, wherein the incubation step c) is carried out in an anaerobic environment and in the absence of light. The method according to any one of claims 15 to 20, wherein the fungi biomass is from a genus selected from one or more of the following; Rhizopus, Neurospora, Aspergillus, Trichoderma, Pleurotus, Ganoderma, Inonotus, Cordyceps, Usti/ago, Tuber, Fusarium, Pennici/lium, Xy/aria, Trametes, or any combination thereof. The method according to any one of claims 15 to 21, wherein the fungi biomass is from the species Aspergi//us oryzae, Rhizopus oryzae, Fusarium graminareum, Cordyceps militaris, Cordyceps sinensis, Tuber me/anosporum, Tuber magnatum, Pennici//ium camemberti, Neurospora intermedia, Neurospora sitophi/a, Xy/aria hypoxion, or any combination thereof The method according to any one of claims 15 to 22, wherein the fat and/or oil comprises or consists of one or a combination of the following oils/fat; canola oil waste canola oil, linseed oil, sunflower oil, olive oil, waste olive oil, coconut fat, shea butter, peanut oil, corn oil, soybean oil, sesame oil, palm oil, echium oil, evening primrose oil, borage oil, mustard oil and walnut oil. The method according to any one of claims 15 to 22, wherein the fat and/or oil has a fish origin, an algal origin and or is of animal origin or has a chemical composition and structure analogue to animal fat. The method according to any one of claims 15 to 24, wherein the fat or oil added into the liquid substrate media in step a) in claim 15 or wherein the fat or oil added into the liquid media in step c) in claim 16 comprises lipophilic sensory-enhancing molecules, such as lipophilic flavours and lipophilic taste enhancers. The method according to any one of claims 15 to 25, wherein the fat or oil added into the liquid substrate media in step a) in claim 15 or wherein the fat or oil added into the liquid media in step c) in claim 16 has a melting point of 10°C or less, optionally 5°C or less.