SE2250075A1

SE2250075A1 - Fungi-based fat tissue

Info

Publication number: SE2250075A1
Application number: SE2250075A
Authority: SE
Inventors: Anna Kristina Sofia Karlsson; Joan Lluch Casarramona; Paulo Gonçalves Teixeira; Ramkumar Balachandran Nair
Original assignee: Mycorena Ab
Priority date: 2022-01-28
Filing date: 2022-01-28
Publication date: 2023-07-29
Also published as: WO2023144148A1

Abstract

The present document relates to a fungi-based fat tissue comprising fungal biomass, fat and water. The present document also discloses methods for producing such fungi-based fat tissue and products comprising it.

Description

Technical field The present invention relates compositions which can be used to replace fat, such as animal fat, such as animal fat tissue. The compositions comprise fungal biomass, fat and water. The present document also discloses methods for producing such fungal-based fat tissue compositions and food products comprising them.

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

Livestock rearing not only emits massive quantities of greenhouse gases due to its excessive use of land, water and resources, but also contributes to deforestation, biodiversity loss, eutrophication, and a range of other climate-related issues. Second, excessive consumption of animal-based protein is associated with a range of detriments to health and wellbeing that include but are not limited to higher prevalence of obesity, and elevated risks of cancer and cardiovascular disease. ln addition, the unsustainable practices that prevail in many parts of meat and dairy manufacturing contribute to increased risks of zoonosis as well as antibiotic resistance. ln recent years, these issues have led to a heavily increased demand for meat resembling food products ('meat replacements') comprised of protein sources of non-animal origin ('alternative protein'). These forces have spilled over into the segment for fish as well and consumers are increasingly also looking for fish replacements based on alternative protein. The food manufacturing industry has responded by innovating heavily within the area, outputting large quantities of products that are perceived as capable of meeting the emerging needs of the market. Typically, these products are made using plant-based protein SOU FCGS. 106928 While it is apparent that plant-based protein sources have the potential to perform significantly better than meat and fish on factors relating to both nutrition and climate impact, achieving appealing palatability is a challenge. On one hand, creating non-meat and non-fish products that have taste profiles similar to those of meat and fish is difficult. More pressing, however, is the issue of texture. Most raw materials of alternative protein are provided in non-texturized (e.g. as powder) form such as plant protein isolates or concentrates, meaning that several advanced processing steps and extensive use of additives is required to acquire a meat- or fish-like texture.

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

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

However, even with the alternative protein sources that are available, there is still a need for a non-animal based, high-nutritious and environmentally friendly food product which has a taste and texture resembling a wide variety of meat- and fish- based food products. One challenge when preparing such products is how to mimic animal fat that has a consistency, ﬂavour and melting behaviour similar to fat tissue in meat. Animal fat tissue is often a complex structure of fat cells, which are essentially large encapsulated droplets of fat, held together in a structure of collagen. This collagen structure does not melt when heated as it is a proteic structure, and so there is a complex effect of partial melting and partially holding 106928 its structure in animal fats. Adding to this effect, animal fats are mostly saturated, which increases their melting point, but at the same time poses health issues as consumption of saturated fats is a significant contributor to cardiovascular diseases and obesity. Further, it is difficult to obtain a non-animal fat that has an appealing texture, mouthfeel and taste, and even more difficult without using heavily saturated fats.

There is thus a need for fat products which can be used to replace animal-derived fat, such as fat in meat and dairy products. Also, there is a need for fat replacement product which have a healthier composition of fats than e.g. animal fat has.

An object of the present invention is thus to overcome or at least mitigate one or more of the problems described herein.

Summary of invention The herein defined problems are overcome or at least mitigated by one or more of the aspects of the invention as disclosed herein.

The present document relates to a fungi-based fat tissue, said fungal-based fat tissue being an oil-in-water emulsion comprising a fungal biomass of food-safe fungi, one or more of fat(s) and water. The fungi-based fat tissue may comprise from about 40 wt% to about 90 wt% fat, based on the total weight of the fungi- based fat tissue. The fat may be saturated, unsaturated and/or a comprise a mixture of saturated, such as hydrogenated, fats and unsaturated fats. For example from about 30 wt% to about 100 wt% of the fats may be unsaturated, based on the total weight of the fat in the fungi-based fat tissue. The fat may e.g. be selected from the group consisting of canola oil, olive oil, sunflower oil, coconut fat, palm oil, peanut oil, soybean oil, algal oil and shea fat.

The fungi-based fat tissue of the present document may comprise from about 0.1 wt% to about 20 wt% fungal biomass, such as from about 0.5 wt% to about 15 wt%, based on the total weight of the fungi-based fat tissue.

The fungi-based fat tissue may comprise at least 1 wt% fungal protein, based on the total weight of the fungi-based fat tissue. At least 5 wt%, of the fungal proteins of the fungal biomass may be fungal protein that is released from the fungal cells. The fungi-based fat tissue may thus comprise both fungal proteins present in the fungal cells and fungal proteins released from the fungal biomass.

The fungi-based fat tissue may comprise from about 15 to about 30 wt% water, based on the total weight of the fungi-based fat tissue.

The fungi-based fat tissue may further comprise one or more additive(s), such as a salt, a flavour and/or a hydrocolloid. The hydrocolloid may e.g. be a cellulose derivative, carrageenan, starch (modified or unmodified), xanthan, guar gum, locust bean gum, pectin, gellan, agar and/or alginate.

The fungi-based fat tissue may comprise salt, such as NaCl, in an amount of from about 0.1 wt% to about 3 wt%, based on the total weight of the fungi-based fat üssue.

The fungi-based fat tissue disclosed herein may not contain any animal-derived constituents.

The droplet size of the fat in the fungi-based fat tissue may be 30 um or less. For example the droplet size of the fat in the oil-in-water emulsion with fungal biomass may be from about 5 um to about 20 um, such as about from 8 um to about 15 um, such as from about 10 um to about 13 um.

The food-safe fungi in the fungi-based fat tissue may be filamentous fungi. For example, the fungi may be of the Zygomycota and/or Ascomycota phylum, excluding yeasts, such as fungi of the genera Rhizopus, Neurospora, Aspergillus, Trichoderma, Pleurotus, Ganoderma, lnonotus, Cordyceps, Ustilago, Tuber, Fusarium, Pennicillium, Xylaria, Trametes, or any combination thereof. For example fungi of the species Aspergillus oryzae, Rhizopus oryzae, Rhizopus oligosporus and Rhizopus microspores, Fusarium graminareum, Cordyceps militaris, Cordyceps sinensis, Tuber melanosporum, Tuber magnatum, Pennicillium camemberti, Neurospora intermedia, Neurospora sitophila, Xylaria hypoxion, or any combination thereof may be used.

The present document also relates to a method for producing a fungi-based fat tissue as defined herein said method comprising the steps of: -mixing and emulsifying a fungal biomass of food-safe fi|amentous fungi with water and one or more fat(s) to obtain an oil-in-water emulsion with fungal biomass; -heat-treating the oil-in-water emulsion with fungal biomass of step a), until a core temperature of from about 70 °C to about 100 °C, such as from about 85 °C to about 95 °C, is reached, to obtain a fungi-based fat tissue.

The fungal biomass may first be mixed with the water before addition of the fat. The fat may be added in liquid form. The fungal biomass used may be dried, such as freeze-dried.

The fungal biomass may be grinded, such as to a mean particle diameter of 0.6 mm or less.

The method for producing a fungi-based fat tissue may further comprise a step of adding an additive as disclosed elsewhere herein.

The heat-treatment may involve dipping in hot water, steaming, pan-frying, and/or heating in microwave, autoclave or oven.

The present document is also related to a fungi-based fat tissue obtained or obtainable by a method for producing a fungi-based fat tissue as defined herein.

The present document is also directed to a food product comprising or consisting of a fungi-based fat tissue as defined herein, such as a meat-replacement product, seafood replacement products, egg replacement product, and/or a dairy replacement product, such as a vegan butter, a vegan spread, and/or a creme cheese replacement product.

The present document is also directed to the use of a fungi-based fat tissue as defined herein as a replacement for animalic fat.

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

Definitions Terms such as fungi-based fat tissue/fat tissue replacement/fat tissue replica/fat tissue analogue/fat replacement/animal fat replacement/animal fat replica/fat tissue analogue and the like may be used interchangeably for the composition of the present document comprising fungal biomass, fat and water as disclosed herein. Also, the term "emulsion" may be used to denote the fungi-based fat tissue of the present document. ln the present document the term "fat" refers to a fatty acid ester, for example an ester comprising glycerol and a fatty acid. Triglycerides are one group of fats that may be used in accordance with the present document, as are diglycerides, monoglycerides and mixes of all these as is common in natural plant fat. The term fat includes oils.

Brief description of drawinqs Fig. 1 shows a flow chart diagram explaining the process of producing a fungal fat tissue, from biomass to finished emulsion gel.

Fig. 2 shows a CLSM image of an emulsion made with liquid canola oil. Staining of the fungal cell wall and lipids. Emulsion before heat treatment.

Fig. 3 shows a CLSM tile image of an emulsion made with liquid canola oil. Staining of the fungal cell wall and lipids. Emulsion before heat treatment.

Fig. 4 shows a CLSM image of an emulsion made with liquid canola oil. Staining of the fungal cell wall, fungal proteins and lipids. Emulsion after heat treatment.

Fig. 5 shows a graph showing how the firmness value of the emulsion correlates to the amount of fat added. The measurements were carried out on non-heat-treated emulsion.

Fig. 6 shows a graph showing how the firmness value of the emulsion correlates to the amount of protein added to the aqueous phase. The measurements were carried out on non-heat-treated emulsion. 70 % fat were used in all trials where the protein content was varied based on percentage of the water phase.

Fig. 7 shows a graph showing the remaining weight (%) of a sample of coconut-, pork-, or fungi-fat after being cooked at elevated temperature in a frying pan.

Fig. 8: Left: image of non-heat-treated emulsion. Right: Emulsion after being cooked at elevated temperature in a frying pan for 11 minutes.

Fig. 9 shows an image showing typical fat marbling lines, created by injecting the fungi fat tissue into a mycoprotein structure.

Fig. 10 shows, from right to left; emulsion made with fungal protein, lentil protein and pea protein, respectively.

Fig. 11 shows the visual stability of the emulsion with an increased salt addition of 0.0, 0.2, 0.3, 0.4 and 0.5 M (left to right). The emulsions were studied after 1 day (A), 7 days (B), 14 days (C), 21 days (D), 31 days (E) and 41 days (F).

Detailed description The present document is directed to a fungi-based fat tissue that can be used as a fat in a food product, e.g. to replace animal fat, in particular animal fat tissue.

As mentioned above, one of the challenges when preparing meat replacement products is that it is very hard to mimic a meat structure. For example, it is very difficult to mimic the fat structure of meat. One problem is that non-animal based fat replacement products do not show the same melting pattern as animal fat tissue does when heated. Animal fat tissue that is heated may partly melt but some of the fat tissue is still intact after cooking in meat products.

The present inventors have now found that an oil-in-water composition based on fungal biomass, fat and water has properties mimicking animal fat tissue. This fungi-based fat tissue has a fatty mouthfeel and a complex melting behavior which allows the structure to hold when heated up. Further, the fungi-based fat tissue is freeze-thaw stable which facilitates its storage and/or handling. Also, the fungi- based fat tissue of the present document has a good stability and does not easily separate into an oil and a water phase when stored. The fungi-based fat tissue of the present document can therefore be used as a fat in a wide range of food products. Also, by preparing a fungi-based fat tissue according to the present document, it is possible to prepare fat products having a healthier composition of fats, as well as reaching the same fatty mouthfeel effect using less total amount of fat in the product.

Composition of the fungi-based fat tissue The fungi-based fat tissue of the present document comprises fungal biomass, fat and water.

The fat in the fungi-based fat tissue of the present document is typically present in an amount of from about 40 wt% to about 90 wt% of fat, such as 40 wt% to about 80, such as from about 40 wt% to about 70 wt%, based on the total weight of the fungi-based fat tissue. lncreasing the amount of fat increases the viscosity and/or firmness of the fungi-based fat tissue. Between 40 wt% and 70% fat, based on the total weight of the fungi-based fat tissue, the firmness increases more or less exponentially. At more than 70 wt% fat, the firmness is relatively constant, but the viscosity may be increased.

The fat may be a saturated and/or an unsaturated fat and/or a combination thereof. Preferably a combination of both saturated and unsaturated fats is used to achieve a composition that has the desired properties when it comes to e.g. mouthfeel and melting behavior. Fats that may be used alone or in combination in the fungi-based fat tissue of the present document include, but are not limited to, canola oil, olive oil, sunflower oil, coconut fat, palm oil, peanut oil, soybean oil, algal oil, shea fat and hydrogenated fats, such as hydrogenated canola oil. Fats 106928 which are at least partially saturated, like coconut oil, shea and hydrogenated fats, can give a higher firmness, spreadability or solid characteristics to the fungi-based fat tissue. However, it may be preferable to add also unsaturated fats, like canola, olive and sunflower oil to stabilize the fungi-based fat tissue. For example, the fungi-based fat tissue may comprise from about 30 wt% to about 100 wt% unsaturated fat, based on the total weight of the fat in the fungi-based fat tissue, such as from about 70 wt% to about 100 wt% unsaturated fat.

The fungi-based fat tissue also comprises fungal biomass. The fungal biomass is preferably used as a whole, i.e. comprises both whole fungal cells and disrupted fungal cells (including e.g. cell walls, proteins and other constituents of the fungal cells). Fungal biomass typically comprises about from 45 to about 70 wt% protein, such as from about 50 to about 60 wt%. For example, at least 5 wt%, such as 7 wt%, 10 wt%, 15 wt% or 20 wt% of the fungal proteins present in the fungal biomass may be released from the fungal cells. Typically, the maximum amount of fungal proteins that is possible to release from the fungal cells is about 80 wt% of the total amount of protein in the fungal biomass. Typically, about a third of the proteins of the fungal biomass may be released. lt is important that the whole fungal biomass is used when preparing the fungi-based fat tissue in order to get the right consistency of the fungi-based fat tissue, as both the fungal proteins and the fungal cell walls etc. contribute to the consistency of the fungi-based fat tissue.

The fungi-based fat tissue of the present document typically comprises from about 0.1 wt% to about 20 wt% fungal biomass based on the total weight of the fungi- based fat tissue, such as from about 0.1 to about 15 wt%, such as from about 0.1 to about 12 wt%, such as from about 0.3 wt% to about 8 wt%, from about 0.5 to about 8 wt%, or from about 0.5 wt% to about 5 wt%. The amount of fungal biomass has to be adjusted depending on the amount of fat of the fungi-based fat tissue so that the desired consistency is obtained.

The amount of fungal protein in the fungi-based fat tissue will vary depending on the amount of fungal biomass used and the amount of the other constituents, but is typically at least 1 wt%, based on the total weight of the fungi-based fat tissue. A higher amount of fungal protein (e.g. due to the use of a higher amount of fungal biomass) increases the firmness of the fungi-based fat tissue. Typically from about 0.5 wt% to about 4 wt% fungal protein is present in the fungi-based fat tissue, such as from about 0.6 wt% to about 3.6, such as about from 2 wt% to about 3 wt%, such as about 2.4 wt%, based on the total weight of the fungi-based fat tissue. The firmness of the fungi-based fat tissue can be varied depending on the intended use of it. Typically, the firmness is from about 100 g to about 2500 g, such as from about 150 g to about 2000 g.

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

The fungi-based fat tissue typically comprises from about 15 to about 30 wt%, such as from about 15 to about 20 wt% water, based on the total weight of the fungi-based fat tissue.

The fungi-based fat tissue may also comprise one or more additive(s), such as salt(s), a ﬂavour(s) and/or a hydrocolloid(s). Such additives may e.g. improve the consistency of the fungi-based fat tissue and/or its taste. For example, addition of salt (NaCl) to the fungi-based fat tissue may stabilize the fungi-based fat tissue so that it does not separate into an aqueous and a fat phase. Salt, such as NaCl, may typically be added in an amount of up to 3 wt%, such as from about 0 to about 2.5 wt%, such as from about 0.1 wt% to about 2.5 wt%, such as from about 0.3 to about 2 wt%, such as from about 0.2 wt% to about 2 wt%, 0.2 wt% to about 1.5 wt%, such as from about 0.2 wt% to about 1 wt%, such as from about 0.35 wt% to about 2 wt%, such as from about 0.35 wt% to about 0.88 wt%, based on the total weight of the fungi-based fat tissue.

Examples of hydrocolloids that may be used according to the present document include, but are not limited to, a cellulose derivative (e.g. methyl cellulose), carrageenan, starch (modified or unmodified), xanthan, guar gum, locust bean gum, pectin, gellan, agar and/or alginate. Hydrocolloids may e.g. be used in order to be able to prepare a fungi-based fat composition comprising less fat. For example, starch (modified and/or unmodified) can be used to create a fungi-based fat tissue with less than 30 wt% fat, based on the total weight of the fungi-based fat tissue.

The droplet size of the fat in the fungi-based fat tissue is typically 30 um or less. Typically, the size is not less than 1 um. The size of the fat droplets may e.g. be from about 5 um to about 20 um, such as about from 8 um to about 16 um, such as from about 10 um to about 14 um. The droplet size of the fat in the fungi-based fat tissue can be adjusted by the method for producing the fungi-based fat tissue. Homogenization may e.g. be used to decrease the size of the fat droplets. A smaller droplet size is generally desirable as this may increase the emulsion stability. Also, a smaller droplet size may enhance the palatability (i.e. give better mouthfeel) of the fungi-based fat tissue.

The fungi-based fat tissue of the present document may consist of fungal biomass, water, fat(s) and optionally one or more additives as defined herein. lt is possible to prepare the fungi-based fat tissue of the present document without any animal derived ingredients, thus enabling the preparation of a product which is fully vegan. 106928 12 Method for preparing a fungi-based fat tissue The present document also discloses a method for producing a fungi-based fat tissue, such as the fungi-based fat tissue of the present document. Such a method comprises the steps of: a) mixing and emulsifying a fungal biomass of food-safe filamentous fungi with water and one or more fat(s) to obtain an oil-in-water emulsion with fungal biomass; b) heat-treating the oil-in-water emulsion with fungal biomass of step a), until a core temperature of about 70-100 °C, such as 85-95 °C, is reached, to obtain a fungi-based fat tissue.

The fungal biomass, water and fat may be added at the same time and then mixed. lt is also possible to first mix the fungal biomass and the water and then add the fat and continue mixing. The mixing may be performed by any method that allows an oil-in-water emulsion to be obtained. For example, homogenization may be used for mixing. The mixing should be performed so that the fat and water are mixed well enough to form an oil-in-water emulsion. Depending on how vigorously the mixing is performed smaller or larger droplets of fat in the emulsion may be obtained. Vigorous mixing provides smaller droplets of fat.

The fat may be added in liquid or solid form. lt may be preferred to melt a fat which is solid at the temperature used when preparing the fungi-based fat tissue before mixing it with the fungal biomass and water. The fat(s) may thus preferably be added in liquid form (naturally liquid or melted) in the method for preparing the fungi-based fat tissue. lf a melted fat is used, it preferably has a maximum temperature of 65 °C when used in the method for preparing a fungi-based fat üssue.

The fungal biomass may be fresh fungal biomass. Alternatively, dried fungal biomass may be used, such as e.g. freeze-dried fungal biomass. One advantage with the use ofdried fungal biomass is that the fungal biomass can be prepared and stored until use thus not necessitating the preparation of the fungal biomass in 106928 13 close connection with the preparation of the fungi-based fat tissue. Also, using dry fungal biomass may be preferred as it is easier to control the amount of water to be added. Further, it may be easier to grind (see below) the fungal biomass if it is dry.

However, in terms of process efficiency and cost, it may be preferred to use fresh fungal biomass, as this will allow omitting the step of drying the fungal biomass.

The fungal biomass used in the preparation of the fungi-based fat tissue may be grinded, e.g. to a mean particle diameter of 0.6 mm or less. The use of grinded fungal biomass may be advantageous as it may provide a smoother final product. Also, the release of protein from the fungal biomass may be increased if the fungal biomass used is grinded Any method commonly used for growing fungi may be used to produce the fungal biomass used in the present document. The fungal biomass may e.g. be obtained by growing fungi by liquid or solid fermentation. For example, the fungi may be grown under aerobic submerged fermentation conditions in a closed fermentation vessel with a liquid substrate medium with stirring. The culture media used for growing the fungi may advantageously contain a carbon source, nitrogen source, phosphates and sulphates, and a trace metal solution to enable growth of the fungi and obtaining of the biomass. The bioreactor conditions may be kept at e.g. a pH between 4.0 and 6.0, with an aeration of at least 0.1 vvm and stirred using propeller blades. The growth can e.g. be done in a batch mode, in which fungi are harvested from the production tank after a 24 h process or until less than 5 % of the remaining carbon source is present, or as a continuous process, in which biomass is removed at a constant rate that matches growth and nutrient feed rate, or a semi-batch mode where biomass is partially harvested from one or several reactor and then such reactor(s) are filled with new media to continue fungal growth. Typically, the fungal biomass is dewatered, such as by filtering or pressing, before it is used to prepare the fungi-based fat tissue.

An additive as disclosed elsewhere herein may be added during the preparation of the fungal biomass. Such an additive is preferably added before or during the 106928 14 mixing of the fungal biomass, water and fat, e.g. the additive(s) may be added to the fungal biomass and/or water before addition of the fat.

After the oil-in-water emulsion comprising fungal biomass, water and fat is prepared, the emulsion is heat treated. The heat-treatment is performed until the core temperature of the oil-in-water emulsion is from about 70 °C to about 100 °C, such as from about 85 °C to about 95 °C. The heat-treatment is preferably performed so that the temperature does not exceed these temperatures in any part of the oil-in-water emulsion. The heat treatment is performed in any manner that results in the desired core temperature and may e.g. be performed by dipping in hot water, steaming, pan-frying, and/or heating in microwave, autoc|ave or oven. lt is preferred to perform the heat treatment in a manner so that the core temperature is quickly reached in order to avoid destabilization and separation of the oil-in-water emulsion. The heat-treatment step is important to obtain the desired consistency, such as firmness, of the fungi-based fat tissue.

Details regarding the amount and constitution of the fungal biomass, water, fat and other constituents, such as additives, are found elsewhere in this document.

The fungi-based fat tissue may be used directly for consumption or incorporated into food products as disclosed elsewhere herein. The method for producing a fungi-based fat tissue according to the present document may thus also comprise a step of incorporating said fungi-based fat tissue in a food product.

The present document is also directed to a fungi-based fat tissue obtained or obtainable by the method for preparing a fungi-based fat tissue disclosed herein.

Food products comprising the fungi-based fat tissue The fungi-based fat tissue disclosed in the present document can be used in food products to provide these with e.g. an appealing texture, mouthfeel and/or taste. For example, the fungi-based fat tissue may be used as a replacement for animal derived fat, such as fat tissue in meat or diary fat. 106928 The present document is therefore also directed to a food product comprising or consisting of a fungi-based fat tissue as defined herein. Such food products include, but are not limited to, meat-replacement products (such as a replacement for e.g. beef, pork, chicken, fish, or seafood, such as fish), egg replacement products, and/or a dairy replacement products, such as a vegan butter, vegan spreads, and/or creme cheese replacement product.

Meat-replacement products may e.g. be based on plant material, fungal material, insect material, bacteria protein, yeast protein and/or cultured meat cells as the protein source.

The fungi-based fat tissue may be provided e.g. in the form of flakes, spreads, as a liquid composition, powders, solid blocks. These forms could be used directly for consumption or used for preparing other food product by incorporating them into a food product, such as a meat-replacement product. The fungi-based fat tissue according to the present document may e.g. be provided as a fresh or a frozen product. lt is advantageous to be able to provide a fat that is freeze-thaw stable, which the fungi-based fat tissue of the present document is. Further, it is an advantage that the present fungi-based fat tissue so easily is customizable to widely spread apart applications, such as meat-replacement products and spreads.

The present document is therefore also directed to the use of a fungi-based fat tissue as defined herein as a replacement for animalic fat. The fungi-based fat tissue of the present document can e.g. be incorporated into a meat-replacement product as a layer of fat mimicking animal fat in meat, see Figure 9.

The fungi-based fat tissue of the present document may thus be used e.g. to prepare a meat substitute with fat, such as lard, bacon or a steak.

The fungi-based fat tissue of the present document has a complex melting behavior mimicking that of animal fat in e.g. meat. This e.g. means that it still, at least partly, holds together when heated up. This is very advantageous when preparing food products intended to be heated before consumption as the fat stays 106928 16 at least partly intact during heating and does not fully melt away. Providing such a fat product based on non-animal ingredients has previously been a challenge.

The fungi-based fat tissue of the present document also allows the preparation of fat wherein saturated fats can be replaced with unsaturated fats, thus providing a healthier product. Saturated fats have been linked to health risks, while unsaturated fats have been considered a healthier choice. However, saturated fats are generally firmer while the unsaturated fats often are liquid already at room temperature. Unsaturated fats are thus not suitable to incorporate into products where a solid fat structure is desirable. The present fungi-based fat tissue solves this problem as it has quite a firm structure.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims Experimental section Example 1: Creation of fungi-based fat tissue Production of funqal biomass A fungal spore suspension of the filamentous fungi Rhizopus oligosporus was prepared by flooding a PDA plate culture 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 liquid cultivations. Fungi cultures 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. 1 mL of spore suspension (10 ^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).

Sterilization 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. Upon sterilization, a volume of 30 L of fungi culture obtained from a 16-24 h rich media preculture was used to inoculate 300 L of media in a 400 L stirred-tank bioreactor using media comprised of glucose, ammonium sulphate, potassium 106928 17 phosphate, calcium chloride, and a trace mineral solution. The pH was adjusted to 4.0-5.5 with 5M NaOH. Fermentation conditions were kept at pH 4.0 using NH3 as a base for pH titration, an air flow of 120 L/min (0.6 vvm) and a temperature of 30- 35 °C were kept constant with a stirring of 200 rpm. The fermentation process was carried for 24 h and biomass was harvested after this period. 50 L from this culture was used to inoculate a volume of 5 m3 in a 6 m3 bioreactor and the process was repeated for an additional 24 h. Processing of fungal biomass into a fungi-based fat tissue Fungal biomass was obtained by harvesting the fungal mycelium through a metal sieve. The obtained fungal biomass was washed thoroughly in cold water to remove any remains of fermentation media. The fungal biomass was pressed to remove as much excess water as possible, shredded into pieces (about 2 cm size) and put in a freezer (-18 °C) until completely frozen.

The fungal biomass was then transferred to a freeze dryer (Christ Alpha 2-4 LSCplus, Germany) where the shelf temperature was set to -10 °C and the pressure did not exceed 3 mbar during the drying process. The drying process took about 30-56 h depending on sample amount.

The dry fungal biomass was pulverized into a powder using a blender (Bosch Multitalent 8, Germany) followed by a manual mortar and pestle grinding step. The powder was sieved through a mesh of size 0.6 mm and was stored in air-tight plastic bags until further use. .2 g dry fungal biomass powder was rehydrated in 28.9 g water. 79.6 g canola oil was added slowly during the homogenization process using a high shear homogenizer (CAT x120, Germany) at up to 33 000 rpm. The emulsion was transferred to a piping bag and extruded into a sieve which was placed in a 97 °C water bath. The emulsion was heat-treated until the core reached a temperature of 90 °C, ensuring that the structured was completely gelled. Excess water was removed, the gelled emulsion was let to cool down and was then stored in a freezer at -18 °C over night. The entire process can be seen in the flow diagram presented in Figure 1. 106928 18 The frozen emulsion was cut into small flakes (up to 5 mm size) and were stored frozen until further used. Samples of the emulsion were analysed by fluorescent microscopy using the method explained below.

Confocal microscopy A confocal laser scanning microscopy (CLSM) equipment consisting of a Zeiss LSM 710 (Zeiss, Germany) attached to a Zeiss Axio lmager.Z microscope was used for the imaging. A small droplet of the emulsion, either non-heat treated or after gelling, was placed on a microscope glass slide and the components present in the samples were visualised with two staining combinations. The first staining combination included staining of fungal cell walls (cellulose and chitin) and lipids by adding 40 ul of 0.01 % (w/v) Calcofluor White (Fluorescent brightener 28, Aldrich, Germany) and 40 ul 0.1 mg/mL (w/v) Nile Red (Merck, Germany). The second staining combination included staining of fungal cell walls (cellulose and chitin), lipids and protein by adding 40 ul of0.01 % (w/v) Calcofluor White (Fluorescent brightener 28, Aldrich, Germany), 40 ul 0.1 mg/mL (w/v) Nile Red (Merck, Germany) and 40 ul of 0.1 mg/mL (w/v) Fluorescein (FITC) (Merck, Germany) on top of the sample surface. Stained samples were examined on the glass and were covered with a cover slip. Diode laser line of 405 nm was used for excitation of Calcofluor and emission was collected at 425-480 nm. Argon laser line of 488 nm was used for excitation of FITC and emission was collected at 495- 570 nm. HeNe laser line 543 nm was used for excitation of Nile Red, and emissions were collected at 571-620 nm and 650-710 nm, respectively. Images were assembled of the optical sections taken using a 40x objective (Zeiss EC Plan-Neofluar, numerical aperture of 0.75) to the depth of 13-55 um with 0.33 um z step using ZEN software (Zeiss). ln general, the confocal microscopy images confirmed that a protein stabilised oil- in-water emulsion was created. The emulsion is further stabilised by the fibre content which increased the viscosity of the aqueous phase. Before heat- treatment the average droplet size was about 11.98*_f3.84 um in an emulsion made with 100 % liquid canola oil. Upon heating, the protein denatures, gelling the emulsion and encapsulating the oil droplets in a network structure. The average 106928 19 droplet size seems to be smaller in the gelled structure. Figure 2 and 3 (first staining combination) shows how larger pieces of the fungi cell wall embed the lipid droplet structure, indicating that these acts as a supporting structure for the network, contributing to the stability and hindering the emulsion from collapsing during elevated temperatures. The droplets in Figure 2 (non-heat treated emulsion), are seen to be much closer packed together than after the gelling occurs, Figure 4. Lipid droplets closely packed seems to be connected with a smooth, creamy texture of the emulsion, while the less dense droplet structure results in a texture which is airier and spongier. Smaller droplets are generally associated with a nicer mouthfeel.

Figure 4 (second staining combination) shows lipid droplets stabilized by the fungal proteins surrounding them. Arrow A indicates a bright white circle, representing the stained proteins, surrounding a lipid droplet, shown in dark by arrow B. The proteins further seem to be partially forming an interconnected structure after heat-treatment of the emulsion. Another feature which can be observed is that the lipid droplets are both further apart, but also the droplet size in the heat-treated emulsion is smaller.

Example 2: Creation of fat structures with different firmness by changing the fungi-based fat tissue composition Different effects of varying compositions of fat and biomass amounts in the emulsion were analysed as the effect in the firmness of the emulsion structure. For this, texture analysis was used as described below.

Texture analysis Texture analysis was carried out using a Stable Microsystems TA.TX Plus-C (UK). A back extrusion test was used to compare the textural properties of emulsions made with varying amounts of fat, protein and salt. Samples of 45 g emulsion were filled into cylindrical glass beakers with a diameter of 55 mm and a height of 78 mm. The beaker was placed in the centre of the base plate of the texture analyser and the cylindrical probe (50 mm diameter) was immersed into the sample using a pre-test speed of 1 mm/s, to a depth of 15 mm at a test speed of 1.5 mm/s. The force at maximum penetration depth was recorded as the firmness.

Figure 5 shows how the firmness of the emulsion varies by the amount of added fat. Five different emulsions with varying amounts of fat to water phase were prepared and the firmness were assessed by the texture analysis method previously described. The addition of biomass was kept at 8 % of the water phase in all the samples, consequentiy, the total protein content of the emulsion was being Iowered as the fat content was increased. At around 70 % fat, the emulsion reached a saturation threshold where the firmness no longer increased by an increased fat addition. The measurements were made on emulsions prepared like explained in Example 1, but before heat-treatment.

Also, different emulsion samples were made as explained in Example 1, each sample using 70 % fat and a different amount of fungal biomass in the water phase. Figure 6 shows how the firmness is increasing by an increase in protein addition as percentage of the water phase. At above 10 % protein addition, the water phase is very dense, and emulsification becomes more difficult. The average firmness for a non-heat-treated emulsion made like in Example 1 was around 620 g. This value can be compared to the value obtained by doing the same measurement of firmness on a commercially available vegan mayonnaise (Hellmans) which was 630 g.

After heat-treatment the measured firmness value of the emulsion was increased to about 2000 g. This value is closer to e.g. margarine which had value of about 2300.

Example 3: Analysing freeze-thaw stability of the created fungi-based fat üssue A fungal emulsion was prepared as explained in Example 1, with the modification that 15 wt% of the liquid canola added was replaced by hydrogenated canola fat which was melted prior to the addition to the emulsion. The freeze-thaw stability behaviour of the emulsion was investigated by placing the fungi-fat tissue in a freezer at -18 °C until completely frozen. The sample was then defrosted in room temperature. This procedure was repeated five times for the same sample. Between each cycle the appearance and possibility to heat-treat the sample was investigated, both remaining the same for the five cycles. Additionally, less than 5 % of the sample weigh was lost during each thawing cycle.

Example 4: Creating a fat structure with melting behaviour mimicking animal fat tissues A fungal emulsion was prepared as explained in Example 1, but the sample was taken before heat-treatment. The melting behaviour of the emulsion was investigated by recording the weight of a non-heat-treated emulsion sample before cooking, and then heating the sample in a hot pan, at heating plate setting 6-7 out of 9. The sample was taken out for weighing every 60 s, excluding fat and water released into the pan. The same procedure was repeated for a piece of pork fat and for coconut oil (Kung Markatta). The weight-loss was calculated as percentages of weight-loss of total sample and the result, shown in Figure 7 indicates that the melting behaviour of the emulsion is much closer to that of an animal fat tissue than to a plant based saturated fat. Figure 8 shows a fungi-based fat tissue before and after heat-treatment by cooking at elevated temperature in a frying pan for 11 minutes.

Example 5: lncorporation of fungal biomass emulsion (i.e. fungi-based fat tissue) as a fat replacer in a meat-replacer product 55 g of mycoprotein bits similar to minced meat were grinded in a kitchen food processor (Bosch Multitalent 8, Germany) and mixed with 5 g potato flour and 1,5 g of methylcellulose dispersed in 22 g cold water. 1.3 g burnt sugar, 0.85 g baking soda 0.6 g gellaner and 0.28 g of beet juice was added to the food processor to obtain a burger base material. 18 g of the frozen emulsion, in the shape of flakes, was mixed into the burger base by hand. Burger patties were formed and fried in a pan on medium-high heat until the core temperature of the burger had reached 83 °C.

As an example, a test panel of six people were asked to try three different burgers, prepared as described previously. ln the first burger, frozen pieces of the fungal 106928 22 biomass emulsion were used as the source or fat. ln the second burger, frozen pieces of coconut fat were used as the source of fat. ln the third burger, liquid canola oil was used as the source of fat. All panellists selected the fungal fat burger as their favourite, describing it as having a better fatty mouthfeel, visual appearance, and taste than the other two. The fungal fat tissue was also used as to create a fat marbling effect in a mycoprotein structure. 50 g of fungal fat tissue, prepared as in example 1, but replacing the canola with coconut, was injected into a meat-like, fibrous, mycoprotein structure. The sample was let to set in the fridge for one hour, before being cut in smaller pieces. Figure 9 shows a cross-section of the marbled structure. Example 6: Creation of fungi-based fat tissue using heat-treated biomass Fungal biomass was obtained as in Example 1. The obtained fungal biomass was washed thoroughly in 72 °C water for 10-15 min, i.e. a heat-treatment step of the fungal biomass. The fungal biomass was pressed to remove as much excess water as possible, shredded into pieces (about 2 cm size) and put in a freezer (-18 °C) until completely frozen.

The heat-treated fungal biomass was then transferred to a freeze dryer (Christ Alpha 2-4 LSCplus, Germany) where the shelf temperature was set to -10 °C and the pressure did not exceed 3 mbar during the drying process. The drying process took about 30-56 h depending on sample amount.

The dry heat-treated fungal biomass was pulverized into a powder using a blender (Bosch Multitalent 8, Germany) followed by a manual mortar and pestle grinding step. The powder was sieved through a mesh of size 0.6 mm and was stored in air-tight plastic bags until further use. .2 g dry heat-treated fungal biomass powder was rehydrated in 28.9 g water. 79.6 g canola oil was added slowly by using a high shear homogenizer (CAT x120, Germany). No thickening properties were observed in the emulsion, the water and oil phase separated quickly and only a liquid sample remained. The process was repeated several times, with different amounts of biomass added, with the same result. The results show that a standard step in handling biomass for mycoprotein 106928 23 production, heat-treatment of said biomass, is crucial to omit before the emulsification process in the preparation of a functional emulsion gel.

Example 7: Creation of fungi-based fat tissue of fungal protein isolate and non-soluble fibre 4 g of freeze-dried fungal biomass powder was mixed with 22.3 g of water. The mixture was poured into falcon tubes and was centrifuged at a speed of 4000 rpm for 25 minutes. The clear liquid containing the dissolved protein was separated from the non-soluble fiber fraction at the bottom of the tube.

Total protein content of the biomass was determined by the Dumas method and was found to be 52.5 i 5 %. A Bicinchoninic Acid (BCA) protein Assay was used to quantify the protein percentage dissolved in the aqueous phase. A 3.35*_f0,05 % total dissolved protein was found, giving a yield of 35.5 %.

Two emulsions were created as described in Example 1, but for one of them only the protein containing liquid, and for the other only the non-soluble fiber fraction was added to the aqueous phase.

The emulsion made with the protein fraction had good emulsification and heat treatment properties, but the firmness was lower than when complete fungal biomass was used. The emulsion made using the fibre fraction also had good emulsifying properties, but heat treatment was not possible. The conclusion was that using complete fungal biomass is advantageous over using either the protein or fibre fraction on its own.

Example 8: Comparison of fungi-based emulsion with plant-based protein emulsions Three emulsions were created following the procedure in Example 1, but in two of the emulsions the fungal biomass was replaced by the same amounts of plant- based proteins, namely red lentil, and pea. Figure 10 shows images of the three emulsions where the fungal one had improved thickness and higher stability than the other two. Separation of the oil and water phase was seen in the two plant- based emulsion already after 2-3 hours, while no change was observed for the fungi-based emulsion, indicating a better stability. Further, neither one of the other 106928 24 emulsions could be heated up without phase separating and losing its structure completely. This is an example of one of the features which makes the fungal fat very special. Example 9: Salt addition to the fungi-based fat tissue Emulsions were created following the procedure in Example 1. Salt at different concentrations were added to the water phase of the emulsions, and no heat treatment was performed. The effect of the addition was studied with regard to stability of the emulsion. The emulsions were placed in inverted tubes and left to settle for up to 41 days. The tubes were then visually examined after 1, 7, 14, 21, 31 and 41 days. The result is shown in Figure 11 and the concentrations of salt were 0.0, 0.2, 0.3, 0.4 and 0.5 M (left to right). The results indicate that salt, even in low concentrations, might be an important contribution to the stability of the emulsion over prolonged times. Example 10: Addition of hydrocolloids to the fungi-based fat tissue Emulsions were created following the procedure in Example 1, with the exception that starch (corn or Emden ET 50) was blended with the powdered fungal biomass before mixing with the water. The proportions of the components were either 26.87 % water, 5.33 % dried fungal biomass, 2.8 % modified starch and 65 % fat, or 23.93 % water, 4.57 % dried fungal biomass, 1.5 % modified starch and 70 % fat. The effect of the addition was studied by keeping the emulsion non-heat-treated in the fridge for three days. After this time, some coalescence would have been observed for the non-heat-treated emulsion prepared as in Examples 1. The results indicate that the addition of hydrocolloids, even in low concentrations, might contribute to a prolonged stability of the emulsion structure. lt is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. 106928 Unless expressly described to the contrary, each of the preferred features described herein can be used in combination with any and all of the other herein described preferred features.

Claims

1. A fungi-based fat tissue, said fungal-based fat tissue being an oil-in-water emulsion comprising a fungal biomass of food-safe fungi, one or more of fat(s) and water.

2. The fungi-based fat tissue according to any one of the preceding claims, wherein said fungi-based fat tissue comprises from about 40 wt% to about 90 wt% fat, based on the total weight of the fungi-based fat tissue.

3. The fungi-based fat tissue according to any one of the preceding claims wherein said fat comprises a mixture of saturated, such as hydrogenated, fats and unsaturated fats, such as from about 30 wt% to about 100 wt% of unsaturated fats, based on the total weight of the fat in the fungi-based fat tissue.

4. The fungi-based fat tissue according to any one of the preceding claims wherein said fat is selected from the group consisting of canola oil, olive oil, sunflower oil, coconut fat, palm oil, peanut oil, soybean oil, algal oil and shea fat.

5. The fungi-based fat tissue according to any one of the preceding claims, wherein said fungi-based fat tissue comprises from about 0.1 wt% to about 20 wt% fungal biomass, such as from about 0.5 wt% to about 15 wt%, based on the total weight of the fungi-based fat tissue.

6. The fungi-based fat tissue according to any one of the preceding claims, wherein said fungi-based fat tissue comprises at least 1 wt% fungal protein, based on the total weight of the fungi-based fat tissue.

7. The fungi-based fat tissue according to any one of the preceding claims, wherein at least 5 wt%, of the fungal proteins of the fungal biomass is released from the fungal cells.

8. The fungi-based fat tissue according to any one of the preceding claims, wherein said fungi-based fat tissue further comprises one or more additive(s), such as a salt, a flavour and/or a hydrocolloid, such as a cellulose derivative, 106928carrageenan, starch (modified or unmodified), xanthan, guar gum, locust bean gum, pectin, gellan, agar or alginate.

9. The fungi-based fat tissue according to any one of the preceding claims, wherein said fungi-based fat tissue comprises salt in an amount of from about 0.1 wt% to about 3 wt%, based on the total weight of the fungi-based fat tissue.

10. The fungi-based fat tissue according to any one of the preceding claims wherein the fungi are filamentous fungi.

11. The fungi-based fat tissue according to any one of the preceding claims, wherein said food-safe filamentous fungi are of the Zygomycota and/or Ascomycota phylum, excluding yeasts, such as fungi of the genera Rhizopus, Neurospora, Aspergillus, Trichoderma, Pleurotus, Ganoderma, lnonotus, Cordyceps, Ustilago, Tuber, Fusarium, Pennicillium, Xylaria, Trametes, or any combination thereof.

12. The fungi-based fat tissue according to any one of the preceding claims, wherein said fungi are of the species Aspergillus oryzae, Rhizopus oryzae, Rhizopus oligosporus and Rhizopus microspores, Fusarium graminareum, Cordyceps militaris, Cordyceps sinensis, Tuber melanosporum, Tuber magnatum, Pennicillium camemberti, Neurospora intermedia, Neurospora sitophila, Xylaria hypoxion, or any combination thereof.

13. A method for producing a fungi-based fat tissue as defined in any one of claims 1-12, said method comprising the steps of: a) mixing and emu|sifying a funga| biomass of food-safe filamentous fungi with water and one or more fat(s) to obtain an oil-in-water emulsion with funga| biomass; b) heat-treating the oil-in-water emulsion with funga| biomass of step a), until a core temperature of from about 70 °C to about 100 °C, such as from about 85 °C to about 95 °C, is reached, to obtain a fungi-based fat tissue.

14. The method according to claim 13, wherein the fungal biomass is first mixed with said water before addition of said fat.

15. The method according to claim 13 or 14, wherein said fat is added in liquid form.

16. A fungi-based fat tissue obtained or obtainable by a method according to any one of claims 13-

17. A food product comprising or consisting of a fungi-based fat tissue as defined in any one of claims 1-12 or 16, such as a meat-replacement product, seafood replacement products, egg replacement product, and/or a dairy replacement product, such as a vegan butter, a vegan spread, and/or a creme cheese replacement product.

18. Use of a fungi-based fat tissue as defined in any one of claims 1-12 or 16, as a replacement for animalic fat.