EP4701428A1 - A method for preparing a food product and a food product - Google Patents

Abstract

A method for preparing a food product, the method comprising providing first fungal cells producing structural proteins, providing second fungal cells producing lipids, combining the first and the second fungal cells to obtain a mixture, culturing the mixture until the fungal cells area attached together by the structural proteins to obtain a matrix of edible biomass, and harvesting the edible biomass A food product, comprising edible biomass comprising structural proteins produced by first fungal cells and fatty acids produced by second fungal cells, the first and the second fungal cells being attached together by the structural proteins.

Description

A method for preparing a food product and a food product Technical field The present application relates to method for preparing a food product and to a food product. The food product is an alternative to animal-based food products. Background There is a need to find new smart and sustainable food production systems decoupled from lands and climate assuring also biodiversity. In an attempt to find “animal product alternatives”, such as alternatives to meat and cheese, the recent food research has mainly focused on production of isolated pure ingredients, such as beta-lactoglobulin and albumin. A meat substitute is a food product that is analogous to meat in terms of structure, texture or composition and is used and consumed in similar way as meat. Most meat substitutes are plant-based made by extrusion processing of plant proteins obtained from for example soy, wheat, pea, fava or oats, but there exist also plant-fungus-based substitutes, which are made by fermenting soy and filamentous fungi (Rhizopus oligosporus), such as tempeh, or from mycoprotein, such as quorn. However the properties of these products are not fully satisfactory, and the preparation methods may be complex, expensive and environmentally unacceptable. In similar way a cheese substitute is a food product that is analogous to cheese in terms of structure, texture or composition and is used and consumed in similar way as cheese. The existing solutions focus on plant proteins with extrusion processing or single cell protein biomass, such as quorn to create meat-like structure. In these cases additive ingredients, such as lipids, mainly palm oil and coconut butter, or even animal originated binding agents, such as egg protein in the case of quorn, are added to the product if needed. There are still major flavor and texture challenges for preparing meat substitute food products as the prior art solutions are too far away in mimicking nature’s own architecture. For example meat structure is complex and comprises a combination of proteins, lipids, flavor compounds that cannot be successfully replicated with only mechanical shearing of plant proteins. There is a need to provide better alternatives for animal-based food products, especially with better nutritional value, better texture, better taste, such as similar taste to animal-based products, and other properties that are appealing to users. There is also a need to find simpler and economically feasible methods for producing such products. Summary The present invention overcomes drawbacks of prior art and provides a concept for alternative meat or other animal-based food products using microbial cultures. Also the need for cultivating and processing plants is avoided. The desired properties are achieved by co-cultivating various yeast and/or fungal strains, and attaching or clustering the microbial cells together to form a matrix of edible biomass. The microbial strains can produce various structural proteins for optimized texture as well as diverse fatty acids (lipids) to achieve meat-like texture, taste and aroma. In addition, microbial strains producing color compounds may be included for an enhanced eating experience and a matrix scaffold, such as cellulose, may be used in the culture media. The microbial strains can be attached or crosslinked together through a process of cell-to-cell interaction, such as flocculation, and various downstream processing steps can be subsequently applied to obtain the final product. In the present invention proteins and lipids are produced at the same time making the process simple and economically feasible. The sensory properties and texture of the produced materials are more animal product-like than in the existing products. The present application provides a method for preparing a food product, the method comprising -providing first fungal cells producing structural proteins, -providing second fungal cells producing lipids, -combining the first and the second fungal cells to obtain a mixture -culturing the mixture until the fungal cells area attached together by the structural proteins to obtain a matrix of edible biomass, and -harvesting the edible biomass and preferably forming the harvested edible biomass into the food product. The present application also provides a food product, comprising edible biomass comprising structural proteins produced by first fungal cells and lipids produced by second fungal cells, the first and the second fungal cells being attached together by the structural proteins. The main embodiments are characterized in the independent claims. Various embodiments are disclosed in the dependent claims. The embodiments and examples disclosed herein are mutually freely combinable unless otherwise explicitly stated. The present method enables immediately combining the fungal cells contributing to the desired properties of a meat substitute and directly obtaining a stable food product. The obtained food products may be for human use, which set high criteria for the properties. The obtained food product may be also further prepared, such as cooked or fried, and products exhibiting properties desired for human consumption can be obtained. It is thus possible to obtain a meat substitute product for animal-based food products efficiently mimicking most properties of meat, including meat-like texture, taste and aroma, directly from a fast and controllable process. Brief description of the figures Figure 1 shows a schematic illustration of a multicomponent and crosslinked food matrix comprising different yeast cells, proteins, and cellulose. Figure 2 shows a scheme of the plasmid PP_SES_ScFLO5 containing genome integration Flo5 expression cassette. Figure 3 shows morphologies of P. pastoris strains expressing FLO5Mut. Figure 4 shows confirmation and copy numbers of the FLO5 gene expression cassettes in constructed transformants by qPCR. Error bars represent the deviation from two replicates. Copy numbers of native UBC6 and KU70genes of P. pastoris are also presented. Gene PEP4 was used as reference gene. Figure 5 shows results of 72 h cultivation experiments of FLO5 and FLO5Mut expressing strains. Figure 6 shows FLO5 and FLO5Mut strain cultivations before and after sedimentation period. Figure 7 shows mutated Rhodotorula strains selected for FAST-FAME analysis.18 UV mutant strains can be seen in 7A and 10 EMS mutants in 7B. 11th strain in 7B is control strain, a wild type Rhodotorula. Figure 8 shows fatty acid profiles of 6 UV mutated Rhodotorula strains. Only most abundant fatty acids are included in the column charts. Figure 9 shows fatty acid profiles of control strain and UV 13, 15, and 18 strains cultivated on YPD and C/N 75, and analyzed in second batch. Figure 10 shows fatty acid profiles of EMS mutated strains cultivated on YPD and C/N 75 media. Figure 11 shows fatty acid profiles of EMS mutated strains compared to fatty acid profile of beef. Figure 12 shows flocs of the present biomass applied to a support of test equipment for testing hardness Figure 13 shows confocal microscopy images of a cluster of Pichia pastoris expressing Flo5Mut and Rhodotorula cells. A) beta-glucan (cell wall); B) intracellular lipids; C) proteins. Figure 14 shows microscope pictures of cocultivation samples 1, 2 and control with 10x magnification and 23 mm ocular camera. Yeast cells indicated with red arrow, filamentous fungus cells with white arrow. Figure 15 shows microscope pictures with 10x magnification of sample 1 and 3, and 40x magnification of control samples, comparing filament length. Figure 16 shows Sample 3 and Mucor control sample after frying in a pan with rape seed oil. Figure 17 shows firmness of microbial patties as measured with the Texture Analyser. Detailed description In this specification, percentage values, unless specifically indicated otherwise, are based on weight (w/w, by weight, or wt%). If any numerical ranges are provided, the ranges include also the upper and lower values. The open term “comprise” may be limited with a closed term “consisting of” as one option. The sizes or diameters disclosed herein, unless specifically indicated otherwise, refer to the smallest diameter, and may be presented as average or number-average diameter and in the case of small objects may be determined microscopically, such as by using optical or electron microscope, which may be equipped with a dedicated software for analysing and/or outputting results. Meat alternatives, also known as meat analogues, meat substitutes, fake meat, non-meat or alternative proteins, refer to meat-free products, preferably for human consumption, that aim to mimic meat in terms of taste, texture, appearance, and nutritional value. Further, important sensory properties of meat from the perspective of consumers are taste, tenderness, juiciness, color, hardness, and chewiness. Proteins in meat contain all essential amino acids for humans, whereas beef fat is source of essential fatty acids. In addition to macronutrients, meat, especially beef, is rich in vitamins and minerals. So far obtaining meat substitute products with all such properties has been challenging, but in the present invention it was possible to obtain products that could better mimic most of the properties. In the present solution it was found advantageous to use microorganisms for preparing the meat substitutes, especially fungal organisms, such as yeasts and/or fungi. The present application discloses production of complex protein clusters together with lipid producing microbial strains with a whole cell approach, which is different from conventional fermentation processes, such as traditional fermentation, biomass fermentation, and precision fermentation. The products from traditional processes have not been considered as alternatives or microbial-based since microorganisms are only used as active agents in the processes. Compounds obtained from processes such as precision fermentation usually require higher extraction and purification steps compared to whole-cell biomass. In addition, these compounds are presented in microbial cells at lower levels, which effect production volume. Therefore, precision fermented products are used only as high-value additives in plant-based foods or cultivated meat for improving sensory properties. In the present biomass fermentation, a fast-growing microorganism is cultivated and the resulting high-protein content biomass can be completely used as an edible whole-cell final product, whereas methods such as precision fermentation concentrate on producing specific proteins or other tailored products by using microbes as cell factories. Biomass fermentation is well suited for large-scale production since the doubling time of microbial biomass is often just a few hours. Filamentous fungi are an excellent material for meat-like fibrous structures since their hyphae are similar to muscle fibers in length and size. Biomass fermentation may utilize downstream processing to improve the sensory properties of fermented biomass. Therefore biomass fermentation was found to be a suitable approach for the present methods and products. In order to achieve meat mimicking properties, only flavor coming from lipids was found not enough for a complete product. In addition, a proper texture is required. Meat has its unique, highly organized, and crosslinked structure, in which cells and molecules bind to each other. Therefore, meat alternative made of fungal cells should have a similar structure. In the present invention it was found out that by combining two different types of fungal cells with structural proteins it was possible to obtain an edible product exhibiting desired properties. The present application provides a method for preparing a food product, which may be an alternative food product for animal-based food products, such as a meat substitute food product or a cheese substitute food product. The present food products are preferably non-animal based, i.e. they contain no animal cells or tissue, or additives derived from animals. The food product may be raw material, which is used to make a final food product, and/or an intermediate product or material, which can be processed into the final food product. Any of these materials and/or products may be recovered, further processed, shaped, packed, stored, delivered and/or provided to users, which may be for example operators preparing further products, or end users. The method comprises -providing first fungal cells producing structural proteins, and -providing second fungal cells producing lipids. The first and the second fungal cells may be different fungal species. The fungal species or cells may comprise yeast species or cells and/or filamentous fungi species or cells. The first and/or the second fungal cells may be selected to produce the cell products discussed herein, such as the structural proteins and/or the lipids, or any other desired cell products, preferably in increased amounts, or the first and/or the second fungal cells may be modified, such as genetically modified, to produce the cell products. The cells may produce the cell products, such as the cells may be modified to produce the cell products, and/or the cells producing the cell products may be selected to produce the cell products, in substantially higher amounts or at higher level compared to for example wild type cells of the same or corresponding type, and/or the cells may produce such cell products that the wild type cells do not produce. It was found advantageous to produce the lipids by oleaginous fungal cells incorporated in the biomass. In this way the process can be controlled and a food product with the desired meat-like texture, taste and aroma can be obtained. The oleaginous yeast cells take part in the formation of the matrix of edible biomass, i.e. they remain as integral part of the food product, and thus the lipids produced by the oleaginous yeast cells remain bound in the matrix as accumulated intracellular lipids. The obtained product is thus stable and contains the lipids contributing to the meat-like taste and aroma bound inside the oleaginous cells. These properties are especially exhibited after cooking the final edible biomass. This is substantially different from a situation wherein lipids would be added to cell culture, in which case the cells could consume the lipids and the lipids would be properly bound to the formed biomass. Such added lipids could be for example washed away in the processing of the biomass. The method comprises combining the first and the second fungal cells to obtain a mixture. The first and the second fungal cells may be combined before they are cultured to obtain a mixture, or either the first fungal cells or the second fungal cells may be first cultured for a period of time, and subsequently the other of the first fungal cells or the second fungal cells is added to obtain a mixture. If any further types of fungal cells are used, such as third, fourth or further fungal cells, these may be added in any suitable order and/or in combination with one or more of the other type of fungal cells. In one example the method comprises providing or adding the first fungal cells producing structural proteins first, and cultivating them, and after a suitable period of time, such as a period of time sufficient to produce structural proteins in effective amount for obtaining the attaching effect, adding or providing further fungal cells, such as the second fungal cells producing lipids. It was found out that a better structure of the food product could be obtained by this way. The method comprises culturing the mixture to obtain a matrix of edible biomass. The structural proteins are of the type and/or produced in amounts and/or conditions causing attaching the fungal cells together. The culturing is carried out in conditions resulting in attaching the fungal cells together by the structural proteins to obtain a matrix of edible biomass. Such conditions may include production of one or more structural proteins by the first fungal cells producing structural proteins. The conditions may also include adding one or more structural proteins to the mixture, and/or adding one or more compounds capable of enhancing the attachment of the fungal cells together, such as one or more matrix components or compounds, for example in combination with the one or more structural proteins. The conditions may include suitable incubation time, such as incubation time resulting in the attaching the fungal cells together by the structural proteins to obtain a matrix of edible biomass, or incubating until the fungal cells are attached together by the structural proteins to obtain a matrix of edible biomass. The attachment can be detected by a change (increase) in viscosity of the mixture, a change in visual appearance of the mixture, a change in hardness of the mixture and/or any other suitable detectable change. The culturing may be carried out in a culture medium, which is preferably aqueous medium comprising suitable nutrients, buffering agents, salts and the like agents commonly used in culture medium. The culturing may be carried out or continued until a desired viscosity, appearance, hardness, flowability, spreadability and/or the like property is obtained, which indicates the formation of the matrix of edible biomass and/or which characterizes desired properties of the biomass and/or food product. For example increase in viscosity, such as gelling, may be detected indicating the formation of the matrix. During culturing the first and the second fungal cells are preferably viable, and the culture and the result may be a substantially homogenous mixture comprising these cells. In general the desired properties may vary between different types of alternative food products. The properties may include properties such as viscoelasticity, flowability, firmness and/or hardness. For example a cheese- like of product may be soft, medium or hard, and meat-like products are usually less softer. A soft cheese-like product may behave like a soft semi- solid material, and it may be spreadable. Desired properties may be also obtained after preparing a final edible product, such as after heating for example in an oven and/or by cooking and/or frying. Such product may form a desired brownish color during cooking/frying, which makes the final edible product more attractive and may provide desired type of taste obtained in the cooking/frying of the biomass. In general the obtained or harvested biomass, or the food product, shall be more viscous than liquid, for example gel, semi-solid or solid, such as having an apparent viscosity over 1 Pa·s, preferably 2 Pa·s or more, such as 5 Pa·s or more measured at 22+1ºC at a shear rate of 0.1 s^-1, 1 s^-1, 10 s^-1, 100 s^-1, 1000 s^-1 or 10000 s^-1, for example, with a rotational stress controlled rheometer using plate geometry. AR-G2 rheometer (TA Instruments, UK) has been used in tests. These values may correspond to spreadable or other soft food products. A rheometer may have a dedicated software configure to output desired measurement results, such as viscosity, shear viscosity, yield stress, and the like. For cheese-like products the apparent viscosity may be 5 Pa·s or more, such as 10 Pa·s or more or 20 Pa·s or more. For meat-like products the viscosity may be higher, such as 500 Pa·s or more, or 1000 Pa·s or more. A hardness of the food product may be over 0.5 N, such as 1 N or more, 2 N or more, 3 N or more, 5 N or more, or 10 N or more. Hardness can be determined by using a uniaxial compression test, for example with a texture analyser, such as TA.XTPlus, Stable Micro Systems Ltd., UK, which may include dedicated software for analyzing and/or outputting the results. With this device the measurement can be carried out with a cylindrical probe (P/20P) with a diameter of 20 mm, which may be used to compress individual flocs. The compression may be carried out at a speed of 2 mm/s until a strain of 50% is achieved. The maximum force exerted during the compression process is measured and recorded as the hardness of the flocs. The flowability and spreadability of the food product can be estimated using shear flow and/or squeeze flow test methods. Firmness of the product, such as in the form of patties or the like samples with suitable thickness, may be determined by using a texture analyser, for example to perform a puncture test to analyse the firmness of the fresh patties. The samples may be punctured with a 4 mm cylindrical probe at a speed of 2 mm/s to 50% of their initial height and the force required for compression was recorded. Measurements may be carried out at five different positions on each sample. The maximum force measured during the test is defined as the firmness. The firmness of the product may be in the range of 2.5–5.5 N, such as in the range of 3.0–5.2 N, for example 3.5–5.0 N, 2.5–4.6 N, or 3.5 N or more, or 4.0 N or more. The culturing may be carried out in conditions, wherein growth parameters are optimized for one or more of the used fungal cells. For example if one type of fungal cells are first cultured, the conditions may be optimized for these fungal cells, and when other type of fungal cells are added, the conditions may be changed to be optimized for the both, or all, fungal cells. If the fungal cells are combined before they are cultured, the conditions may be optimized for all the fungal cells. The culturing may be carried out in a suitable reactor, which may be a bioreactor of a known type, such as single-use, multi-use, multi-parallel, benchtop, or microbial bioreactor or fermenter with culture vessels manufactured out of plastic, glass, or stainless steel, and volumes from a few milliliters (lab scale) to thousands of liters (pilot and manufacturing scale). The reactor may be equipped with means for controlling temperature, one or more mixers, one or more aeration means, an inlet for cells, an inlet for liquid, such as culture medium, and an outlet for the cultured cells or the edible biomass and preferably means for harvesting the cells/biomass. Growth parameters including pH, aeration, temperature, composition, and carbon-to-nitrogen (C/N) ratio of medium, and cultivation time can affect the growth of the fungal cells, and the production of the cell-derived products. For example the growth parameters may have impact on lipid accumulation and composition of oleaginous organisms. One or more of the growth parameters and/or reactor parts may be controlled, such as monitored and adjusted, which may be carried out automatically and/or semi-automatically. The method may comprise harvesting and preferably forming the edible biomass into the food product. This may include recovering the cells, i.e. the edible biomass from the culture medium and/or the reactor, for example by filtering, centrifuging, sedimenting, precipitating, pressing, using vacuum, and/or by any other suitable dewatering, concentrating and/or other separating means. The method may comprise further forming the harvested edible biomass into a food product, such as a food product having a shape. The forming may include one or more steps required to obtain a desired product, which may be intermediate product or final product. The harvested biomass is usually concentrated during the harvesting. The biomass may be washed, for example by suspending the biomass into water or aqueous medium, and again separating the liquid from the biomass. The obtained biomass, such as the washed biomass, may be further processed, such as further concentrated, for example by pressing or using the other concentrating means, and/or formed into a suitable form, preferably by a suitable forming means, such as shaping into a suitable and/or desired form or shape. The shape may be a shape of a product such as a patty, a block, a ball, spreadable mass and/or the like. The present products are preferably products with a shape and/or form, which is enabled by the formed matrix. Other supporting material and/or agents may not be needed and/or may not be present. However agents or material such as spices, salt, colored agents and/or the like used to enhance taste and/or appearance may be included and/or added. It is also possible to include the biomass or the food product into a skin to form sausage-like products. An intermediate food product or a final food product may be formed at this point, or it can be formed later. For example the biomass may be extruded with an extruder or other forming means to obtain a food product comprising elongated shapes, which may resemble minced meat. The biomass may be also applied into one or more molds to obtain blocks or other forms having a desired shape. The biomass may be also shaped with other methods, such as by additive manufacturing (3D printing by using a suitable device), which enables preparing a variety of different final food products, for example from one or more types of biomasses obtained by the present method, such as from two or more types. The properties of the obtained biomass(es) can be adjusted according to needs, such as to provide biomass with desired properties, including viscosity, hardness, flowability and/or the like, which may enable controlling the process of forming the final products, such as the additive manufacturing process, wherein the biomass may act as or may be part of the 3D printing ink used in the process. From the method, a food product is obtained, comprising biomass comprising structural proteins produced by first fungal cells and lipids produced by second fungal cells, the first and the second fungal cells being attached together by the structural proteins. In the food product the cells may be partly or fully inactivated, so they may no longer produce the cell products, but the produced structural proteins and fatty acids remain in the product. The first and the second fungal cells can be identified from the product, for example based on the cell products present in the product and/or based on other markers, such as genetic markers, which may be recognized by using suitable methods known in the art. The present method comprising culturing the mixture until the first and the second fungal cells are attached together by the structural proteins to obtain a matrix of edible biomass, harvesting the edible biomass, and preferably forming the harvested edible biomass into a food product, enable immediately combining the fungal cells contributing to the desired properties of a meat substitute and directly obtaining a stable food product. There is no need to separately add further components of the food product during forming the edible biomass, such as lipids. The food product may be further processed, such as cooked and/or fried, such as in vegetable oil. In tests different types of food product could be obtained by controlling the process, which food products could resemble food products such as patties, fried cheese or nuggets, cottage cheese, and/or the like. Also food products comprising fluffy mass could be obtained. The properties of the final food product, which may be prepared to be consumed immediately, can be adjusted by selecting suitable fungal cells, process conditions and/or additives, such as by adding spices, herbs, flours, dietary fibers, vegetables and the like additives commonly used in cooking and/or food production, and which may have an impact to the flavor, and/or structure, texture and/or other properties of the food product. Preferably all the additives are non-animal based. The flavor as used herein may refer to taste and/or to aroma. Examples of fungal cells, such as the first fungal cells, include genera and species from the fungal kingdom, including yeast, such as order Saccharomycetales, such as class Saccharomycetes, including but not limited to genera Saccharomyces, Kluyveromyces, Candida, Pichia, Eremothecium, Kazachstania, Yarrowia, Zygosaccharomyces, or Schizosaccharomycetes, such as species Saccharomyces cerevisiae, Saccharomyces pastorianus, Kluyveromyces lactis, Candida krusei (Pichia kudriavzevii), Pichia pastoris (Komagataella pastoris), Pichia kudriavzevii, Eremothecium gossypii, Kazachstania exigua, Yarrowia lipolytica, Zygosaccharomyces lentus, or Schizosaccharomyces pombe; filamentous fungi, such as classes Eurotiomycetes, including but not limited to Aspergillus or Penicillium, such as species Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, Penicillium chrysogenum, and others; Sordariomycetes, including but not limited to Trichoderma or Myceliophthora, such as species Trichoderma reesei (Hypocrea jecorina), Myceliophthora thermophila, and others; or order Mucorales, such as genus Mucor, for example species Mucor circinelloides, Mucor indicus, and others. Examples of filamentous fungi include dimorphic fungi, such as ones belonging to the order Mucorales or to genus Mucor, for example Mucor circinelloides, or genus Rhizopus, for example Rhizopus stolonifer or Rhizopus oligosporus. The filamentous fungi can be used for controlling the properties of the food product, such as texture. Rhizopus producing longer filaments/fibers/fibrils can provide more flexible and/or viscous structure, while Mucor producing shorter filaments/fibers/fibrils can produce more fluffy structure. These properties may further contribute to properties such as ability to bind water, mouldability, cooking and/or frying properties and/or other properties having impact to the properties of the final food product. Especially Rhizopus was found to efficiently bind water, even after oven cooking of the final mass. In one example the first fungal cells are selected from one or more of Saccharomyces, Pichia, Rhizopus and Mucorales. The first fungal cells produce one or more types of structural proteins. The first fungal cells may be based on or comprise a suitable fungal genus. In one embodiment the first fungal cells comprise cells of one or more Pichia genus, such as Pichia pastoris species. The first fungal cells may also comprise cells of one or more Saccharomyces genus, such as Saccharomyces cerevisiae or Saccharomyces pastorianus species. Saccharomyces cerevisiae has an ability to grow on low-priced and simple culture media with high growth rate as unicellular. It exhibits flocculation ability, where yeast cells aggregate together and sediment out of solution. Compared to Saccharomyces cerevisiae, Pichia pastoris has properties that are advantageous for expression systems. Pichia pastoris is able to grow up to a very high cell density, which makes it excellent organisms for large-scale production with high yields. Pichia pastoris generally possess higher production yields and better secretory capacity with more uniform glycosylation. The structural proteins cause and/or facilitate the cell-cell adhesion utilized in the present methods and products, which can be also called as aggregation and/or crosslinking. The structural proteins may include one or more suitable structural proteins and/or polypeptides, which may include natural and/or engineered proteins and/or polypeptides. The structural protein may originate from the first fungal cell, or it may be derived from another organism, such as from a different fungal cell. A structural protein may be defined for example as a protein that possesses a characteristic amino acid sequence or motif that repeats and forms a scaffold and/or contributes to the mechanical properties of a living organism, a cell, or material. A structural protein may be for example a cell wall protein, such as a cell-cell adhesion protein, which structural proteins may be called as flocculation proteins, including lectin-like proteins, such as flocculins, or other suitable proteins or biopolymers, such as synthetic biopolymers. Examples of other suitable proteins and polypeptides include elastomeric proteins and elastin-like polypeptides or proteins, such as resilin, elastin, silk fibroin and mussel adhesive proteins. Some yeast cells have ability to form structures and crosslinking networks. It was found out that this phenomenon, flocculation, can be used as one crosslinking method between fungal cells in the present multicomponent food matrix. Flocculation may be described as one phenotype of controlled cell adhesion, wherein yeast cells aggregate to form flocs and sediment rapidly out of solution. These macroscopic cell clusters may comprise thousands of yeast cells adhered together. This yeast cell adhesion known as flocculation depends on specific cell adhesion proteins called flocculins or Flo proteins. These proteins, like other fungal adhesins share a common three domain structure composed of C- and N- terminal domains and highly repetitive central domain. Lectin-like Flo proteins are present on the cell wall of flocculent yeast cells and bind to mannose residues present on the cell wall of adjacent cells promoting lectin-like cell-cell adhesion. The carboxyl terminal domain contains a glycosylphosphatidylinositol anchor that allows binding of flocculin to the surface of the cell, whereas the amino terminal domain functions as carbohydrate recognition domain to bind mannans present at the cell wall of adjacent cells. In between these terminals, there is a glycosylated serine- and threonine-rich central domain containing large number of highly repetitive tandem repeats. Presence of calcium is required since Ca²⁺ ions allow the correct conformation and activation of flocculins. Flocculation in for example Saccharomyces cerevisiae is strongly influenced by the expression of FLO genes, such as FLO1, FLO5, FLO8, FLO9, FLO10 and FLO11. Among FLO genes, FLO1, FLO5, FLO9 and FLO10 share sequence similarity, are located adjacent to telomeres and are known to promote flocculation. For example, FLO5, FLO9 and FLO10 show 96%, 94%, and 58% similarity with FLO1. Especially the expression of FLO1 and FLO5 leads to strong flocculation. FLO genes are long sequences containing numerous DNA tandem repeats. These repetitive and dynamic sequences are responsible for recombination events during DNA replication allowing rearrangements both between and within FLO genes. Therefore, yeast cells show variable flocculation properties as they are able to quickly adapt their phenotype to the new environments. The removal or addition of tandem repeats results in different sized novel flocculins. The length of the Flo protein has an effect on flocculation phenotype since longer flocculins seem to promote stronger flocculation. In order to achieve a recombinant strain possessing strong flocculation phenotype, a stable construct with a long FLO gene containing various tandem repeats is required. This is in general challenging due to the various internal tandem repeats of FLO genes. Co-flocculation, also called mutual flocculation, is a heterotypic aggregation process between flocculent and non-flocculent yeast strain, if the non- flocculent strain has flocculation receptors. When these strains are mixed in the presence of Ca²⁺ ions, flocs are formed when non-flocculent cells interact with flocculent cells, and culture sediments out of solution. This phenomenon can be utilized in the present invention. Flocculation can be measured by various methods and flocculence can be determined based on different criteria like strength of the formed bonds, morphologies of the flocs, and extent and rate of sedimentation. However, most measuring methods are based on determining the extent and rate of sedimentation and flocculation is mainly quantified by protocols based on the Helm’s sedimentation test. These tests are based on counting of free cells in a flocculating culture, which are compared to the total cell number after deflocculation or before flocculation. In most cases non-flocculating yeast strains exhibit flocculation rates between 0% and 15%, whereas strongly flocculating yeast strains will show flocculation in the range of rates 90– 100%. In one embodiment the structural proteins comprise flocculin protein, such as a flocculin protein of Saccharomyces cerevisiae, for example FLO1 and/or FLO5 protein, such as FLO5 protein comprising the amino acid sequence of SEQ ID NO: 1. The mutant FLO5M protein disclosed herein comprising the amino acid sequence of SEQ ID NO: 2 may be also used. In one example the first fungal cells, such as cells of Pichia pastoris, are modified to express a flocculin protein of Saccharomyces cerevisiae. In one embodiment the structural proteins comprise one or more proteins selected from elastin-like proteins and polypeptides (ELP). A general structure of polymeric ELPs may be (VPGXG)n, where the monomeric unit is Val-Pro-Gly-X-Gly, and the "X" denotes a variable amino acid that can have consequences on the general properties of the ELP, such as the transition temperature (T_t). These proteins and polypeptides aggregate at physiological temperatures due to interactions between hydrophobic domains in a process called coacervation, or can be crosslinked via their lysine residues. Therefore they can be used as structural proteins causing and/or facilitating cell-cell adhesion in the present methods and products. The structural proteins may be able to bind to one or more compounds capable of enhancing the attachment of the fungal cells together, such as one or more matrix components or compounds, which may be polymeric compounds, such as natural compounds for example cellulose or the like natural polymers. In such case the structural proteins may comprise one or more matrix compound/component-specific binding proteins. The matrix compound or component may comprise for example cellulose, which can be bound by a cellulose-binding protein. This is analogous mechanism to flocculation, as flocculins contain mannose binding domain(s) which interact with poly-mannosylated structures of the cell walls (cell-wall proteins). In one embodiment the structural proteins comprise one or more proteins selected from cellulose binding proteins, such as proteins comprising cellulose binding domains, In such case the method may comprise culturing the first fungal cells in the presence of cellulose. The cellulose may be provided or added to the culture in a suitable form and in suitable amount resulting in formation of a desired matrix in combination with the cellulose binding proteins. The second fungal cells produce one or more lipids, such as one or more of fatty acids, for example monoglycerides, diglycerides or triglycerides (triacylglycerides), phospholipids, fats, waxes, sterols, fat-soluble vitamins or any combinations thereof. Fatty acids may be saturated and/or unsaturated, for example monounsaturated and/or polyunsaturated. The fungal cells may be modified to produce desired lipids and/or in desired amounts. The produced lipids may be produced in increased amounts compared to corresponding wild type fungal cells, and/or the produced lipids may be lipids not produced by corresponding wild type fungal cells. The fungal cells may be selected to produce desired lipids and/or in desired amounts. In one embodiment the second fungal cells comprise one or more oleaginous yeast cells, such as yeast cells selected from one or more genera Apiotrichum, Rhodotorula, Yarrowia, and Cutaneotrichosporon (former Cryptococcus). Also Mucor, such as Mucor circinelloides, can represent oleaginous second fungal cells suitable for the present purposes, however preferably used with different fungal cells as first fungal cells. Using oleaginous fungal cells producing relatively high amount of intracellular lipids enabled obtaining enough fatty acids to obtain a desired meaty taste and flavor of the food product. The oleaginous yeast genus Rhodotorula (also known as Rhodosporidium) is advantageous for the present purposes. It includes carotenoid producing red pigmented yeasts. Several Rhodotorula species are oleaginous yeasts which are able to accumulate intracellular lipids between 40–70% (w/w). The lipids synthesized by Rhodotorula species can be employed as a source of nutritionally valuable fatty acids. In addition, Rhodotorula species have a biotechnological advantage over other oleaginous yeasts since they are able to grow on a wide range of inexpensive substrates and possess great tolerance toward inhibitory compounds found in biomass hydrolysates. Among Rhodotorula species, Rhodotorula glutins, Rhodotorula graminis and Rhodotorula toruloides can be considered as excellent lipid and carotenoid producers for the present applications. Microbial lipid production by yeasts has many advantages compared to traditional lipid production including animal fats and vegetable oils, for example a short life cycle, simple growing requirements, and independency of requirements for place, season, and climate, which also makes microbial lipid production preferred for the sustainable production for lipids for the present purposes. Microbial production of polyunsaturated lipids, such as fatty acids, is preferable, for example when it is not desired to use current animal- and plant based sources of polyunsaturated lipids. Many polyunsaturated lipids are essential fatty acids which have health-promoting effects. Microbial lipids, also known as single cell oils (SCOs), can be produced by the oleaginous microorganisms. Term oleaginous refers to their ability to synthesize and accumulate intracellular lipids to over 20% of their dry cell weight (DCW). Advantages of yeasts in microbial lipid production include their rapid accumulation of lipids into discrete lipid droplets without production of endotoxins. Generally, the triacylglyceride (TAG) content of the lipid in oleaginous yeasts is around 80% (w/w) to 90% (w/w). Fatty acid profile of oleaginous yeasts varies between species and strains and depends on the composition of the culture medium. The most abundant fatty acids in oleaginous yeasts are palmitoleic acid (C16:1), oleic acid (C18:1), palmitic acid (C16:0), stearic acid (C18:0), linoleic acid (C18:2). Examples of fatty acids that the second fungal cells may produce include one or more of C4:0, C6:0, C8:0, C10:0, C12:0, C14:0, C16:1n7, C16:1n9, C16:0, C17:1, C18:1n9, C18:2n6, C18:3n3, C19:1n9, C24:0. In one example the second fungal cells produce oleic acid C18:1n9 (such as about 60–70%), palmitic acid C16:0 (such as about 8–12%), linoleic acid C18:2n6 (such as about 8–10%), stearic acid C18:0 (such as about 3–7%), and/or alpha-linolenic acid C18:3n3 (such as about 3–7%). In one example the second fungal cells produce 10- nonadecenoic acid C19:1n9, preferably in increased amount compared to wild type cells, and/or less alpha-linolenic acid C18:3n3. In one example the second fungal cells produce one or more of butyric acid (C4:0), hexanoic acid (C6:0), octanoic acid (C8:0) decanoic acid (C10:0), and dodecanoic acid (C12:0). Preferably the lipids, or the fatty acids, disclosed herein are produced in increased amount compared to wild type cells. Since the lipid profile of yeast lipids does not share similarity to that of meat lipids or other food oils, the fungal cells may be modified to produce desired lipids. In one embodiment the lipids comprise one or more saturated fatty acids, such as palmitic acid and/or stearic acid. In one embodiment the lipids comprise one or more unsaturated fatty acids, such as disclosed herein. The lipids may also comprise one or more of saturated fatty acids and one or more of unsaturated fatty acids. The produced fatty acids may be selected from one or more or short chain fatty acids, medium chain fatty acids, and long chain fatty acids, and combinations thereof, saturated and/or unsaturated. In one example the fatty acids are selected from short and medium chain fatty acids, such as C4–C12 fatty acids, C4–10 fatty acids, C4–C8 fatty acids, C6–C12 fatty acids or C6– C10 fatty acids. In one example the second fungal cells produce one or more fatty acids selected from plant fatty acids and animal fatty acids. The plant fatty acids may be selected from one or more of omega-3 fatty acids and omega-6 fatty acids, for example from α-linolenic acid, eicosapentaenoic acid and/or docosahexaenoic acid. In one example the second fungal cells produce one or more fatty acids selected from one or more animal fatty acids, which are dairy lipids comprising saturated short and medium chain fatty acids, such as one or more of C4:0 to C12:0. Genetic engineering enables for example modification of key pathways in lipid synthesis resulting in yeast strains producing lipids more efficiently. By engineering enzymes such as desaturases or elongases, conversion processes of fatty acids can be altered, which may result in triacylglycerides (TAGs) with unconventional fatty acid compositions. Genetic engineering enables also to generate yeast or filamentous fungus strains producing short chain fatty acids, for example by engineering enzymes such as fatty acid synthases (FAS) and/or diacylglycerol transferases (DGAT). The modifications can be carried out by using one or more suitable methods, which are known for a skilled person. Random mutagenesis in yeasts can be performed by using physical or chemical mutagens, which alters the DNA of the organism, followed by selection of mutants with the desired features. In physical mutagenesis microbial cells are exposed to radiation including ultraviolet (UV), X-rays and ion beams. Ionizing radiation changes the structure of the genetic material, whereas errors introduced by UV light include base pair transitions or deletions. In chemical mutagenesis, cells are treated with chemicals that react with DNA and lead to mutations. Most commonly used mutagens for improving the lipid content of oleaginous yeasts are alkylating agents, such as ethyl methane sulphonate (EMS) and methylnitronitro-soguanidine (MNNG). Compared to genetic engineering, random mutagenesis is inexpensive and straight forward and requires less information about the yeast’s genotype and biochemical pathways. In addition, strains generated via random mutagenesis do not require the strict regulatory process of genetically engineered microorganisms. On the other hand, in random mutagenesis is not possible to predict which type of mutation would yield an improvement in particular strain. This method also results in generation of hundreds of mutated colonies, making the screening of mutants challenging and laborious process. Growth parameters can have an effect on lipid accumulation and composition of oleaginous organisms. Since ideal growth conditions vary between species and strains, optimization of environmental parameters individually for each oleaginous strain is necessary. Fatty acid profiles vary depending on the growth phase of the cultures. C/N ratio, especially the amount of nitrogen, affects lipid accumulation since nitrogen limitation induces a cascade of reactions leading to the formation of continuous supply of acetyl-CoA and enough NADPH allowing the accumulation of lipids. In addition, production costs can be reduced by cultivating microbes on alternative carbon sources, such as agro-industrial waste streams. The first or the second fungal cells may be selected or modified to produce one or more further cell product(s), such as a compound producing color, antioxidant, flavor, and/or other property disclosed herein. In one example the second fungal cells produce one or more of these compounds. It is also possible to include third fungal cells producing such cell products. In one embodiment the fungal cells produce a colored compound, which may be a pigment. The colored compound may provide suitable color for the food product, such as it may imitate meat color and/or it may mask other colors, which makes the food product more acceptable for a user. The colored compound may comprise for example one or more carotenoids and/or anthocyanins, which can also enhance the nutritional value of the food product. Examples of carotenoids, also called as tetraterpenoids, include carotenoids in classes xanthophylls, which contain oxygen, for example lutein and zeaxanthin; and carotenes, which are hydrocarbons containing no oxygen, for example α-carotene, β-carotene, and lycopene. Examples of anthocyanins include glycosides of cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin. The fungal cells used in the method are preferably GRAS types (generally regarded as safe) and/or QPS types (qualified presumption of safety). In one example GRAS yeast species Pichia pastoris is used to produce structural proteins such as elastin-like proteins and cellulose binding protein domains. In one example oleaginous yeasts such as Apiothricum, Rhodotorula, Yarrowia, or Cutaneotrichosporon are used for fatty acid (lipid) production. The oleaginous yeasts may have undergone extensive mutagenesis and screening to obtain the optimal flavor profile. The matrix formation can be achieved through the process of flocculation or "clumping together". For example flocculin proteins, such as mainly FLO5 from Saccharomyces cerevisiae and/or its mutant variant, can be expressed in Pichia pastoris to achieve a strong flocculation phenotype. In an optimized system, the oleaginous yeasts and other strains in the mixture are included in the matrix through co-flocculation during optimized co-cultivation. The present application also provides use of any of the fungal cells or combinations thereof for preparing edible biomass and/or any of the food products disclosed herein, preferably with any of the methods disclosed herein. The present application also provides use of the edible biomass for preparing a food product. Examples Example 1 In the present invention a multicomponent meat alternative was created from various yeast cells by biomass fermentation. A food matrix was obtained that mimics meat in forms of taste and texture. Different microbial cells producing different texturizing and flavoring compounds essential for meat-like characteristics were investigated and could be crosslinked together. One aim was to crosslink oleaginous Rhodotorula cells producing desired fatty acids for meaty flavor, and P. pastoris cells producing structural proteins for optimal meaty texture together through a process of flocculation. To obtain meaty flavor, one aim was to create a Rhodotorula strain with a similar fatty acid profile to that of meat trough random mutagenesis and cultivation experiments. For achieving flocculation interactions between Rhodotorula and P. pastoris cells, P. pastoris strain was created expressing Saccharomyces cerevisiae flocculin proteins able to cause strong flocculation phenotype. Flocculation as a crosslinking method between microbial cells was investigated and developed. Cocultivation of wild type Saccharomyces cerevisiae and Rhodotorula species + S pastorianus, Rhizopus stolonifera + Rhodotorula species, and Apiotrichum brassicae and Mucor circinelloides and Rhodotorula also provided promising results. It was found that in addition to flocculins, Pichia pastoris could express other proteins and binding domains, such as elastin-like proteins responsible for textural characteristics, and cellulose-binding domains to crosslink cellulose that could be added as texturizing material to the biomass. In addition, heme proteins could be expressed to obtain meaty color and flavor. Therefore, P. pastoris was chosen over S. cerevisiae since it has higher production yields and better capability for heterologous protein expression. Figure 1 shows a schematic illustration of the multicomponent food matrix. Materials and methods 1.1 Strains, genes, and plasmids P. pastoris was used as a protein production host for expressing FLO genes. For lipid mutagenesis part, oleaginous yeast strain from Rhodotorula species was utilized. All yeasts strains used in this example are described in Table 1. Table 1. Strain Organism Description Function H5659 P. pastoris Non-flocculating Expression of FLO P. pastoris genes in order to strain construct flocculating P. pastoris strain C-00393 Rhodotorula Oleaginous Lipid mutagenesis yeast P. pastoris Laboratory To test flocculation strain S. Laboratory To test flocculation cerevisiae strain Synthetic FLO5 genes, FLO5 and FLO5Mut, were ordered from GenScript. Compared to the sequence of wild type FLO5 (SEQ ID NO: 1), sequence of FLO5Mut (SEQ ID NO: 2) contains two missense point mutations, which are demonstrated to cause strong flocculating phenotype. Alignment of amino acid sequences of FLO genes are presented in Table 2. Properties of FLO genes studied in this work are listed in Table 3. Table 2. Amino acid sequences of FLO5 gene and its mutant variant FLO5Mut FLO5 1 MTIAHHCIFLVILAFLALINVASGATEACLPAGQRKSGMNINFYQYSLKDSSTYSNAAYM 60 FLO5M 1 MTIAHHCIFLVILAFLALINVASGATEACLPAGQRKSGMNINFYQYSLKDSSTYSNATYM 60 FLO5 61 AYGYASKTKLGSVGGQTDISIDYNIPCVSSSGTFPCPQEDSYGNWGCKGMGACSNSQGIA 120 FLO5M 61 AYGYASKTKLGSVGGQTDISIDYNIPCVSSSGTFPCPQEDSYGNWGCKGMGACSNSQGIA 120 FLO5 121 YWSTDLFGFYTTPTNVTLEMTGYFLPPQTGSYTFSFATVDDSAILSVGGSIAFECCAQEQ 180 FLO5M 121 YWSTDLFGFYTTPTNVTLEMTGYFLPPQTGSYTFSFATVDDSAILSVGGSIAFECCAQEQ 180 FLO5 181 PPITSTNFTINGIKPWDGSLPDNITGTVYMYAGYYYPLKVVYSNAVSWGTLPISVELPDG 240 FLO5M 181 PPITSTNFTINGIKPWHGSLPDNITGTVYMYAGYYYPLKVVYSNAVSWGTLPISVELPDG 240 FLO5 241 TTVSDNFEGYVYSFDDDLSQSNCTIPDPSIHTTSTITTTTEPWTGTFTSTSTEMTTITDT 300 FLO5M 241 TTVSDNFEGYVYSFDDDLSQSNCTIPDPSIHTTSTITTTTEPWTGTFTSTSTEMTTITDT 300 FLO5 301 NGQLTDETVIVIRTPTTASTITTTTEPWTGTFTSTSTEMTTVTGTNGQPTDETVIVIRTP 360 FLO5M 301 NGQLTDETVIVIRTPTTASTITTTTEPWTGTFTSTSTEMTTVTGTNGQPTDETVIVIRTP 360 FLO5 361 TSEGLITTTTEPWTGTFTSTSTEMTTVTGTNGQPTDETVIVIRTPTSEGLITTTTEPWTG 420 FLO5M 361 TSEGLITTTTEPWTGTFTSTSTEMTTVTGTNGQPTDETVIVIRTPTSEGLITTTTEPWTG 420 FLO5 421 TFTSTSTEVTTITGTNGQPTDETVIVIRTPTSEGLITTTTEPWTGTFTSTSTEMTTVTGT 480 FLO5M 421 TFTSTSTEVTTITGTNGQPTDETVIVIRTPTSEGLITTTTEPWTGTFTSTSTEMTTVTGT 480 FLO5 481 NGQPTDETVIVIRTPTSEGLISTTTEPWTGTFTSTSTEVTTITGTNGQPTDETVIVIRTP 540 FLO5M 481 NGQPTDETVIVIRTPTSEGLISTTTEPWTGTFTSTSTEVTTITGTNGQPTDETVIVIRTP 540 FLO5 541 TSEGLITTTTEPWTGTFTSTSTEMTTVTGTNGQPTDETVIVIRTPTSEGLITRTTEPWTG 600 FLO5M 541 TSEGLITTTTEPWTGTFTSTSTEMTTVTGTNGQPTDETVIVIRTPTSEGLITRTTEPWTG 600 FLO5 601 TFTSTSTEVTTITGTNGQPTDETVIVIRTPTTAISSSLSSSSGQITSSITSSRPIITPFY 660 FLO5M 601 TFTSTSTEVTTITGTNGQPTDETVIVIRTPTTAISSSLSSSSGQITSSITSSRPIITPFY 660 FLO5 661 PSNGTSVISSSVISSSVTSSLVTSSSFISSSVISSSTTTSTSIFSESSTSSVIPTSSSTS 720 FLO5M 661 PSNGTSVISSSVISSSVTSSLVTSSSFISSSVISSSTTTSTSIFSESSTSSVIPTSSSTS 720 FLO5 721 GSSESKTSSASSSSSSSSISSESPKSPTNSSSSLPPVTSATTGQETASSLPPATTTKTSE 780 FLO5M 721 GSSESKTSSASSSSSSSSISSESPKSPTNSSSSLPPVTSATTGQETASSLPPATTTKTSE 780 FLO5 781 QTTLVTVTSCESHVCTESISSAIVSTATVTVSGVTTEYTTWCPISTTETTKQTKGTTEQT 840 FLO5M 781 QTTLVTVTSCESHVCTESISSAIVSTATVTVSGVTTEYTTWCPISTTETTKQTKGTTEQT 840 FLO5 841 KGTTEQTTETTKQTTVVTISSCESDICSKTASPAIVSTSTATINGVTTEYTTWCPISTTE 900 FLO5M 841 KGTTEQTTETTKQTTVVTISSCESDICSKTASPAIVSTSTATINGVTTEYTTWCPISTTE 900 FLO5 901 SKQQTTLVTVTSCESGVCSETTSPAIVSTATATVNDVVTVYPTWRPQTTNEQSVSSKMNS 960 FLO5M 901 SKQQTTLVTVTSCESGVCSETTSPAIVSTATATVNDVVTVYPTWRPQTTNEQSVSSKMNS 960 FLO5 961 ATSETTTNTGAAETKTAVTSSLSRFNHAETQTASATDVIGHSSSVVSVSETGNTMSLTSS 1020 FLO5M 961 ATSETTTNTGAAETKTAVTSSLSRFNHAETQTASATDVIGHSSSVVSVSETGNTMSLTSS 1020 FLO5 1021 GLSTMSQQPRSTPASSMVGSSTASLEISTYAGSANSLLAGSGLSVFIASLLLAII 1075 FLO5M 1021 GLSTMSQQPRSTPASSMVGSSTASLEISTYAGSANSLLAGSGLSVFIASLLLAII 1075 Table 3. FLO genes Name Strain Gene Description Length Origin FLO5 Synthetic, FLO5 Encodes lectin- 3228 bp GenScript S288C like cell wall S. cerevisiae protein flo5 as model organism FLO5Mut Synthetic, FLO5 Base 188 3228 bp GenScript S288C converted to A S. cerevisiae Base 605 as model converted to C organism Encodes lectin- like cell wall protein flo5 FLO5 genes were ordered ready cloned into B9928 P. pastoris expression vector from GenScript. Ordered plasmids are listed in Table 4. Table 4. Plasmids ordered from GenScript. Plasmid Backbone Insert Insert encoding PP_SES-ScFLO5 B9928 FLO5 Flo5 PP_SES- B9928 FLO5MUT Flo5-Mut ScFLO5MUT The plasmid B9928 was used as the vector backbone for FLO5 and FLO5Mut expression strains. Both FLO5 and FLO5Mut expression constructs contained origin of replication (ORI), a constitutive promoter An_201cp preceded by eight binding sites (BS), an ampicillin resistance (amp) gene for replication and cloning in E. coli, URA3 marker gene for replication and cloning in P. pastoris, terminator, and NotI restriction sites for linearization. Figure 2 shows a scheme of the plasmid PP_SES_ScFLO5 containing genome integration Flo5 expression cassette. The used synthetic expression system (SES) is functional in a broad spectrum of fungal species without the need for host-dependent optimization. The SES consists of two expression cassettes, the first providing a weak, but constitutive level of a synthetic transcription factor (sTF), and the second enabling strong, at will tunable expression of the target gene via an sTF-dependent promoter. 1.2 Growth media and chemicals Growth media The P. pastoris, S. pastorianus and Rhodotorula strains were cultivated in yeast extract-peptone-dextrose (YPD) consisting of 20 g/l bacto peptone, 10 g/l yeast extract, and 20 g/l glucose in DDIW. P. pastoris H5659 strains were cultivated in YPD containing 1 g/l Uridine. P. pastoris transformants were plated on synthetic complete drop-out medium lacking uracil (SCD-URA) plates composed of 20 g/l agar, 50 ml/l Synthetic Complete stock (-uracil), 50 ml/l 40% glucose, and 100 ml/l 10x YNB-stock. Rhodotorula mutants were plated on YPD plates composed of YPD medium with 1,5% agar, and on YPD + 0.1% oleic acid + 0.1% Tween 80 plates composed of YPD medium with 20 g/l agar, 5% glucose, 0.1% oleic acid, and 0.1% tween80. Rhodotorula mutants were cultured also on C/N 75 media containing 0.139 g/l NH₄Cl, 1.5 g/l yeast extract, 3.2 g/l KH₂PO₄, 1 g/l MgSO₄*7H₂O, 4 μg/l sterile biotin, and 85 ml/l 40% glucose. Yeast strains were cultured at +30°C and liquid cultures with 220 rpm shaking. Chemicals For the ethyl methanesulfonate (EMS) mutagenesis, following chemicals were used: 0.1 M sodium phosphate (NaH₂PO₄) buffer pH 7, 1.206 g/ml EMS (Sigma-Aldrich), and 5% sodium thiosulfate (Na₂S₂O₃) buffer. For the flocculation assay, 50 mM EDTA, 4 mM CaCl₂, and flocculation solution containing 4 mM CaCl₂, 6.8 g/l sodium acetate, 4.05 g/l acetic acid and 4% (v/v) ethanol pH 4.5, were used. 1.3 Transformation of P. pastoris Plasmids PP_SES-ScFLO5 and PP_SES-ScFLO5MUT were transformed into P. pastoris H5659 strain. Preparing yeast cells for transformation The strain was grown on YPD+URA (1 g/l) medium overnight at 30°C with 220 rpm shaking until OD₆₀₀ of 1 was reached. The cells were prepared for transformation by the lithium acetate (LiAc) method. The culture was transferred to a 50 ml Falcon tube and cells were spun down for one minute at 4000 rpm. The pellet was resuspended to 50 ml DDIW, and cells were spin down for one minute at 4000 rpm. The pellet was resuspended to 50 ml freshly made 1xTE+0.1 M LiAc and cells were spun down for one minute at 4000 rpm. Remaining pellet was resuspended in 100 µl of 1xTE+0.1 M LiAc. Preparing plasmids for transformation Plasmids PP_SES_ScFLO5 and PP_SES_ScFLO5MUT were digested with FastDigest NotI restriction enzyme (Thermo Scientific) to release the linear expression cassettes with selection markers for subsequent genome integration. Both digestion reactions included 5 µl FastDigest 10X buffer (Thermo Scientific), 1 µl NotI, 7 µg plasmid, and DDIW up to a total volume of 50 µl. Plasmids were digested at +37°C for one hour and NotI was inactivated at +80°C for 5 minutes. Transformation SS-DNA (herring sperm DNA) was heated for 5 min at +95°C and cooled on ice for 5 minutes. A master mix for one transformation containing 400 µl 50% PEG, 50 µl 10xTE, 50 µl 1M LiAc and 10 µl SS-DNA was prepared.400 µl of master mix was added to 50 µl of cell suspension. 7 µg of NotI-digested plasmid was added and vortexed to mix. Transformation solution was incubated at +30°C for 30 minutes and at +42°C for 20 minutes. Cells were centrifuged for 30 seconds at 140000 rpm. Supernatant was removed and cell pellet was resuspended to 200 µl of DDIW and plated on SCD-URA plates. Plates were incubated for 4 days at +30°C. Screening of transformants Transformants containing the correct DNA constructs were identified using quantitative PCR (qPCR). qPCR was performed with Roche Light Cycler SYBR Green I Master kit according to manufacturer’s instructions. Analysis was done with LightCycler 480 II Standard SYBR Green I protocol (Roche, Switzerland). Five FLO5 and five FLO5Mut strains were selected for screening. Primers used for detection are shown in Table 5. Table 5. Primers used for qPCR. Primer name Sequence Detects SEQ ID NO: ScFLO5_qPCR_F1 GACTACTATCACTGGGACGA FLO5 3 ScFLO5_qPCR_R1 ATGTTACGCTGGATGAAATGAC FLO5 4 ScFLO5_qPCR_F2 GCAACTGTACCATCCCTGAC FLO5 5 ScFLO5_qPCR_R1 CTCGTGAAAGTCCCTGTCCA FLO5 6 Pp_UBC6_qPCR_F ACCACCGGATCTATAAGCAC UBC6 7 Pp_UBC6_qPCR_R ATAGCACCTTCAGCAGTATCAG UBC6 8 Pp_ku70 F GGTACTTTGAAGAACAATGCGG KU70 9 Pp_ku70 R TTCTTGATGTGGATCTTTCCGA KU70 10 Pp_pep4 F GAATTAGATCTCCTTGACGAACC PEP4 11 reference Pp_pep4 R ATTCAGCATATTCGGATCCCA PEP4 12 reference 1.4 Cultivation of P. pastoris strains Small scale cultures of the constructed strains were prepared in order to analyze the flocculation phenotypes of P. pastoris strains. The cultivations were done as shake flask cultures in 250 ml Erlenmeyer flasks with culture volumes of 50 ml. Five screened FLO5 and FLO5Mut strains were cultivated, and each flask was inoculated with approximately same number of cells. 1.5 Flocculation assay Flocculation ability of individual yeast strains and mixed cultures were measured by flocculation assay. Flocculation of the yeast strains was evaluated by measuring the optical density of yeast suspensions after shaking using a modified Helm’s assay. Yeast cells were grown in 50 ml YPD medium over weekend at +30°C with shaking. Cultures were harvested and washed twice with 50 mM EDTA (pH 7.0) and diluted in the same buffer to an OD600 of 0.4. Mixed yeast strain cultures were prepared by culturing strains separately, harvesting, and washing cells with EDTA, and then mixing cells in appropriate ratios in presence of calcium.1 ml of diluted cell suspension was transferred into six 2 ml Eppendorf tubes, from which three was marked as “flocculation” and three as “control”. For flocculation tubes, cells were harvested and washed once with 1 ml 4 mM CaCl2 solution and resuspended in 1 ml of flocculation buffer containing 4 mM CaCl2, 6.8 g/l sodium acetate, 4.05 g/l acetic acid, and 4% (v/v) ethanol (pH 4.5). For “control” tubes, cells were harvested and resuspended in 1 ml of 50 mM EDTA (pH 7.0). Tubes were vortexed and allowed to settle for 10 minutes. After sedimentation period, 200 µl samples were taken from just below the meniscus and dispersed in 800 µl 50 mM EDTA. The absorbance at 600 nm was measured using a spectrophotometer and percentage of flocculation was determined from the difference in absorbance between control and flocculation tubes according to equation (1). All analyses were performed as triplicate. 1.6 UV and EMS mutagenesis of Rhodotorula UV Mutagenesis One inoculation loop of Rhodotorula cells were scraped from plate and resuspended to 1 ml of 0.9% NaCl. Cell suspension was diluted to 1:10000 and plated on YPD + 0.1% oleic acid + 0.1% Tween 80. Plates, excluding control plates, were exposed to different amounts of UV in completely dark room. Plates were protected from light and incubated at +30°C for 2–3 days until colonies appeared. Colonies were counted and number of colonies on UV treated plates were compared to the number of colonies on control plates. Amount of UV radiation that gave a cell survival of 5% was selected for use. EMS Mutagenesis Yeast strain was inoculated into 50 ml of YPD medium using a 250 ml flask and cultured at +30°C for 12 h with shaking. Cell density was determined with cell counter (LUNA II^TM Automated Cell Counter,). An amount of culture corresponding to 1 x 10⁸ cells was transferred into 15 ml Falcon tubes. Cells were centrifuged and washed with 5 ml DDIW and 5 ml 0.1 M sodium phosphate (NaH2PO⁴) buffer, and resuspended in 1.7 ml sodium phosphate buffer. Under a laminar hood, 50 µl EMS was added to each tube, except the non-mutagenized control, and the cells were incubated on a roller at 30°C for varying time points between 30 min and 2 h 30 min. At each time point, 8 ml of sterile 5% sodium thiosulfate was added to inactivate the EMS and to stop the mutagenesis. Cells were centrifuged and resuspended to 9 ml DDIW. Cell suspensions were diluted to 1:1000 and plated on YPD agar for 2–3 days until colonies appeared. Colonies were counted and number of colonies on EMS treated plates were compared to the number of colonies on control plates. Time point that gave a cell survival of 20% was selected for use. Screening of colonies When colonies appeared on UV and EMS mutagenesis plates, 20 colonies from each plate were randomly selected for screening. Colonies with different phenotype, like abnormal color, were always selected for screening. Lines from each colony were spread first on YPD + 0.1% oleic acid + 0.5% Tween80 plate and then on YPD plate without any supplements, in order to find colonies that are only able to grow on presence of oleic acid. 1.7 Cultivation of Rhodotorula strains Small scale cultures of the obtained Rhodotorula mutant strains were prepared in order to analyze their fatty acid compositions. The cultivations were done as shake flask cultures in 250 ml Erlenmeyer flasks with culture volumes of 50 ml. Strains were cultivated both on YPD and C/N 75 medium in duplicates. The medium composition of YPD and C/N 75 are described in section 1.2. For YPD cultures, each flask was inoculated with one loop of cells and cultures were incubated for 24 h at 30°C. For C/N 75 cultures, flasks were inoculated with starting OD₆₀₀ of 0.2 and cultures were incubated for 6 days at +30°C. 1.7.1 Collecting and drying of samples Cultures were transferred into 50 ml Falcon tubes, cells were centrifuged for 5 minutes at 4000 rpm, and pellets were washed twice with DDIW. Pellets were stored at -80°C until freeze drying. Frozen samples were freeze-dried with Christ Alpha 2-4 LSCBasic freeze-dyer for 24 h at 0.5 mbar. 1.8 Fatty acid profile analysis The analysis of fatty acid profiles of yeast biomass was performed by GC- MS. Samples of 5 mg of dried cell biomass samples were spiked with 20 µg of glyceryl triheptadecanoate and heptadecanoic acid. The samples were esterified by using 750 µl acidic methanol (with 3 N HCl) and heating for 45 min at +85°C. The fatty acid methyl esters were extracted into 1 ml of hexane by shaking for 1 minute. 1 µl of the separated hexane phase was injected into an Agilent 7890 GC coupled to 5975C MSD for gas chromatography- mass spectrometry analysis. Transport gas was helium with a constant flow rate of 1.2 ml/min. The injector temperature was +240°C and splitless mode was used. An HP-FFAP column (25 m × 0.25 mm × 0.33 μm) was used with a temperature program from +40°C (1.5 min) to +240°C (15°C/min, stay for 9 min). Interface temperature was +240°C and ion source temperature was +150°C. The mass spectra were recorded over a 35-600 atomic mass unit range at 3 scans/s. 1.9 Microscopy imaging Cell mass samples were investigated by confocal laser scanning microscopy (CLSM) using stains specific for beta-glucan, lipids, and protein. For staining and imaging, an aliquot of each sample was transferred in a 1 mm deep silicone well with diameter of 9 mm placed on a microscopy slide. Beta- glucan, lipids and protein present in the samples were stained by adding 100 µl of aqueous solution containing 0.01% (w/v) Calcofluor White (Fluorescent brightener 28, Aldrich), 0.005 % (w/v) Nile Red (N3013, Sigma; [103]) and 0.02% (w/v) Fast Green FCF (F7258, Sigma-Aldrich; [104]), respectively. After staining (5 min, +4°C), sealed samples covered with a sealed cover slip were imaged using CLSM equipment consisting of a Zeiss LSM 710 (Zeiss, Jena, Germany) attached to a Zeiss Axio Imager.Z microscope. Diode laser line of 405 nm, argon laser line of 514 nm and diode laser line of 633 nm were used for excitation of Calcofluor, Nile Red and Fast Green, and emissions were collected at 425-475 nm, 590-620 nm, and 640-720 nm, respectively. Images were assembled of the optical sections taken using a 63x objective (Zeiss LCI Plan-Neofluar, numerical aperture of 1.3) to the depth of 5.6-8.0 μm with 0.4 μm z step using ZEN software (Zeiss). The final CLSM micrographs, in which beta-glucans appear cyan, lipids yellow and protein red, were reconstructed by superimposing three emission images. Representative images were selected for the report. 2 Results 2.1 Phenotype investigation of P. pastoris strains expressing FLO5 and FLO5Mut Direct observation Two different P. pastoris strains expressing flocculins were constructed in this work, one with FLO5 and the other expressing its mutant variant FLO5Mut. When colonies of transformed P. pastoris strains appeared on SCD-URA plates, the flocculation phenotype was observed immediately. The transformed colonies showed higher adherence between cells and towards the plate compared to colonies of the untransformed control strain. In addition, the morphologies of transformants were more structured compared to smooth morphology of control colonies. Morphologies of the colonies can be seen in Figure 3. No differences between FLO5 and FLO5Mut transformed colonies could be observed. qPCR FLO5 gene copy numbers of five FLO5 and five FLO5Mut strains were confirmed by qPCR. All the ten strains were tested with FLO5 gene primers and reference gene primers. Used primers are listed in Table 5. The copy numbers of the FLO5 and FLO5Mut genes are presented in Figure 4. According to qPCR results, all transformed strains contained a correct gene construct. Copy numbers of FLO5 and FLO5Mut genes slightly varied between strains, while most of the strains contained single copies. Cultivation experiments Flocculation ability and phenotypes of the expression strains and control strain were investigated by cultivating the strains in YPD medium for 72 hours. As predicted, the control strain was not flocculating. All ten expression strains showed an ability to flocculate, but the flocculation phenotype, especially the size of the formed flocs, varied between strains. Strain FLO5_c2 had the weakest flocculation phenotype and barely flocculated, whereas many strains showed strong flocculation potential similar to each other. For example, all FLO5 strains flocculated similarly excluding the barely flocculating strain FLO5_c2. Among the strains expressing FLO5Mut, FLO5Mut_c1, c2, and c5 showed similar flocculating phenotype forming bigger and discrete flocs, whereas strains FLO5Mut_c3 and c4 formed notably smaller flocs. Overall, P. pastoris strains expressing FLO5Mut, a mutated variant of FLO5, formed bigger and more discrete flocs compared to strains expressing wild type FLO5. In Figure 5 different flocculation phenotypes of P. pastoris expression strains are presented. Difference in phenotypes between FLO5 and FLO5Mut strains was easiest to observe after a small sedimentation time. After sedimentation, FLO5 flocs sedimented to the bottom of the flask and formed a microbial mat, in which individual flocs were difficult to detect, whereas FLO5Mut flocs remained detached after a sedimentation and rarely formed microbial mats. This phenomenon can be seen in Figure 6. Since the aim was to obtain as strongly flocculating strain as possible, FLO5_c1 and FLO5Mut_c5 were selected for further use based on the phenotype investigations. 2.2 Flocculation measurements Flocculation assay was performed in order to investigate the extent of flocculation between strains. Flocculation ability is presented as percentage of flocculation comparing the free cells in a culture to the total number of cells in a culture. Table 6 shows typical flocculation abilities of non-flocculating, slightly flocculating, medium flocculating, and strongly flocculating strains. Typically, non-flocculating yeast strains exhibit flocculation rates between 0% and 15%, whereas strongly flocculating yeast strains will show flocculation in the range of rates 90–100%. Most industrial brewing yeasts flocculates somewhere between 40% and 90%. Table 6. Flocculation abilities of differently flocculating strains. Flocculation ability % Non-flocculating 0–15 Slightly flocculating 15–40 Medium flocculating 40–90 Strongly flocculating 90–100 2.2.1 Flocculation Flocculation ability of six different yeast strains were tested. The main focus was to study flocculation ability of Flo5 and Flo5Mut expression strains compared to their parental strain, P. pastoris H5659, not expressing Flo protein and to investigate whether there is a difference. The flocculation ability of P. pastoris H5659 was tested and as predicted the strain did not flocculate. Conversely, both Flo5 and Flo5Mut expression strains showed strong flocculation ability, and there were no notable differences in flocculation percentages between expression strains. Flo5 P. pastoris exhibited flocculation ability of 96.5% and Flo5Mut 97.5%. However, when treated with EDTA, Flo5 flocs disintegrated more clearly than FLO5Mut flocs. Table 7 presents results from flocculation assays of P. pastoris control strain and expression strains. Table 7. No. Strain Percentage of Flocculation flocculation (%) ability 1 P. pastoris H5659 control 2.4% ± 1% Non-flocculating 2 P. pastoris Flo5 96.5% ± 0.5% Strongly flocculating 3 P. pastoris Flo5Mut 97.5% ± 0.5% Strongly flocculating Another main interest was to study co-flocculation between Rhodotorula and P. pastoris expression strains. Therefore, flocculation ability of Rhodotorula was tested and as predicted the strain did not flocculate. To compare the co- flocculation ability between different yeast species (Rhodotorula, P. pastoris, S. cerevisiae), two other laboratory strains, P. pastoris H5085 and S. cerevisiae H4579 were selected as control strains for flocculation and co- flocculation experiments. P. pastoris H5085 showed a slight flocculation capacity, whereas S. cerevisiae H4579 was non-flocculating. Flocculation percentages of P. pastoris control, S. cerevisiae control, and Rhodotorula are presented in table 8. Table 8. No. Strain Percentage of Flocculation flocculation (%) ability 4 Rhodotorula 13.55% ± 1.75% Non-flocculating 5 P. pastoris control H5085 33.05% ± 0.35% Slightly flocculating 6 S. cerevisiae control 12.5% ± 1.3% Non-flocculating H4579 2.2.2 Co-flocculation In order to investigate co-flocculation, yeast strains were mixed together in ratios 1:1 and 5:1 in presence of calcium and flocculation abilities of mixed cultures were measured by flocculation assay. Since there was no notable difference in flocculation ability between P. pastoris FLO5 and FLO5M, FLO5Mut was selected for co-flocculation experiments. Results of co- flocculation measurements are presented in tables 9 and 10. Table 9. Co-Flocculation measurements. Strain 1 and strain 2 mixed in ratio 1:1. (n=2) Strain 1 Strain 2 Percentage of flocculation (%) P. pastoris H5659 Rhodotorula 8.5% (n=1) control P. pastoris Flo5Mut P. pastoris control 87% ± 0% P. pastoris Flo5Mut S. cerevisiae control 93.5% ± 2,5% P. pastoris Flo5Mut Rhodotorula 53% ± 2% Table 10. Co-Flocculation measurements. Strain 1 and strain 2 mixed in ratio 5:1. (n=2) Strain 1 Strain 2 Percentage of flocculation (%) P. pastoris Flo5Mut P. pastoris control 94% (n=1) P. pastoris Flo5Mut S. cerevisiae control 96% (n=1) P. pastoris Flo5Mut Rhodotorula 60% ± 1% 2.2.4 Microscopy Figures 13 A–C show confocal microscopy images (40 x objective) showing of a cluster of Pichia pastoris expressing Flo5Mut and Rhodotorula cells. A) beta-glucan (cell wall); B) intracellular lipids; C) proteins A = beta-glucan, B= lipids, C= proteins. 2.3 UV and EMS mutagenesis of Rhodotorula In order to find correct mutants producing more saturated fatty acids (C16: 0 and C18:0) and less (poly)unsaturated fatty acids (e.g. C18:1n9 and C18:2n6), UV mutagenesis was performed. Different amounts of UV radiation were tested to achieve a cell survival of approximately 5%, and 400 s x 1000 µJ was selected for use. Since no correct mutants were found via UV mutagenesis, the stronger EMS treatment was selected as a mutagenesis method. EMS treatment times from 30 min to 3 h were tested to find out a time point that gave a cell survival of 20%.1.5 h EMS treatment time fulfilled the criteria and was selected for use. Approximately 1500 UV colonies and 1500 EMS colonies were screened, but all screened colonies were able to grow on YPD without any supplements. Therefore, the selection based on the inability to grow in the absence of oleic acid could not be used and color was used as selection criteria for further analysis. In addition to colonies with abnormal color, colonies with color similar to that of wild type Rhodotorula were selected for analysis to test if the color correlated with fatty acid profile or lipid productivity. A total of 18 UV mutants and 10 EMS mutants were selected for further analysis. Selected strains can be seen in Figure 7. Lipid Analysis Batch 1 Rhodotorula mutant strains were cultivated, and fatty acid profiles analyzed in two different batches. First, 20 UV mutants of Rhodotorula were cultivated on YPD and fatty acid profiles were analyzed. Mutant strains 9 and 10 did not grow. Figure 8 presents some examples of fatty acid profiles of UV mutants. Most of the UV mutated Rhodotorula strains shared a fatty acid profile similar to UV mutant 1, comprising mostly oleic acid C18:1n9 (60–70%), palmitic acid C16:0 (10%), linoleic acid C18:2n6 (10%), stearic acid C18:0 (5%), and alpha-linolenic acid C18:3n3 (5%). Fatty acid profiles of UV 5, UV 12, UV 13, and UV 15 were most different from others since they contained higher amounts of linoleic and alpha-linolenic acids and lower amount of oleic acid. Fatty acid methyl ester (FAME) content of all strains was quite low varying from 2.28% to 4.50%. No correlation between color and fatty acid composition or FAME content were detected. Batch 2 In the second batch, wild type Rhodotorula strain, UV mutants 13, 15, and 18, and 10 EMS mutated strains were cultivated both on YPD and C/N 75 media, and fatty acid compositions were analyzed. To confirm the results of first batch, UV mutants 13, 15 and 18 were cultivated and analyzed again. Cultivations were performed in two different media in order to see how media composition effects on fatty acid profiles and FAME content of strains. Figure 9 presents fatty acid composition of wild type strain and UV mutated strains 13, 15, and 18 cultivated on YPD and C/N 75. Interestingly, fatty acid profiles of UV 13, 15 and 18 strains were different between first and second batch. In the second batch, all strains displayed a fatty acid profile very similar to that of control strain. In addition, strains cultivated and analyzed in second batch did not contain alpha-linolenic acid but contained 10-nonadecenoic acid C19:1n9. However, effectiveness of media composition on fatty acid profiles could be detected. Cultivation on C/N 75 media increased C16:0 content and decreased C18:1n9 content of strains compared to strains cultivated on YPD. The same effect could be seen when comparing EMS mutated strains cultivated on YPD and C/N 75 media. Figure 10 presents fatty acid profiles of some of these strains. All EMS mutated strains had a similar fatty acid profile to that of control strain cultivated in the same media, except of strain EMS 9 that had similar fatty acid profile both on YPD and C/N 75. Compared to the target fatty acid profile, fatty acid profile of beef, EMS strains cultivated on C/N 75 media showed highest similarity. Fatty acid profiles of EMS mutated strains compared to that of beef is presented in Figure 11. 2.3.1 Influence of media composition on FAME content Cultivation on C/N 75 media increased FAME content of strains approximately by 335%. For example, FAME content of wild type Rhodotorula strain increased from 3.84% to 16.69% when cultivated on C/N 75. FAME contents of strains can be seen in Table 11. Table 11. FAME content of Rhodotorula mutants and wild type strains cultivated on YPD and C/N 75 media. Rhodotorula Sample Cultivation Media FAME content (%) Wild type (control) YPD 3.84 UV mutants YPD 3–6 EMS mutants YPD 3–6 Wild type (control) C/N 75 16.69 UV mutants C/N 75 14–17 EMS mutants C/N 75 13–17 Evaluation of flocculation FLO5_c2 had the weakest flocculation phenotype, which is supported by the qPCR results. Otherwise there were no obvious differences in expression between single copy and multi copy strains since for example FLO5Mut5 multicopy and single copy FLO5Mut1 flocculated similarly. Flocculation varied between strains regardless of whether FLO5 or FLO5Mut was expressed. Mutated variant of FLO5 formed bigger flocs, but no difference was detected in flocculation percentage. Great excess of non-flocculant cells resulted in the absence of flocculation, such as ratios of 3:1 to 5:1. Non-flocculent cells were not strictly trapped inside the flocs but rather interacted with them. From all FLO genes, FLO1 is known to promote more cell-cell adhesion between different yeast species, whereas overexpression of FLO5 leads to a species-specific adhesion. In a mixture of FLO1 expressing cells and cells that do not express FLO1, the latter are expected to make up the outer layer of cells in yeast flocs. 2.3.2 Hardness test Pichia flocs cocultivated with Rhodotorula were tested for hardness. Figure 12 shows flocs applied to a support of the test equipment and ready to be compressed with the cylindrical probe seen above. The hardness of the flocs was evaluated using a uniaxial compression test conducted with a Texture Analyser (TA.XTPlus, Stable Micro Systems Ltd., UK). A cylindrical probe (P/20P) with a diameter of 20 mm was employed to compress individual flocs. The compression was carried out at a speed of 2 mm/s until a strain of 50% was achieved. The maximum force exerted during the compression process was measured and recorded as the hardness of the flocs. Individual floc pieces had hardness value (maximum force under uniaxial compression at constant strain) approximately three times higher (6.7±0.9 vs 2.2±0.2 N) than raw mince-meat which was shaped similarly to flocs. However, when cluster of flocs was measured with the same method the force value was 3.5 N. 3 Conclusions It was confirmed that yeasts can be successfully used for preparing meat substitute food products. Flocculation is helpful for separation, collecting biomass, and crosslinking if also other proteins/compounds/adhesins are expressed, such as Pichia expressing flocculins and other proteins. Rhodotorula mutants obtained by genetic engineering are useful for obtaining desired mutants and to improve lipid productivity. Example 2: Preparation and characterization of microbial patties Cultivation of microbial biomass Cocultivation of microbial strains with the following combinations (Table 12) were performed in shake flasks. Strains were grown on cultivation medium containing 10 g/l yeast extract, 20 g/l bacto peptone, 30 g/L gelatine and 20 g/l glucose. Gelatine was used to increase homogenic filamentous growth and could be replaced with agar. Yeast strains were inoculated with approximately same number of cells (one loop of cell material from plate), and filamentous fungi were inoculated with 2*10⁵ spores per ml. Cell material of Pichia pastoris was first mixed with 1 ml of 0.9% NaCl solution, then mixture was added to the flask. Cocultivated strains were inoculated to the same 250 ml Erlenmeyer flasks containing 50 ml cultivation medium and grown in 30 °C with 200 rpm for 72h. Table 12. Cocultivation of yeast and filamentous fungal strain in following combinations. *Oleaginous (lipid producing) strain. Sample Yeast strain Filamentous fungal strain 1 Pichia pastoris FLO5mut H6194 Mucor circinelloides D-96563 * 2 Saccharomyces pastorianus A-62020 Mucor circinelloides D-96563 * 3 Pichia pastoris FLO5mut H6194 Rhizopus stolonifera D-96584 Control none Mucor circinelloides D-96563 * Microscopy of biomass Biomass was microscoped using a Nikon Labophot microscope with x10 magnification and equipped with a 23 mm ocular camera (Dino-Lite) to compare cell growth between samples. Figure 14 showed Pichia yeast cells incorporated with filament growth of Mucor in sample 1, while Saccharomyces cells were not. Filament length is also seen to be longer for Rhizopus compared to Mucor (Figure 15.). Initial frying trial Sample 3 (Pichia+Rhizopus) and Mucor control sample was fried in a pan with rape seed oil (Figure 16). Sample 3 resulted in a crispy, juicy looking patty. Preparation of patties Biomass of each shake flask was collected by filtering under vacuum onto a Whatman GF/B glass fibre filter paper. Biomass was poured into a round cutting mould (ø 2.8 cm) placed on the filter paper, creating a round shape of the biomass. Biomass was washed with 50 ml of RO-water. Produced biomass patties were further cut with a smaller round cutting mould (ø 2.2 cm) to remove the higher edges. 8 replicate shake flasks of each sample created 8 replicate patties. Patties of sample 1, 2 and control Mucor had even surfaces, which is important for texture analysis, and were therefore chosen for further characterization. Characterisation of patties The following samples were characterized: 1: Pichia pastoris + Mucor circinelloides 2: Saccharomyces pastorianus + Mucor circinelloides Control: Mucor circinelloides Texture A Texture Analyser (Stable Micro Systems Ltd.) was used to perform a puncture test to analyse the firmness of the fresh patties. The patties (n=4) were punctured with a 4 mm cylindrical probe at a speed of 2 mm/s to 50% of their initial height and the force required for compression was recorded. Measurements were carried out at five different positions on each patty. The maximum force measured during the test was defined as the firmness. The Saccharomyces+Mucor patties showed the highest firmness (Figure 17). The firmness of the Pichia+Mucor patties was somewhat higher than that of the Mucor control sample. It was noted that firmness is affected by the dry matter content of the samples. Moisture loss during heating The patties (n=4) were heated in an oven at 155ºC for 10 min (on filter paper) and weighed. The moisture loss was calculated as the ratio of the sample weight before heating to that after heating (x 100). The Pichia+Mucor patties showed the lowest and the Saccharomyces+Mucor patties the highest moisture loss during heating (Table 13). The moisture loss is affected by the water holding capacity of the sample matrix. A higher water holding capacity means that a lower amount of moisture is lost, which could help improve the juiciness of a food product. It was noted that Saccharomyces+Mucor patties were thinner than the others, which could have facilitated the moisture loss. Table 13. Moisture loss of microbial patties during heating. Sample Moisture loss (%) Pichia + Mucor 61.3 ± 0.9 Saccharomyces + Mucor 69.8 ± 2.0 Mucor 66.0 ± 2.0 Colour The color of the patties was measured with a Minolta before and after heating the patties in an oven at 155ºC for 10 min (as described above). The measurements were performed with a handheld colorimeter (Minolta Chroma meter, CR-200). L* (lightness), a* (green-red) and b* (blue-yellow) values were recorded according to the CIELAB color space system. The measurements were taken from the top surface of the patties (n=4) at three points of each patty. Heating caused an increase in especially the b* value (Table 14), the change being the greatest with the Saccharomyces+Mucor patties, followed closely by the Pichia+Mucor patties. The increase in the b* value means that yellowness of the patties increased a bit, possibly due to slight heat-induced browning. Table 14. Color of patties before and after heating in an oven at 155ºC for 10 min. Sample Before/after L* a* b* heating Pichia + Mucor before 80.5 ± 3.5 1.1 ± 0.5 8.7 ± 4.1 after 82.8 ± 4.2 1.0 ± 0.3 15.0 ± 4.0 Saccharomyces before 79.9 ± 3.0 0.9 ± 0.5 10.1 ± 6.0 + Mucor after 80.2 ± 4.5 1.0 ± 0.6 19.7 ± 6.0 Mucor before 83.6 ± 2.5 1.5 ± 0.2 13.1 ± 2.7 after 82.3 ± 3.7 1.0 ± 0.3 15.0 ± 4.0

Claims

Claims: 1. A method for preparing an alternative food product for animal-based food products, the method comprising -providing first fungal cells producing structural proteins causing and/or facilitating cell-cell adhesion, -providing oleaginous second fungal cells producing lipids, the oleaginous fungal cells having ability to synthesize and accumulate intracellular lipids to over 20% of their dry cell weight, -combining the first and the second fungal cells to obtain a mixture -culturing the mixture until the first and the second fungal cells are attached together by the structural proteins to obtain a matrix of edible biomass, and -harvesting the edible biomass. 2. The method of claim 1, comprising forming the harvested edible biomass into a food product, such as a food product having a shape. 3. The method of claim 1 or 2, comprising providing or adding the first fungal cells producing structural proteins first, and cultivating them, and after a period of time sufficient to produce structural proteins in effective amount for obtaining an attaching effect, providing or adding the oleaginous second fungal cells producing lipids. 4. An alternative food product for animal-based food products, comprising edible biomass comprising structural proteins causing and/or facilitating cell-cell adhesion produced by first fungal cells and lipids produced by oleaginous second fungal cells, the oleaginous fungal cells having ability to synthesize and accumulate intracellular lipids to over 20% of their dry cell weight, the first and the second fungal cells being attached together by the structural proteins. 5. The method of any of claims 1–3 or the food product of claim 4, wherein the fungal cells comprise yeast cells and/or filamentous fungi cells. 6. The method of any of claims 1–3 or 5 or the food product of any of claims 4–5, wherein the first fungal cells comprise cells of one or more of Saccharomyces, Kluyveromyces, Candida, Pichia, Eremothecium, Kazachstania, Yarrowia, Zygosaccharomyces, or Schizosaccharomycetes, such as species Saccharomyces cerevisiae, Saccharomyces pastorianus, Kluyveromyces lactis, Candida krusei (Pichia kudriavzevii), Pichia pastoris (Komagataella pastoris), Pichia kudriavzevii, Eremothecium gossypii, Kazachstania exigua, Yarrowia lipolytica, Zygosaccharomyces lentus, or Schizosaccharomyces pombe; or Aspergillus or Penicillium, such as species Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, or Penicillium chrysogenum; Trichoderma or Myceliophthora, such as species Trichoderma reesei, Myceliophthora thermophila; Mucor, such as species Mucor circinelloides or Mucor indicus, or Rhizopus, such as species Rhizopus stolonifer or Rhizopus oligosporus. 7. The method of any of claims 1–3 or 5–6 or the food product of any of claims 4–6, wherein the structural proteins comprise flocculin protein, such as FLO1 and/or FLO5 protein. 8. The method of claim 7 or the food product of claim 7 wherein the flocculin protein comprises a flocculin protein of Saccharomyces cerevisiae. 9. The method of any of claims 1–3 or 5–8 or the food product of any of claims 4–8 wherein the structural proteins comprise one or more proteins and polypeptides selected from elastin-like proteins and polypeptides. 10. The method of any of claims 1–3 or 5–9 or the food product of any of claims 4–9, wherein the structural proteins comprise one or more cellulose binding proteins, such as proteins comprising cellulose binding domains, preferably wherein the method comprises culturing the first fungal cells in the presence of cellulose. 11. The method of any of claims 1–3 or 5–10 or the food product of any of claims 4–10, wherein the second fungal cells comprise one or more yeast cells selected from genera Apiotrichum, Rhodotorula, Yarrowia, and Cutaneotrichosporon. 12. The method of any of claims 1–3 or 5–11 or the food product of any of claims 4–11, wherein the second fungal cells produce one or more lipids selected from plant lipids and animal lipids, such as one or more lipids selected from short and medium chain fatty acids. 13. The method of claim 12 or the food product of claim 12, wherein the one or more lipids comprise one or more saturated fatty acids, such as palmitic acid and/or stearic acid. 14. The method of claim 12 or the food product of claim 12, wherein the one or more lipids comprise one or more plant fatty acids, such as selected from one or more of omega-3 fatty acids and omega-6 fatty acids, for example selected from α-linolenic acid, eicosapentaenoic acid and/or docosahexaenoic acid. 15. The method of any of claims 1–3 or 5–14 or the food product of any of claims 4–14, where the fungal cells produce a colored compound, such as a pigment, for example one or more carotenoids and/or anthocyanins.