Novel food

Abstract

The present invention relates to a composition comprising thermophilic fungal biomass for use as an ingredient in food, beverages, pet food, or animal feed. Due to its nutritional composition, thermophilic fungal biomass can be used as a source of protein and / or amino acids, including essential amino acids, lipids, oleic acid and linolenic acid, dietary fiber, vitamins such as choline and vitamin E, and minerals such as iron. Thermophilic fungal biomass is available as a compressed biomass cake or as a dry powder. Therefore, thermophilic fungal biomass is suitable as an ingredient in meat substitutes and hybrid meat products. In the form of a dry powder, thermophilic fungal biomass can be easily mixed with other ingredients to produce bakery products such as bread or soup.

Description

Field of the Invention

[0001] The present invention relates to the field of food supplements and to the field of replacements for use in both human food and animal feed. Background of the Invention

[0002] There is an increasing demand for edible products that can provide a high protein content derived from non-animal sources. Driven by the growing awareness of personal health, edible products containing non-animal source components such as protein and fiber are considered healthier alternatives to animal protein-based products. In particular, there is an increasing demand for edible meat substitutes that mimic meat in their composition and texture but consist of non-animal components, which can reduce reliance on animals such as cows and reduce the carbon footprint associated with such animals.

[0003] Single cell protein (SCP) is an interesting alternative to meat protein and is a source of protein in many other food applications such as breakfast cereals, bread, pasta, dairy products, ice cream, chocolate and soups. When fungal SCP is produced, it can be manufactured from sugar-rich crops in a more sustainable way compared to meat production, because SCP produces more tons of protein per hectare compared to meat production and has lower nitrogen emissions. One way to produce a dietary source of protein for human food or animal feed is to generate "single cell protein" (SCP) by means of fermentation (Suman et al., 2015, Int J.Curr.Microbiol.Appl.Sci., Vol. 4, No9, pp. 251-262). Fermentation in this context is understood to be the microbial conversion of carbon-rich feed into a protein-rich product consisting of microbial cells such as bacteria, yeast or fungi. The use of SCP as animal feed and food ingredient offers the further advantage that microbial cells have a high content of essential amino acids. Furthermore, fungal cells in particular can be extremely rich in trace elements and vitamins, making the fermented feed very nutritious.

[0004] SCPs are already used in food for human consumption. For example, Quorn (trademark) contains mycoproteins produced as SCPs by the fermentation of the fungus Fusarium venenatum, and contains vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), and biotin (www.mycoprotein.org). Quorn is available in a variety of forms, i.e., meat-like forms, such as sausages, cutlets, burgers, patties, and strips.

[0005] One problem with SCP production is the concentration of SCP-biomass produced in the fermentation broth, particularly in cases of liquid fermentation with bacteria or yeast. Another problem is the need for expensive enzymes to convert inexpensive polymer carbon sources into monomeric fermentable sugars. Furthermore, when using mesophilic bacteria for SCP production, sterilization fermentation conditions must be applied to prevent infection, which leads to prohibitive operational costs due to high capital investment and energy demands (WO2018 / 029353). Some of these problems have been explored by enabling the use of non-sterilization conditions using liquid fermentation with thermophilic fungi at high temperatures and acidic pH, and by selecting thermophilic fungi that combine the desirable properties of good growth at high temperatures and acidic pH with a sieved form and high protein content (WO2018 / 029353).

[0006] However, in addition to the meat-like forms currently available, there remains a need for non-animal protein sources that offer a wider range of applications.

[0007] [Overview of the prefecture] In a first aspect, the present invention relates to the use of a composition comprising thermophilic fungal biomass as a source of at least one of the following: a) proteins and / or amino acids, b) lipids, c) dietary fiber, d) choline and / or vitamins and e) minerals, in at least one of the following products: food, beverages, pet food and feed products.

[0008] In preferred uses of the present invention, a composition containing thermophilic fungal biomass is a cake that can be obtained by a method comprising: a) growing a strain of thermophilic fungus in liquid culture in a culture medium; b) recovering fungal biomass from the culture medium by at least one of sieving, filtration and decantation to produce a biomass cake, thereby preferably having a dry matter concentration of at least 12(w / v)% of the sieved, filtered or decanted biomass cake; and c) optionally further drying the biomass cake by pressing out any remaining water to obtain a biomass cake having a dry matter concentration of at least 20%; or a composition containing thermophilic fungal biomass is a dried powder that can be obtained by flash drying the biomass cake obtained in claim 2 and further drying it with warm air, preferably by freeze-drying under vacuum, to a biomass powder with a moisture content of 7(w / w)% or less.

[0009] In preferred use according to the present invention, fungal strains include Rasamsonia, Talaromyces, Penicillium, Acremonium, Humicola, Paecilomyces, Chaetomium, Rhizomucor, Rhizopus, Thermomyces, Myceliophthora, Thermoascus, and Thielavia. A strain of a fungal genus selected from the group consisting of Mucor, Stibella, Melanocarpus, Malbranchea, Dactylomyces, Canariomyces, Scytalidium, Myriococcum, Corynascus, and Coonemeria, more preferably the genus is Rhizomucor, more preferably the strain is Rhizomucor pusillus, most preferably the strain is Rhizomucor pusillus CBS143028, or a single colony isolate or a derivative thereof.

[0010] In preferred uses of the present invention, the protein and at least one of the amino acids comprises an amino acid selected from the group consisting of aspartic acid, asparagine, threonine, serine, glutamic acid, glutamine, proline, glycine, alanine, citrulline, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine, cysteine, methionine, and tryptophan, preferably an essential amino acid, more preferably an essential amino acid selected from the group consisting of threonine, valine, isoleucine, leucine, phenylalanine, lysine, histidine, methionine, and tryptophan, most preferably found in vegetarian and vegan foods. A more preferred use of the present invention is: a) the biomass of thermophilic fungi is used in vegetarian or vegan foods as a (dietary) source of at least one of proteins, amino acids, and essential amino acids; b) the biomass of thermophilic fungi is used as a (dietary) source of citrulline in foods, food supplements, or beverages for sports enthusiasts; or c) the biomass of thermophilic fungi is used in pet foods as a source of proteins, amino acids, and essential amino acids, preferably as a substitute for standard animal or plant sources of proteins and amino acids, and more preferably as the sole source of proteins and / or amino acids.

[0011] In a preferred use of the present invention, the biomass of thermophilic fungi is used as a source of dietary fiber to enrich the fiber content of foods selected from i) meat substitutes, ii) hybrid foods containing meat and non-meat ingredients, iii) carbohydrate-rich foods, iv) bakery products made from refined flour, and v) pet food, preferably the dietary fiber containing chitosan. In a more preferred use of the present invention, the biomass of thermophilic fungi is used as a source of dietary fiber in human or pet food to provide satiety to humans or pets who need weight management or weight loss, preferably the dietary fiber containing chitosan.

[0012] In a preferred use according to the present invention, the biomass of thermophilic fungi is used as a source of choline in vegan or vegetarian foods, or in pet foods, preferably cat or dog foods.

[0013] In a preferred use according to the present invention, thermophilic fungal biomass is used as a source of at least one of retinol, vitamin E, riboflavin, pyridoxine, and folic acid, and preferably, the biomass is used as a retinol source in cat food.

[0014] In a preferred use of the present invention, the biomass of thermophilic fungi is used as an iron source, preferably as a substitute for soybeans as an iron source, and more preferably as a substitute for soybeans as an iron source in a meat substitute.

[0015] In a preferred use of the present invention, the biomass of thermophilic fungi is used as a source of at least one of monounsaturated fatty acids and polyunsaturated fatty acids, and preferably, the biomass thermophilic fungi is used as a source of at least one of oleic acid and linoleic acid.

[0016] In a preferred use according to the present invention, thermophilic fungal biomass is used to be incorporated into food, beverage, pet food and feed products in either cake or powder form at a maximum level of 70 (w / w)% and a minimum level of at least 1 (w / w)%. In preferred uses according to the present invention, the biomass of thermophilic fungi is: a) a meat substitute comprising 42 (w / w) or less of thermophilic fungal biomass in cake form, or 14 (w / w) or less of thermophilic fungal biomass in powder form; b) a hybrid meat product comprising 52 (w / w) or less of thermophilic fungal biomass in cake form, or 17.3 (w / w) or less of thermophilic fungal biomass in powder form; c) a cereal-based food comprising 30 (w / w) or less of thermophilic fungal biomass in cake form, or 10 (w / w) or less of thermophilic fungal biomass in powder form. Albase food, d) ready-to-eat meals containing 16(w / w) or less of thermophilic fungal biomass in cake form, or 5.3(w / w) or less of thermophilic fungal biomass in powder form, e) ready-to-eat soups containing 27(w / w) or less of thermophilic fungal biomass in cake form, or 9(w / w) or less of thermophilic fungal biomass in powder form, and f) food for sports enthusiasts containing 70(w / w) or less of thermophilic fungal biomass in cake form or powder form, to be used as an ingredient for food for sports enthusiasts.

[0017] In a second aspect, the present invention relates to a food comprising a composition containing the biomass of a thermophilic fungus as defined herein, a) a meat substitute comprising 42 (w / w) or less of thermophilic fungal biomass in cake form, or 14 (w / w) or less of thermophilic fungal biomass in powder form, b) a hybrid meat product comprising 52 (w / w) or less of thermophilic fungal biomass in cake form, or 17.3 (w / w) or less of thermophilic fungal biomass in powder form, c) a cereal-based food comprising 30 (w / w) or less of thermophilic fungal biomass in cake form, or 10 (w / w) or less of thermophilic fungal biomass in powder form The present invention relates to foods that are: d) cereal-based foods containing fungal biomass; e) ready-to-eat meals containing 16% (w / w) or less of thermophilic fungal biomass in cake form, or 5.3% (w / w) or less of thermophilic fungal biomass in powder form; e) ready-to-eat soups containing 27% (w / w) or less of thermophilic fungal biomass in cake form, or 9% (w / w) or less of thermophilic fungal biomass in powder form; and f) foods for sports enthusiasts containing 70% (w / w) or less of thermophilic fungal biomass in cake form or powder form.

[0018] [Description of the invention] definition definition Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure belongs. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. In fact, the present invention is by no means limited to these methods.

[0019] For the purposes of this invention, the following terms are defined below.

[0020] As used in the present invention, the term "and / or" indicates that one or more of the described cases may occur individually or in combination with at least one of the described cases up to all of the described cases.

[0021] When used in this invention, a specific value accompanied by "at least" means that specific value or more. For example, "at least 2" is understood to be the same as "2 or more," i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.

[0022] When the words "about" or "approximately" are used in relation to a number (for example, about 10), it preferably means that the value may be more than or less than 0.1% of the given value (of 10).

[0023] The term "unicellular protein" is abbreviated as "SCP" and is understood herein to mean biomass essentially consisting of cells of organisms that exist in a unicellular or single-cell state, including unicellular bacteria, yeasts, fungi, or algae, and this biomass, preferably in a dried form, is suitable as a dietary source of protein or protein supplements in human food or animal feed.

[0024] In this specification, "fungus" is defined as a eukaryotic microorganism, including all species of the subdivision Eumycotina (Alexopoulos et al., 1962, Introductory Mycology, John Wile & Sons, Inc., New York). The term "fungus" thus includes both filamentous fungi and yeasts. In this specification, "filamentous fungi" is defined as a eukaryotic microorganism including all filamentous forms of the subdivisions Eumycotina and Oomycota (Hawksworth et al., Ainsworth and Brisby's Dictionary of the Fungi, 7th edition, Commonwealth Mycological Institute, Kew, Surrey, 1983). Filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation, and carbon metabolism is obligate aerobic. The thermophilic fungi used in this invention are fungi that grow at temperatures of at least 45°C, and occasionally above 56°C.

[0025] The terms “fermentation” or “fermentation process” are defined herein broadly, according to their common definition used in industry, as any (large-scale) microbial process occurring in the presence or absence of oxygen, comprising the cultivation of at least one type of microorganism, thereby preferably the microorganism producing a useful product at the expense of consuming one or more organic substrates. Thus, the term “fermentation” herein has a much broader definition than its stricter scientific definition, which defines it as a limited microbial process in which microorganisms extract energy from carbohydrates in the absence of oxygen. Similarly, the term “fermentation product” is defined herein broadly as any useful product produced in a (large-scale) microbial process occurring in the presence or absence of oxygen.

[0026] The term "TOTOX value," an abbreviation for total oxidation value, is understood to indicate the oxidative load to which a fat has been exposed. The TOTOX value is a number of no magnitude used to describe the total oxidative load to which a fat has been exposed. This value is calculated as the sum of the anisidine values ​​and twice the peroxidation value. The anisidine value (AnV) is a measure of aldehyde production during the oxidation of a fat. This value represents the absorption of the fat that reacted with p-anisidine (a common name for p-methoxyaniline) under specific conditions. This is used to characterize the oxidation history of a fat because aldehydes usually originate from the oxidation of unsaturated fatty acids. The peroxidation value (PV) is a measure of the degree of oxidation of a fat or oil. This value indicates the quality of the oxidizing agent, usually a hydroperoxide, that releases iodine from potassium iodide under specific conditions. PV is expressed as milliequivalents of reactive oxygen species per kg of fat (Beare-Rogers et al., Lexicon of lipid nutrition (IUPAC Technical Report), 2009, DOI:10.1351 / pac200173040685). Oils with a TOTOX value of less than 80 have been described as having a good aroma rating (JP2014054248). Detailed description of the invention

[0027] The inventors have found that biomass derived from thermophilic filamentous fungi such as Rhizomucor pusillus strain, produced through a fermentation process in which simple carbohydrates and inorganic nitrogen sources are converted into high-nutrient biomass during that time, can be compressed (referred to as "cake") and finally dried to obtain a powder. The fungal biomass thus obtained is considered a novel food within the European Union (EU) if its source, for example Rh. pusillus, has no history of human consumption within the EU. For example, fungal biomass in cake form and powder form can be used as materials in various food categories at different usage levels. Thanks to its texture, the fungal biomass of the present invention becomes a versatile material for many foods. Fungal biomass in cake form represents a valuable material for meat substitutes and hybrid meat products. Fungal biomass in its powder form can be easily mixed with other materials and, thanks to its powdery texture, can be used to produce bakery products such as bread or soup. Further, the fungal biomass of the present invention can be used as a substitute for or to improve the nutritional value of feed materials of animal and plant origin.

[0028] In addition, the inventors have found that the fungal biomass of the present invention contains a number of valuable nutritional components and can thus be used as a dietary source for these components.

[0029] In a first aspect, the present invention thus relates to the use of a composition comprising biomass of a thermophilic fungus as a source of at least one of a) protein and / or amino acids, b) lipids, c) dietary fiber, d) choline and / or vitamins, and e) minerals in one of food, beverage, pet food and feed products. Preferably, the composition is used as a dietary source of at least one of a) protein and / or amino acids, b) lipids, c) dietary fiber, d) choline and / or vitamins, and e) minerals in one of food, beverage, pet food and feed products. The term "dietary source" as used herein refers to the consumption of a product containing a particular dietary component.

[0030] A composition containing thermophilic fungal biomass for use according to the present invention is preferably obtained or may be obtained by one of the following methods.

[0031] In one embodiment, the composition containing the biomass of a thermophilic fungus is a cake obtained or obtainable by a method comprising: a) growing a strain of thermophilic fungus in liquid culture in a culture medium; b) recovering the fungal biomass from the culture medium by at least one of sieving, filtering and decanting to produce a biomass cake, thereby preferably having a dry matter concentration of at least 12(w / v)% of the sieved, filtered or decanted biomass cake; and c) optionally further drying the biomass cake by pressing out any remaining water to obtain a biomass cake having a dry matter concentration of at least 20%.

[0032] In another embodiment, the composition containing thermophilic fungal biomass is a dried powder obtained or obtainable by the method specified above, further comprising step d), which involves grinding the biomass cake obtained in b) or c) above and further drying it with warm air, by freeze-drying, preferably under vacuum, or by flash-drying, to a biomass powder with a moisture content of preferably 7(w / w)% or less. More preferably, the moisture content is 5(w / w)% or less.

[0033] Methods, conditions, and media for growing thermophilic fungi in liquid culture in a culture medium are described in detail in WO2018 / 029353 or U.S. Patent No. 2019174809, which are incorporated herein by reference. Preferably, the method comprises the step of growing thermophilic fungi in a culture medium containing a fermentable carbon-rich feed, a nitrogen source, and further components necessary for fungal growth. Preferably, in step a), the fungi are grown in liquid culture. Preferably, in step a), the fungi are grown under non-sterilizing conditions. Preferably, in step a), the fungi are grown at a temperature of 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55°C or higher. Preferably, in step a), the fungus is allowed to grow at pH levels of 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, or less than 2.5.

[0034] In one embodiment of the method of the present invention, a strain of thermophilic fungus is preferably grown or cultured in a chemically defined medium. The term "chemically defined" is understood to refer to a fermentation medium that essentially consists of chemically defined components, i.e., a fermentation medium in which the chemical composition of essentially all chemical substances used in the medium is known.

[0035] Therefore, in one embodiment, the thermophilic fungal strain is cultured in a chemically defined medium consisting of a carbon source, a nitrogen source, and further components necessary for fungal growth.

[0036] The carbon source preferably includes or consists of at least one of carbohydrates and organic acids. Preferably, the carbohydrate includes at least one source from glucose, fructose, galactose, xylose, arabinose, rhamnose, fucose, galactose, and mannose, of which glucose and fructose are preferred, with glucose being the most preferred. For example, suitable carbohydrate carbon sources including a source of glucose and / or fructose include maltose, isomaltose, maltodextrin, starch, glucose syrups (e.g., corn syrup such as HCFS), inverted (sucrose or sugar beet) sucrose, crude starch, liquefied starch (e.g., liquefied using alpha-amylase, e.g., Liquozyme (Novozymes) or Veretase (BASF)), inulin, raffinose, melibiose, and stachyose. Organic acids that may be included in the carbon source include lactic acid, acetic acid, galactoturonic acid, and gluconic acid.

[0037] The nitrogen source in the chemically defined culture medium used in the method of the present invention preferably comprises or consists of at least one of the following: uric acid, ammonia, nitrate, ammonium salts such as ammonium sulfate, ammonium phosphate, and ammonium nitrate, and amino acids such as glutamate and lysine. More preferably, the nitrogen source is selected from the group consisting of ammonia, ammonium sulfate, and ammonium phosphate. Most preferably, the nitrogen source is ammonia.

[0038] Further components necessary for fungal growth include catalytic elements, such as those that are components of enzymes or enzyme cofactors. These elements include, for example, magnesium, iron, copper, calcium, manganese, zinc, cobalt, molybdenum, selenium, and potium. In addition to the structural catalytic elements mentioned above, cations, such as potassium and / or sodium, are preferably present to function as counterions and control intracellular pH and molar osmotic pressure. A suitable mineral composition for the chemically defined culture medium of the present invention is described in U.S. Patent No. 20140342396A1, which is incorporated herein by reference.

[0039] Compounds that may be optionally included in (chemically defined) culture media for fungal growth are chelating agents, such as citric acid, and buffers, such as monopotassium phosphate and dipotassium phosphate, calcium carbonate, and vitamins. Buffers are preferably added only when handling methods without external pH control. Vitamins refer to a group of structurally unrelated organic compounds that may be necessary for the normal metabolism of thermophilic fungi. Fungi are known to vary widely in their ability or inability to synthesize the vitamins they require. Vitamins should only be added to the fermentation media of fungal strains that are incapable of synthesizing the vitamins. Typically, chemically defined fermentation media for lower fungi, such as Mucorals, may require supplementation with one or more vitamins. Higher fungi often do not have vitamin requirements. The vitamins are selected from the group consisting of thiamine, riboflavin, pyridoxal, nicotinic acid or nicotinamide, pantothenic acid, cyanocobalamin, folic acid, biotin, lipoic acid, purines, pyrimidines, inositol, choline, and hemin. In preferred embodiments, however, the thermophilic fungi grown in the method of the present invention are strains that do not require the presence of any vitamins in the chemically defined culture medium. In fact, the inventors have found that species of the genus Rhizomucor, such as strains Rhizomucor psyllus CBS143028, Rhizomucor miehei CBS143029, and Rhizopus species CBS143160, do not require any vitamins even when grown on mineral culture media.

[0040] In a more preferred embodiment, the fungus is grown in step a) in the culture medium specified herein in the presence of an antifoaming agent. Antifoaming agents are generally well known in the art (see, for example, https: / / en.wikipedia.org / wiki / Defoamer). Antifoaming agents are chemical additives that reduce and inhibit foam formation in liquids by industrial methods, such as fermentation broth. Strictly speaking, antifoaming agents remove existing foam, while antifoaming agents prevent further foam formation, but the terms “antifoaming agent,” “antifoaming agent,” and “defoaming agent” are used interchangeably herein. Antifoaming agents suitable for use in the fermentation method of the present invention are preferably at least one of oil-based antifoaming agents, polyalkylene glycol-based antifoaming agents, and silicon-based antifoaming agents. Examples of such ingredients include vegetable oils or mineral oils, as well as animal fats, polypropylene glycol (PPG) or polyethylene glycol (PEG), and Antifoam C100K (Basildon Chem.Comp.Ltd., Abingdon, Oxford, United Kingdom). The defoaming agent is preferably a food-grade defoaming agent. A preferred food-grade defoaming agent is a clean-label defoaming agent. A preferred defoaming agent for use in the methods of the present invention includes or consists of vegetable oils, preferably edible vegetable oils. Preferred (edible) vegetable oils for use as defoaming agents in the methods of the present invention are oils selected from the group consisting of canola oil, coconut oil, corn oil, cottonseed oil, olive oil, palm oil, palm kernel oil, linseed oil, peanut oil, safflower oil, soybean oil, sunflower oil, and high-oleic sunflower oil.

[0041] In one embodiment, the thermophilic fungus grown to produce a composition containing the biomass of a thermophilic fungus according to the present invention is preferably a filamentous fungus. Preferred thermophilic fungi for use in the present invention are strains of fungi selected from the group consisting of Lasamsonia, Talaromyces, Penicillium, Acremonium, Fumicola, Paechilomyces, Chaetomium, Rhizomucor, Rhizopus, Thermomyces, Mikeliophtra, Thermoasuchus, Tierravia, Mucor, Stivera, Melanocarpus, Malbranchea, Dactyromyces, Canariomyces, Scytheridium, Myriococum, Corinasuchus, and Koonemeria. More preferably, thermophilic fungi include Rasamsonia composticola, Talaromyces emersonii, Rhizomucor miehei, Rhizomucor pusilus, Thermomucor indica-seudaticae, Thielavia terricola, Thielavia terrestris, and Thermoascus thermophilus. The strains are selected from the group consisting of (thermophilus), and among them, strains of Lasamsonia composticola CBS141695 (deposited on July 29, 2016), Thermomucor indicae-ceudaticae CBS143027 (deposited on July 21, 2017), Rhizomucor miehei CBS143029 (deposited on July 21, 2017), Rhizomucor pusilus CBS143028 (deposited on July 21, 2017), Thermoasuchus thermophilus CBS528.71 and Tierrawia terrestris CBS546.86 are preferred.

[0042] In preferred embodiments, the thermophilic fungus is a strain of the class Zygomycete, among which the family Mucoraceae is preferred. More preferably, the thermophilic fungus is a strain of a genus selected from the genera Mucor, Rhizomucor, and Rhizopus, among which the genus Rhizomucor is preferred. Most preferably, the thermophilic fungus is a strain of a species selected from the species R. endophyticus, R. miehei, R. pakistanicus, R. tauricus, R. variabilis, and R. psyllus, among which the species Rhizomucor psyllus is preferred. Preferred strains of the aforementioned thermophilic fungi for use in the present invention include Rhizomucor psyllus CBS143028, Rhizomucor meihei CBS143029, and Rhizopus species CBS143160 (deposited on August 11, 2017), deposited at the Westerdijk Institute for Mycological Diversity, Uppsalalaan8, 3584CT Utrecht, Netherlands (formerly known as Centraalbureau voor Schimmelcultures, CBS) under the rules of the Budapest Convention, of which Rhizomucor psyllus CBS143028 is the most preferred.

[0043] In one embodiment, the method of the present invention is a batch method, and more preferably, at least step a) of the method is carried out as a batch method. In a preferred embodiment, however, the method of the present invention, or preferably at least step a) of the method, is carried out as a feed batch method, a repeated feed batch method (where a portion of the fermentation broth is harvested each time), or a continuous method. In one embodiment, the method in step a) is a carbon-limited method, and preferably, the carbon source is supplied at a growth limit rate by continuous or intermittent supply.

[0044] In one embodiment, following fermentation, the fungal biomass may be pasteurized at a temperature of at least 60°C, as described in the concurrently pending application EP20161554.9. Preferably, the biomass is pasteurized at a temperature of at least 70°C for a maximum of 45 minutes. In a preferred embodiment, pasteurization is carried out in an inline heating unit preferably comprising a pipe heater, heating block, or steam infusion element, more preferably a steam infusion element, where the inline heating unit optionally comprises a mixing element such as a static mixer. Preferably, pasteurization is carried out for a maximum of 5 minutes, preferably about 0.5 to about 3 minutes, more preferably about 1 to 2 minutes. Preferably, pasteurization is carried out at a temperature of at least 74°C, preferably at least 80°C, more preferably at least 86°C. Preferably, the microbial count of the pasteurized biomass is log at least 7, preferably at least 11, more preferably at least 12 compared to the supplied biomass before pasteurization. 10 It has a decrease. In a preferred embodiment, the biomass flows through an inline heating unit having a residence time of about 1 to 2 minutes at a temperature of about 86°C, where the pH of the biomass is preferably up to 4.5, more preferably up to 3.5.

[0045] In one embodiment, an antioxidant is added to the biomass. Preferably, the antioxidant is added before pasteurization of the biomass, as described in, for example, concurrent application with EP20161606.7. Preferably, the antioxidant is a free radical scavenger, e.g., carnosol; carnosic acid; rosemary extract; flavan-3-ol, e.g., epicatechin, epigallocatechin, epigallocatechin gallate (EGCG), epicatechin gallate; flavonoid, e.g., kaempferol, quercetin or myricetin; green tea extract; butylated antioxidant, e.g., butylated hydroxytoluene (BHT), butylated hydroxyanisole or tert-butylhydroquinone (TBHQ); gallic acid; gallic acid ester, e.g., propyl gallate; carvone; caffeic acid ester, e.g. The biomass composition is at least one of the following: methyl caffeate, ethyl caffeate, phenethyl caffeate (CAPE), or rosmarinic acid; spearmint extract; vitamin E, e.g., tocopherol or tocotrienol; vitamin A, e.g., retinol, retinoic acid, or retinal; provitamin A, e.g., carotenoids, β-carotene, lutein, zeaxanthin, or echinenone; quinolone antioxidants, e.g., ethoxyquin; or seaweed extract; or oxygen reducing agents, e.g., ascorbic acid or acerola extract; or chelating agents, e.g., citric acid or EDTA. Preferably, the resulting biomass composition has a TOTOX value of less than 50, preferably less than 35, and more preferably less than 20, where the TOTOX value preferably remains less than 50, more preferably less than 40, for at least 6 weeks.

[0046] Next, the fungal biomass is preferably separated from the fermentation medium. Therefore, in one embodiment, a method for preparing a composition containing thermophilic fungal biomass preferably further includes the step of recovering the fungal biomass (grown in step a) from the medium by at least one of sieving, filtration, and decantation. More preferably, the biomass is recovered from the medium by at least one of rotary drum filtration, filter press, belt filter, decanter centrifugation, and sieving. Preferably, the biomass is recovered by sieving on a sieve or screen having pores of 100, 200, 300, 400, or 500 μm, or pores of 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2 mm. The biomass may be recovered on the sieve or screen by at least two, three, or four consecutive rounds of sieving, thereby applying smaller diameter pores in each subsequent round of sieving. For example, a first round sieve with a pore diameter of 2 mm, followed by rounds of 1, 0.5 and / or 0.1 mm. Optionally, the dry matter concentration of the sieved, filtered, or decanted biomass (cake) is further increased by further removal of water, i.e., drying. The biomass cake may be further dried, for example, by pressing out most of the remaining water using compressed air with pneumatic pressure and / or mechanical compression using, for example, a belt press or screw press. The biomass cake thus obtained preferably has a dry matter concentration of at least 12, 15, 20, 25, 30, 35, 40, 45, or 5 (w / w)%.

[0047] In another embodiment, the biomass can be compressed into a cake, and the cake can optionally be crushed or extruded to allow for drying, preferably air-drying. Preferably, the particle size of the compressed mycelial biomass cake is reduced by physical means to allow for (more efficient) drying of the compressed cake. This can optionally be done by extruding the mycelial cake through holes having a diameter of 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, or 2 mm using an extruder known in the art. Alternatively, the particle size of the cake can be reduced by a combination of crushing and sieving. As a crushing step, any type of mill known in the art, such as a knife mill or a hammer mill, can be used. To obtain a homogeneous particle size in the pressed and ground cake, larger particles still present after grinding can be removed before drying by sieving with pore diameter sizes of 0.5, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, or 3 mm sieves. The resulting ground cake is preferably considered to have a particle size between 1 mm and 3 mm before drying. Drying of the extruded or ground cake is preferably carried out at a temperature of 30-70°C. Hot air is then used to gently and cost-effectively dry the cake in a belt dryer or fluidized bed dryer. High-temperature steam drying (e.g., >80°C) is preferably avoided because it may negatively affect the digestibility of proteins by denaturing amino acids, baking, and even chemical decomposition through the Maillard reaction. Alternatively, the extruded or ground powder may be dried under vacuum in a freeze-drying process or by flash drying. The biomass obtained in this way, in the form of a dry powder, preferably has a moisture content of 5%, 4%, 3%, 2%, or 1% (w / w) or less.

[0048] Thermophilic fungal biomass in cake form may be frozen and stored at -18°C, while powder form can be stored at room temperature.

[0049] Proteins and amino acids In one embodiment, the composition comprising the biomass of a thermophilic fungus of the present invention is used as a (dietary) source of protein. Preferably, the protein comprises amino acids selected from the group consisting of aspartic acid, asparagine, threonine, serine, glutamic acid, glutamine, proline, glycine, alanine, citrulline, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine, cysteine, methionine, and tryptophan.

[0050] In one embodiment, the composition containing the biomass of a thermophilic fungus of the present invention is used as a (dietary) source of amino acids. In a preferred embodiment, the amino acids are selected from the group consisting of aspartic acid, asparagine, threonine, serine, glutamic acid, glutamine, proline, glycine, alanine, citrulline, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine, cysteine, methionine, and tryptophan.

[0051] Proteins represent the primary source of nitrogen and are essential amino acids in the human diet. Both are fundamental for tissue growth and maintenance.[3] The main sources of protein in the human diet are animal-derived (meat, fish, eggs, dairy products) or plant-derived, such as legumes, nuts, and cereal products. However, animal-derived proteins are among the highest quality in terms of amino acid composition. While all amino acids are important in the human nutrient, some cannot be produced in humans and can therefore only be introduced through diet. These amino acids are defined as essential amino acids. The amino acids that are essential for humans are threonine, valine, isoleucine, leucine, phenylalanine, lysine, histidine, methionine, and tryptophan.[3]

[0052] In one embodiment, the composition containing the biomass of a thermophilic fungus of the present invention is used as a (dietary) source of protein containing essential amino acids. Preferably, the essential amino acids are selected from the group consisting of threonine, valine, isoleucine, leucine, phenylalanine, lysine, histidine, methionine, and tryptophan.

[0053] In one embodiment, the composition containing the biomass of a thermophilic fungus of the present invention is used as a (dietary) source of essential amino acids. Preferably, the essential amino acids are selected from the group consisting of threonine, valine, isoleucine, leucine, phenylalanine, lysine, histidine, methionine, and tryptophan. More preferably, the composition containing the biomass of a thermophilic fungus of the present invention is used as a (dietary) source of all essential amino acids.

[0054] Citrulline malate is available and widely used by sports enthusiasts as a product to improve athletic performance and muscle recovery. Supplementation with L-citrulline has been shown to improve vascular function and, therefore, improve athletic performance and reduce muscle fatigue after exercise [4]. In addition to its high protein content and the presence of all essential amino acids, the fungal biomass of the present invention represents a good material for sports enthusiast foods thanks to its high citrulline content. In one embodiment, a composition comprising biomass derived from thermophilic fungi is used as a (dietary) source of citrulline. Preferably, the composition is used in sports enthusiast foods, food supplements or beverages.

[0055] The high protein content of the fungal biomass of the present invention makes it a good substitute for animal protein in vegetarian and vegan foods. In one embodiment, the thermophilic fungal biomass is therefore used in vegetarian or vegan foods as a (dietary) source of at least one of the proteins, amino acids, and essential amino acids as defined herein.

[0056] fiber In one embodiment, the composition containing the biomass of a thermophilic fungus according to the present invention is used as a (dietary) source of dietary fiber.

[0057] Dietary fiber is defined as indigestible carbohydrates that play a vital role in the function of the human gut. The adequate intake (AI) of dietary fiber for adults (both genders) for normal bowel movements has been set at 25 g / day according to the EFSA DRV. Dietary fiber intake higher than 25 g / day in adults has been shown to reduce the risk of coronary artery disease and type 2 diabetes and to help maintain weight. Furthermore, a high daily intake of dietary fiber has been shown to be associated with a lower risk of colorectal cancer [1]. Dietary fiber is fermented by bacteria residing in the large intestine, which play an important role in the microbial composition of the gut microbiota. Microbial fermentation of dietary fiber can supply many other micronutrients that can be absorbed by the human gut, such as vitamins and short-chain fatty acids (SCFAs) [2]. The main sources of dietary fiber in the human diet are whole grain cereals, vegetables, fruits, and potatoes.

[0058] The thermophilic fungal biomass of the present invention has a relatively high content of dietary fiber (10.6 g of dietary fiber / 100 g or 37% dry matter). The fungal biomass of the present invention is therefore an excellent source of dietary fiber for use in food, beverages, pet food, or feed products.

[0059] In particular, the high fiber content makes it possible to add dietary fiber to foods that are generally low in fiber. One example of this is the meat substitute made from fungal biomass of the present invention, in which the proteins of the thermophilic fungal biomass can replace the animal proteins contained in meat products, while at the same time, the fungal biomass enriches the meat substitute with dietary fiber, which is essentially absent in meat products.

[0060] In one embodiment, the thermophilic fungal biomass of the present invention is therefore used as a source of dietary fiber in foods that are meat substitutes or hybrid meat products. Due to its texture, the cake form of the thermophilic fungal biomass is particularly suitable as an ingredient for meat substitutes and hybrid meat products.

[0061] Similarly, dietary fiber and protein are essential micronutrients in pet food. The main companion animals, dogs and cats, have different requirements for dietary fiber and protein. Furthermore, the essential amino acids required in pet diets differ from those required in human diets. Recently, protein sources for pet food have been expressed as animal-derived materials (poultry, cattle, pigs, fish, eggs) or plant-based materials, such as corn, rice, beans, and soybeans. In addition, dietary fiber is important for the quality of stool, weight management, and satiety in both pets and humans [5].

[0062] In one embodiment, the thermophilic fungal biomass of the present invention is therefore used as a source of dietary fiber to enrich the fiber content of food. Foods that will be enriched with dietary fiber may be selected from i) meat substitutes, ii) hybrid foods containing meat and non-meat ingredients, iii) carbohydrate-rich foods, iv) bakery products made from refined flours, and v) pet food. More specifically, foods that will be enriched with dietary fiber may be selected from pizza and pizza-like dishes, savory pies and tarts, ready-to-eat soups, pasta and similar products, premixes (dried) for baking products, biscuits, cakes, pancakes, bread and rolls, pizza bases (cooked), gluten-free bread, porridge (dried / ready to eat), cereal bars, cereal flakes and similar products, muesli and breakfast cereal mixes, sports foods, weight-loss foods, dairy ice cream and similar products, fermented dairy products, chocolate and chocolate products.

[0063] In one embodiment, the thermophilic fungal biomass of the present invention is therefore preferably used as a source of dietary fiber in human or pet food to provide a feeling of fullness to humans or pets who need weight management or weight loss.

[0064] Micronutrients The thermophilic fungal biomass of the present invention has a high choline content (Table 8). Although choline can be newly synthesized by the human body, it still represents a basic component of the human diet because the amount newly produced is not always sufficient to meet the appropriate intake of 400 mg / day for adults (both sexes) [9]. In contrast, choline is an essential vitamin for dogs and cats. For adult cats, the minimum required choline nutrient level is 60 mg / kg metabolic body weight (BW), while for adult dogs it is 45 mg / kg metabolic BW

[11] . Therefore, the thermophilic fungal biomass of the present invention is a good source of choline for human and animal nutrition and can be used as a choline source, for example, in vegan foods, as a substitute for animal products such as eggs, dairy products and meat, which are generally good sources of choline.

[0065] In one embodiment, the thermophilic fungal biomass of the present invention is used as a (dietary) source of choline in vegan or vegetarian foods, or in pet food, preferably cat or dog food.

[0066] In addition to choline, the thermophilic fungal biomass of the present invention contains other vitamins essential for humans and / or animals, such as retinol (vitamin A), vitamin E, riboflavin (vitamin B2), pyridoxine (vitamin B6), and folic acid (vitamin B9 / B11) (see Table 8).

[0067] Vitamin E is known to have antioxidant activity.

[13] Vitamin B2, also known as riboflavin, is involved in a variety of reactions in the human body.

[14] Meanwhile, vitamin B6 plays an important role in different metabolic pathways, including amino acid metabolism, monocarbon metabolism, glycogenolysis and gluconeogenesis and heme synthesis.

[15]

[0068] In one embodiment, the thermophilic fungal biomass of the present invention is therefore used as a (dietary) source of at least one of retinol (vitamin A), vitamin E, riboflavin (vitamin B2), pyridoxine (vitamin B6), and folic acid.

[0069] Vitamin A plays an important role in the light conversion mechanism of the eye in both humans and animals

[12] . Vitamin A is an essential vitamin, especially for cats

[11] . Therefore, in a preferred embodiment, the thermophilic fungal biomass of the present invention is used as a (dietary) source of retinol in cat food.

[0070] mineral The thermophilic fungal biomass of the present invention has high concentrations of different minerals (Table 10). This is due to minerals added during the fermentation process, which are necessary for fungal growth. This means that the mineral composition of Rhizomucor pusilus biomass can be modified and / or standardized based on its use in different food or feed products.

[0071] In one embodiment, the composition containing the biomass of a thermophilic fungus of the present invention is used as a (dietary) source of minerals selected from the group consisting of calcium, magnesium, phosphorus, potassium, zinc, iron, copper, and manganese.

[0072] The iron (Fe) content in the fungal biomass of the present invention was particularly high, representing a limiting factor for intake assessment according to EFSA DRV. The amount of iron used in the fermentation of the fungal biomass of the present invention had to be further reduced to reach a final concentration of 14 mg / kg (Table 10).

[0073] The thermophilic fungal biomass of the present invention can therefore be considered a valuable iron source in other plant-based products from which meat substitutes are made, such as soybeans (see Table 11). Soybean products are typically included in vegetarian or vegan diets due to their high iron content. However, soybeans are not suitable for everyone's diet in the case of allergies. Soybeans are actually considered one of the major food allergens. The thermophilic fungal biomass of the present invention can be used to replace soybeans as an iron source in vegetarian and vegan diets. Thus, in one embodiment, the thermophilic fungal biomass is used as an iron source. In a preferred embodiment, the thermophilic fungal biomass is used to replace an iron source. In a more preferred embodiment, the thermophilic fungal biomass is used to replace soybeans as an iron source, and more preferably, the thermophilic fungal biomass is used to replace soybeans as an iron source in meat substitutes.

[0074] Lipids The thermophilic fungal biomass of the present invention contains approximately 8 (w / w)% fat on a dry matter basis, of which approximately 16-22 (w / w)% consists of saturated fatty acids, approximately 50-56 (w / w)% consists of monounsaturated fatty acids (mostly oleic acid), and approximately 27-28 (w / w)% consists of polyunsaturated fatty acids (mostly linoleic acid). In one embodiment, the biomass thermophilic fungus is therefore used as a source of at least one of monounsaturated fatty acids and polyunsaturated fatty acids. More preferably, the biomass thermophilic fungus is used as a source of at least one of oleic acid and linoleic acid.

[0075] Food, beverages, pet food and animal feed In a further embodiment, the present invention relates to food, beverage, pet food and feed products containing biomass derived from the thermophilic fungus of the present invention.

[0076] The thermophilic fungal biomass of the present invention, present in either the cake or powder described herein, can be used as an ingredient in different food categories with different usage levels. The maximum usage levels of the thermophilic fungus of the present invention in different food categories (Table 1) have been established based on the European Food Safety Authority (EFSA) Dietary Reference Intakes (DRVs) for macronutrients and micronutrients contained in the thermophilic fungus of the present invention, and further, with respect to intake assessments based on the background diets of different European populations.

[0077] In one embodiment, thermophilic fungal biomass is incorporated into at least one food or beverage at a level below or equal to the maximum food use level for thermophilic fungal biomass, based on the European Food Safety Authority (EFSA) Dietary Reference Intakes (DRV).

[0078] Therefore, thermophilic fungal biomass is used to be incorporated into food, beverage, pet food and feed products in either cake or powder form at a maximum level of 70, 65, 60, 55, 52, 50, 45, 42, 40, 35, 30, 25, or 20 (w / w)%. Preferably, the food, beverage, pet food and feed products contain at least 1, 2, 5, 10, or 15 (w / w)% of thermophilic fungal biomass in either cake or powder form.

[0079] In one embodiment, thermophilic fungal biomass is used as a material for a meat substitute, where preferably the meat substitute contains 42(w / w) or less of thermophilic fungal biomass in cake form, or 14(w / w) or less of thermophilic fungal biomass in powder form. Therefore, in one embodiment, the present invention relates to a food product which is a meat substitute containing 42(w / w) or less of thermophilic fungal biomass in cake form, or 13(w / w) or less of thermophilic fungal biomass in powder form. The meat substitute preferably contains at least 1, 2, 5, 10 or 15(w / w)) of thermophilic fungal biomass in cake form, or at least 0.5, 1, 2 or 5(w / w)) of a powder.

[0080] In one embodiment, thermophilic fungal biomass is used as an ingredient for a hybrid meat product, where a portion of the meat content is replaced with other protein sources, which are typically of plant origin. Preferably, the hybrid meat product contains 52(w / w)% or less of thermophilic fungal biomass in cake form, or 16(w / w)% or less of thermophilic fungal biomass in powder form. Thus, in one embodiment, the present invention relates to a food product which is a hybrid meat product containing 52(w / w)% or less of thermophilic fungal biomass in cake form, or 16(w / w)% or less of thermophilic fungal biomass in powder form. Preferably, the hybrid meat product contains at least 1, 2, 5, 10, or 15(w / w)% of thermophilic fungal biomass in cake form, or at least 0.3, 0.6, 1.5, 3(w / w)%, or 4.5(w / w)% of a substance in powder form.

[0081] In one embodiment, thermophilic fungal biomass is used as an ingredient in the bakery industry, for example, as a substitute for or in addition to cereal (or leguminous) flour. Such cereal and / or leguminous flours include, but are not limited to, cereal flours (e.g., wheat, spelt, barley, rye, horrah sorghum, emmer wheat, einkorn wheat, rye (containing gluten)), cereal flours (e.g., oats, corn, millet, sorghum, quinoa, rice (gluten-free)), leguminous flours (e.g., chickpea flour, soybean flour), and flours from tubers (e.g., potato flour, tapioca flour). While an example of a bakery product is bread, the thermophilic fungal biomass can also be used as an ingredient for other cereal-based foods, such as pasta and similar products, premixes (dried) for baking products, biscuits, cakes, pancakes, porridge (dried / ready to eat), cereal bars, cereal flakes, muesli, bread, gluten-free bread, and pizza bases (cooked). Preferably, the bakery product contains 10 (w / w) or less of thermophilic fungal biomass in powder form as a substitute for or in addition to the cereal (or leguminous) flour disclosed herein. Thus, in one embodiment, the present invention relates to a bakery product containing 10 (w / w) of thermophilic fungal biomass as a substitute for or in addition to the cereal (or leguminous) flour disclosed herein. The bakery product preferably contains at least 1, 2, 5, or 10 (w / w) of thermophilic fungal biomass in powder form as a substitute for or in addition to the cereal (or leguminous) flour disclosed herein.

[0082] Preferably, the cereal-based food contains 33(w / w) or less of thermophilic fungal biomass in cake form, or 10(w / w) or less of thermophilic fungal biomass in powder form. Therefore, in one embodiment, the present invention relates to a food that is a cereal-based food containing 33(w / w) or less of thermophilic fungal biomass in cake form, or 10(w / w) or less of thermophilic fungal biomass in powder form. The cereal-based food preferably contains at least 1, 2, 5, 10 or 15(w / w)% of thermophilic fungal biomass in cake form, or at least 0.3, 0.6, 1.5, 3 or 4.5(w / w)% of biomass in powder form.

[0083] In one embodiment, the thermophilic fungal biomass is used as an ingredient in ready-to-eat meals, such as savory pies, pizza-like dishes, savory tarts, soups, and porridges. Preferably, the ready-to-eat food contains 16(w / w) or less of thermophilic fungal biomass in cake form, or 5.3(w / w) or less of thermophilic fungal biomass in powder form. Therefore, in one embodiment, the present invention relates to a ready-to-eat food that contains 16(w / w) or less of thermophilic fungal biomass in cake form, or 15.3(w / w) or less of thermophilic fungal biomass in powder form. Preferably, the ready-to-eat food contains at least 1, 2, 5, or 10(w / w)% of thermophilic fungal biomass in cake form, or at least 0.5, 1, or 2(w / w)% of biomass in powder form.

[0084] In one embodiment, the thermophilic fungal biomass is used as an ingredient in a ready-to-eat soup, preferably containing one or more of water, stock vegetables, meat, fruit, pasta, seasonings, aromas, flavorings, and stabilizers. Preferably, the soup contains 25 (w / w) or less of thermophilic fungal biomass in cake form, or 7.5 (w / w) or less of thermophilic fungal biomass in powder form. Therefore, in one embodiment, the present invention relates to a food product which is a soup, preferably a ready-to-eat soup, containing 25 (w / w) or less of thermophilic fungal biomass in cake form, or 7.5 (w / w) or less of thermophilic fungal biomass in powder form. The soup product preferably contains at least 1, 2, 5, 10, or 15 (w / w) of thermophilic fungal biomass in cake form, or at least 0.5, 1, 2, or 5 (w / w) of the biomass in powder form.

[0085] In one embodiment, thermophilic fungal biomass is used as an ingredient in sports enthusiast foods. Sports enthusiast foods may be, for example, protein shakes or protein bars containing one or more of the following: protein sources (e.g., whey protein, soy protein, legume protein), flavorings, stabilizers, sugars, colorings, and finally minerals and vitamins. Preferably, sports enthusiast foods contain 70 (w / w) or less of thermophilic fungal biomass in cake form or powder form. Therefore, in one embodiment, the present invention relates to sports enthusiast foods, preferably protein shakes or protein bars, containing 70 (w / w) or less of thermophilic fungal biomass in cake form or powder form. Sports enthusiast foods preferably contain at least 1, 2, 5, 10, 20, or 40 (w / w) of thermophilic fungal biomass in either cake form or powder form.

[0086] [Table 1]

[0087] In addition to its use in food, the thermophilic fungal biomass of the present invention is a valuable alternative material for feed products such as pet food, thanks to its structure (both cake and powder) and nutritional value. Therefore, in one embodiment, thermophilic fungal biomass is used as an ingredient in pet food. As used herein, the term “pet” means a domestic or domesticated animal, including but not limited to domestic dogs, cats, ferrets, rabbits, and pigs. Pet food is understood herein to mean a composition intended for oral consumption that meets one or more nutritional needs of a pet. Pet food may be selected from treats, chews, biscuits, gravies, supplements, toppers, and combinations thereof. Pet food may or may not be nutritionally balanced and complete. The thermophilic fungal biomass represents a good source of dietary fiber, protein, and essential amino acids to be used in pet food. The amount of thermophilic fungal biomass in pet food may be determined based on the different nutritional needs of pets. Some pets may require more attention to their diet due to weight management or weight loss. In this case, thermophilic fungal biomass can provide pets with a feeling of fullness due to its high fiber content. In addition, thermophilic fungal biomass can serve as a protein source substitute for standard animal or plant-based pet food materials. Therefore, in one embodiment, thermophilic fungal biomass is used as a source of protein, amino acids, and essential amino acids in pet food, preferably as a substitute for standard animal or plant-based sources of protein and amino acids in pet food, and more preferably as the sole source of protein and / or amino acids.

[0088] General definition In this document and its claims, the verb “includes” and its combinations are used in their non-restrictive sense, meaning that the items following the word are included, but not excluded from items not specifically mentioned. In addition, references to elements by the indefinite article “a” or “an” do not exclude the possibility of more than one element unless the context clearly requires that there be one or only one element. Thus, the indefinite article “a” or “an” usually means “at least one.” When the word “about” or “approximately” is used with a number (e.g., about 10), it preferably means that the value may be a given value that is more than or less than 5% of that value. As used herein, “subject” means any animal, preferably a mammal, most preferably a human. In preferred embodiments, the subject is non-human.

[0089] In the context of the present invention, a decrease or increase in the parameter to be evaluated means a change of at least 5% in value corresponding to that parameter. More preferably, the decrease or increase in value means a change of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, or 100%. In the latter case, there may no longer be a detectable value associated with the parameter.

[0090] Throughout this document, when percentages are used to express the amount of a substance in a mixture, weight percentages are intended unless otherwise stated or explicitly evident from the context.

[0091] The present invention has been described above with respect to several exemplary embodiments. Modifications and alternative practices are possible of some parts or elements, and features from the embodiments can be combined where possible. All references to the literature and patent documents are incorporated herein by reference. [Examples]

[0092] Example 1: Production of Rhizomucor pusilus biomass in cake or powder form For preliminary culture, Rhizomucor psyllus strain CBS143028 was cultured with the following solutions: KCl 0.17g / L, KH2PO4 1.3g / L, Na2HPO4 0.4g / L, citric acid 0.5g / L, MgSO4.7aq 0.7g / L, FeSO4.7aq 0.03g / L, CaCl2.2aq 0.035g / L, ZnSO4.7aq 0.04g / L, MnCl2.4aq 0.004, CuSO4. Seeds were seeded in 200 ml of a standardized mineral medium at pH 5.5 containing 5 aq 0.0005 gr / L, CoCl 2.6 aq 0.0005 gr / L, Na2B4O 7.10 aq 0.003 gr / L, KI 0.0003 gr / L, Na2MoO 4.2 aq 0.0005 gr / L, 11 g of dextrose per L; 4 g of (NH4)2SO4 per L; and 7.5 g of tartaric acid per L. Preliminary cultures were incubated for 24 hours at 46°C at 200 rpm in an orbital shaker in a 1 L Erlenmeyer flask equipped with a baffled aeration stop. Next, using this preliminary culture, the fungi were inoculated into a fermenter containing the aforementioned standard mineral medium, which had a pH of 3.5, contained 77 g of dextrose per liter as the carbon source, 1.4 g of (NH4)2SO4 per liter as the nitrogen source, and was supplemented with NH3 as a titrant. The fungi were grown in the fermenter in a supply batch mode for a doubling time of 12 hours. Olive oil was continuously supplied to maintain a concentration of 50 ppm.

[0093] Fermentation broths with a dry matter content ranging from 2 to 5 weight percent were concentrated using a vibrating sieve to achieve a minimum dry matter content of 10 (w / w)%. The biomass was then mixed with antioxidants and pasteurized.

[0094] Next, the sieved biomass was compressed using a hydraulic press to obtain Rhizomcol pusilus biomass as a cake with a dry matter content of approximately 29 (w / w)%.

[0095] A portion of the biomass cake was further freeze-dried and pulverized (6000 rpm, 1 mm mesh size) to obtain Rhizomcor pusilus biomass in powder form with a dry matter content of approximately 96 (w / w)%.

[0096] [Table 2]

[0097] Example 2: Nutritional value of Rhizomcor pusilus biomass Due to its lower moisture content (Table 2), the powder derived from Rhizomucor pusilus biomass concentrates to approximately three times the concentration of cake substitutes. The nutritional value of Rhizomucor pusilus biomass is primarily due to its high protein and dietary fiber content (Table 3).

[0098] In addition, all nine essential amino acids are contained in Rhizomucor pusilus biomass (Table 4). This makes Rhizomucor pusilus biomass a highly nutritious protein source in the human diet. It should be noted that the protein content of Rhizomucor pusilus biomass shown in Table 3 is based on the Kjeldahl method, a standard method used to analyze the protein content of different foods. The Kjeldahl method is based on total nitrogen content and uses a standard conversion factor of 6.25 to estimate the protein content in the analyzed food. However, the conversion factor of 6.25 may not be appropriate for certain foods. In Rhizomucor pusilus biomass, the conversion factor may be overestimated due to the presence of other nitrogen sources such as RNA, chitin, and chitosan. For this reason, the true protein content of Rhizomucor pusilus biomass can be better estimated through amino acid analysis. Among the amino acids, Rhizomucor pusilus biomass has a particularly high content of citrulline (Table 4).

[0099] [Table 3]

[0100] [Table 4]

[0101] The dry-state fiber content of Rhizomcol psyllus biomass is extremely high compared to other foods known to be good sources of fiber (Table 5). The fiber content of Rhizomcol psyllus biomass is even higher (up to 95%) than that of mycoprotein, the main source of the Quorn brand meat substitute.

[0102] [Table 5]

[0103] [Table 6]

[0104] Table 6 shows the chitin and chitosan content in various batches of Rhizomucor pusilus biomass. Interestingly, Rhizomucor pusilus biomass contains not only chitin as fiber but also almost equal amounts of the fiber chitosan. Chitosan is a deacetylated form of chitin, and its health benefits are explained for chitosan in animals. It is also expected that human chitosan will be beneficial to health

[16] . About 9.2 (w / w)% of Rhizomucor pusilus biomass is chitosan. This is in contrast to the mycoprotein source Fusarium wenenatum, whose dietary fiber consists of one-third chitin and two-thirds beta-glucan, according to Finnigan et al. (1,3 and 1,6)

[17] .

[0105] Example 3: Nutritional value of meat substitute made from Rhizomcor pusilus biomass Meat substitute hamburgers were prepared with 28(w / w)% Rhizomcor pusilus biomass in cake form containing 21.2% water, 32.3% textured plant protein extruded, 1.3% textured fiber, and chicken flavoring (ingredients were mixed, formed, coated in a cooking oven at 170°C for 8 minutes, roasted at 185°C for 35 seconds, and frozen until the core reached -20°C). The protein content of the meat substitute made with Rhizomcor pusilus biomass (based on Kjeldahl analysis) was slightly lower than that of the vegetarian hamburger (Table 7). It should be considered that the meat substitute with Rhizomcor pusilus biomass evaluated was made with only 28(w / w)% biomass cake content, which is far lower than the maximum level (42% w / w) set for Rhizomcor pusilus biomass when used as a meat substitute. This means that the protein and fiber content of meat substitutes made from Rhizomcor pusilus biomass can be modified depending on the percentage of Rhizomcor pusilus biomass in the final product and other materials mixed with the Rhizomcor pusilus biomass.

[0106] [Table 7]

[0107] Example 4: Micronutrient content of Rhizomucor pusilus biomass Analysis of Rhizomucor pusilus biomass for micronutrients revealed that it is a source of various micronutrients, including different vitamins such as vitamins A, E, and B vitamins, as well as choline (see Table 8).

[0108] [Table 8]

[0109] Rhizomucor pusilus biomass has a particularly high choline content (Table 8). Choline was formerly also known as vitamin B4 [6][7]. Choline plays an important role in the structural integrity of cell membranes and in different metabolic pathways in the human body, such as methyl metabolism, cholinergic neurotransmission, transmembrane signaling, and lipid and cholesterol transport and metabolism [8]. Choline can be newly synthesized by the human body. However, choline still represents a basic component of the human diet because the amount newly produced is not considered sufficient to reach the level of an adequate intake (AI) of 400 mg / day for adults (both genders) [9]. In 1998, choline was recognized as an essential nutrient by the Institute of Medicine (IOM) [8]

[10] . Table 9 shows the choline content of Rhizomucor pusilus biomass compared to the choline content of several types of foods.

[0110] The vitamin A content of Rhizomcor pusillus biomass cake is 0.3 mg / kg (Table 8). However, vitamin A is also thought to originate from materials used during the production of the biomass (e.g., olive oil or antioxidants). However, the vitamin A content of Rhizomcor pusillus biomass produced without the use of olive oil and antioxidants is still 0.1 mg / kg, suggesting that at least some of the vitamin A is produced by fungi during the fermentation process.

[0111] The same thing happens with vitamin E. Vitamin E is naturally present in olive oil (approximately 5.1 mg / 100g according to NEVO tables) and also appears to be present in antioxidants. The vitamin E (dl-alpha-tocopherol acetate) content of Rhizomucor pusillus biomass cake is 2.5 mg / kg (Table 8). However, when Rhizomucor pusillus biomass cake is produced without the use of olive oil and without the addition of antioxidants, it still has a vitamin E content of 22 mg / kg, suggesting that vitamin E is produced by the fungus during the fermentation process. In this regard, it is interesting to note that the vitamin E content of fungal biomass produced without olive oil and antioxidants is even higher than that of biomass produced in the presence of them.

[0112] In addition, Table 8 shows that Rhizomucor pusilus biomass also contains various B vitamins, particularly vitamin B2 (riboflavin), vitamin B6 (pyridoxine), and folic acid (vitamin B11, also known as vitamin B9 in some countries such as France, Germany, and the United States).

[0113] [Table 9]

[0114] Example 5: Mineral content of Rhizomcor pusilus biomass As shown in Table 10, Rhizomucor pusilus biomass contains large amounts of different minerals that are added during the fermentation process and are necessary for fungal growth. The iron (Fe) content in Rhizomucor pusilus biomass is particularly high and was a limiting factor for intake assessment according to EFSA DRV. The iron dosage for Rhizomucor pusilus biomass production must be reduced to a final concentration of 14 mg / kg (Table 10). In addition, zinc was also reduced to a final concentration of 16 mg / kg (Table 10).

[0115] Table 11 presents a comparison of the iron content of Rhizomcor psyllus biomass with that of various other types of food. While some of the meat products in Table 11 contain lower amounts of iron, it should be noted that their bioavailability is higher because the iron is present in a heme-bound form.

[0116] [Table 10]

[0117] [Table 11]

[0118] Example 6: Lipid content of Rhizomucor pusilus biomass As shown in Table 3 above, Rhizomucor pusilus biomass contains approximately 9% (w / w) on a dry matter basis. More detailed analysis showed that, on average, 22.2% (w / w) consist of saturated fatty acids, 50.1% (w / w) consist of monounsaturated fatty acids (mostly oleic acid), and 27.7% (w / w) consist of polyunsaturated fatty acids (mostly linoleic acid). Thus, Rhizomucor pusilus biomass is relatively rich in monounsaturated and polyunsaturated fatty acids.

[0119] References [1] EFSA Panel onDietetic Products, Nutrition, and Allergies (NDA), "Scientific Opinion onDietary Reference Values ​​for carbohydrates and dietary fiber," 2010. [2] P. Sharma, C.Bhandari, S. Kumar, B. Sharma, P. Bhadwal and N. Agnihotri, "Chapter 11 -Dietary Fibers: A Way to a Healthy Microbiome," in Diet, Microbiome andHealth, Handbook of Food Bioengineering, 2018, pp. 299-345. [3] EFSA Panel onDietetic Products, Nutrition and Allergies (NDA), "Scientific Opinion onDietary Reference Values for protein," 2012. [4] T. Suzuki, M.Morita, Y. Kobayashi and A. Kamimura, "Oral L-citrulline supplementationenhances cycling time trial performance in healthy trained men: Double-blindrandomized placebo-controlled 2-way crossover study.," Journal of theInternational Society of Sports Nutrition, vol. 13, no. 6, 2016. [5] FEDIAF(European Pet Food Industry), "Nutritional needs for cats and dogs,"2018. [6] T. Navarra, TheEncyclopedia of Vitamins, Minerals, and Supplements., Infobase Publishing,2004. [7] F. Macdonaldand R. L. Lundblad, Handbook of Biochemistry and Molecular Biology, Fourthedition, CRC Press, 2010. [8] Institute ofMedicine (US) Standing Committee on the Scientific Evaluation of DietaryReference Intakes and its Panel on Folate, Other B Vitamins anch Choline,"Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6,Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline," 1998. [9] EFSA Panel onDietetic Products, Nutrition and Allergies (NDA), "Dietary ReferenceValues for choline," 2016.

[10] S. H. Zeiseland K. A. Da Costa, "Choline: An Essential Nutrient for PublicHealth," Nutrition Reviews, vol. 67, pp. 615-623, 2009.

[11] FEDIAF(European Pet Food Industry), "Nutritional Guidelines For Complete andComplementary Pet Food for Cats and Dogs," 2018.

[12] EFSA Panel onDietetic Products, Nutrition, and Allergies (NDA), "Scientific Opinion onDietary Reference Values for vitamin A," 2015.

[13] EFSA Panel onDietetic Products, Nutrition, and Allergies (NDA), "Scientific Opinion onDietary Reference Values for vitamin E as α-tocopherol,"2015.

[14] EFSA Panel onDietetic Products, Nutrition and Allergies (NDA), "Dietary ReferenceValues for riboflavin," 2017.

[15] EFSA Panel onDietetic Products, Nutrition and Allergies (NDA), "Dietary ReferenceValues for vitamin B6," 2016.

Claims

1. Use in at least one of food, beverage, pet food and feed products of a composition comprising biomass of a thermophilic fungus as a source of at least one of a) choline and b) minerals, wherein the strain of the thermophilic fungus is Rhizomucor pusilus or a single colony isolate thereof.

2. The use according to claim 1, wherein the composition containing the biomass of a thermophilic fungus is a cake obtained in a method comprising the steps of a) growing a strain of the thermophilic fungus in liquid culture in a culture medium, and b) recovering the biomass of the thermophilic fungus from the culture medium by at least one of sieving, filtration and decantation to produce a biomass cake.

3. The use according to claim 2, wherein the sieved, filtered, or decanted biomass cake has a dry matter concentration of at least 12 (w / v)%.

4. The use according to claim 2, wherein the method includes step c) drying the biomass cake by pressurizing out any remaining water to obtain a biomass cake having a dry matter concentration of at least 20%.

5. The use according to claim 2, wherein the composition containing thermophilic fungal biomass is a dried powder obtained by crushing the biomass cake and further drying it to a biomass powder by warm air, freeze-drying, or flash-drying.

6. The use according to claim 4, wherein the biomass cake is dried to a moisture content of 0 to 7 (w / w)%.

7. The use according to claim 1, wherein the strain of thermophilic fungus is Rhizomucor psyllus CBS143028.

8. The use according to claim 1, wherein the strain of thermophilic fungus is a single colony isolate of Rhizomucor psyllus CBS143028.

9. The use according to any one of claims 1 to 8, wherein the biomass of the thermophilic fungus is used as a source of choline in vegan or vegetarian food or in pet food.

10. The use according to claim 9, wherein the pet food is cat or dog food.

11. The use according to any one of claims 1 to 10, wherein the biomass of the thermophilic fungus is used as an iron source, used to replace soybeans as an iron source, or used to replace soybeans as an iron source in a meat substitute.

12. The use according to any one of claims 1 to 11, wherein the biomass of the thermophilic fungus is used to incorporate into food, beverage, pet food and feed products in either the form of cake or powder at a maximum level of 70 (w / w) and a minimum level of at least 1 (w / w).

13. The biomass of the thermophilic fungus is a) Meat substitute comprising 1 to 42 (w / w)% of the biomass of the thermophilic fungus in cake form, or 0.5 to 14 (w / w)% of the biomass of the thermophilic fungus in powder form, b) Hybrid meat products comprising 1 to 52 (w / w)% of the biomass of the thermophilic fungus in cake form, or 0.3 to 17.3 (w / w)% of the biomass of the thermophilic fungus in powder form, c) A cereal-based food comprising 1 to 30 (w / w) of the biomass of the thermophilic fungus in cake form, or 0.3 to 10 (w / w) of the biomass of the thermophilic fungus in powder form, d) A ready-to-eat food product comprising 1 to 16 (w / w) of the biomass of the thermophilic fungus in cake form, or 0.5 to 5.3 (w / w) of the biomass of the thermophilic fungus in powder form, e) A ready-to-eat soup containing 1 to 27 (w / w) of the biomass of the thermophilic fungus in cake form, or 0.5 to 9 (w / w) of the biomass of the thermophilic fungus in powder form, and f) The use according to any one of claims 1 to 12, which is used as an ingredient for one of the foods for sports enthusiasts, comprising 1 to 70 (w / w) percent of the biomass of the thermophilic fungus in the form of a cake or powder.

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