Alternative meat comprising fungal mycelium and method for producing same

Abstract

The present specification relates to an alternative meat comprising fungal mycelium, an alternative meat processed food and feed composition comprising same, and methods for producing same. The fungal mycelium produced by a method according to one aspect has excellent cultivation efficiency, thus enabling mass production, and has the effect of removing specific odors that can be generated during cultivation. Thus, the fungal mycelium has the effect of enabling the production of an alternative meat having excellent taste and texture, and an alternative meat processed food and feed composition comprising same.

Description

Meat substitute containing fungal mycelia and method for producing the same

[0001] The present invention relates to a meat substitute containing fungal mycelia and a method for producing the same.

[0002]

[0003] Alternative meat refers to artificial meat that is designed to resemble real meat and can replace it. It is a rapidly growing field, driven by concerns about ecological destruction, global warming, and animal cruelty caused by meat production. With the growing vegetarian population and growing interest in healthier foods, it is gaining attention as the food of the future.

[0004] Meat alternatives can be broadly categorized into meat made from insects, meat cultured from animal cells, and meat made from plant-based ingredients. While insects are environmentally friendly, they often attract consumer aversion. While animal-cell-cultured meats offer a taste and aroma similar to real meat, they are time-consuming and expensive. Plant-based meat alternatives offer the advantage of being cheaper and faster to produce than animal-cell-cultured meats, but their taste, aroma, and texture are significantly different from those of real meat.

[0005] Recently, mushroom mycelia, which possess a fibrous structure and possess a meat-like flavor, have been attracting attention as a new food ingredient for meat substitutes. However, the unique flavor that can be produced during cultivation and the lack of proper cultivation conditions can lead to low cultivation efficiency, making mass production difficult.

[0006] Therefore, the inventors of the present invention have established suitable cultivation conditions to enable mass production and have conducted repeated research to eliminate the specific odor. As a result, they have developed a method for culturing mushroom mycelia with high productivity. Furthermore, they have completed the present invention by developing a meat substitute with the specific odor removed according to the manufacturing method of the present invention.

[0007]

[0008] One aspect provides a method for producing fungal mycelia, comprising the steps of: pre-cultivating a fungus; inoculating the pre-cultivated fungus into a liquid culture medium to perform a main culture; and filtering the main cultured fungus to recover the mycelia.

[0009] Another aspect provides a method for producing a meat substitute, comprising the step of producing mycelia by the above method.

[0010] Another aspect is to provide a meat substitute comprising mycelia prepared by the above method.

[0011] Another aspect is to provide a processed meat substitute food containing the above meat substitute.

[0012] Another aspect is to provide a feed composition comprising the mycelia or meat substitute.

[0013]

[0014] One aspect provides a method for producing fungal mycelia, comprising the steps of: pre-cultivating a fungus; inoculating the pre-cultivated fungus into a liquid culture medium to perform main cultivation; and filtering the main-cultivated fungus to recover mycelia.

[0015] As used herein, the term "alternative meat" (meat analogue) refers to an artificial meat that resembles real meat in appearance and texture. While cultured meat and edible insects are also used to produce meat alternatives, most meat alternatives are made from plant-based ingredients such as soy protein or wheat gluten, and are therefore also called plant-based meat.

[0016] In this specification, the term "mycelium" refers to a general term for hyphae that grow in a densely entangled state, and is observed in eukaryotic fungi and prokaryotic actinobacteria. In the case of mushroom mycelia, it is known that nutrients and medicinal ingredients are much richer than the fruiting body corresponding to the main body.

[0017] The above fungi may refer to a group of microorganisms including molds, yeasts, and mushrooms.

[0018] In one specific example, the fungus may not be a fungus of the genus Ganoderma.

[0019] The above fungi are Agrocybe, Albatrellus, Amillaria, Agaricus, Bondarzewia, Cantharellus, Cerioporus, Climacodon, Cordyceps, Fistulina, Flammulina, Fomes, Fomitopsis, Fusarium, Grifola, Herecium, Hydnum, Hypomyces, Hypsizygus, Ischnoderma, Laetiporus, Laricifomes, It may be any one or more selected from the group consisting of, but not limited to, species used for food from the genera Lentinula, Lentinus, Lepista, Meripilus, Morchella, Ophiocordyceps, Panelus, Piptoporus, Pleurotus, Polyporus, Pycnoporellus, Rhizopus, Schizophyllum, Stropharia, Tuber, Tyromyces and Wolfiporia.

[0020] Preferably, the fungus may be at least one selected from the group consisting of the genera Lentinus, Flammulina, Lentinula, Morchella and Pleurotus.

[0021] More preferably, the fungus may be a species of the genus Lentinus.

[0022] More preferably, the fungi are selected from the group consisting of Lentinus edodes, Lentinus crinitus, Lentinus tigrinus, Lentinus strigosus, Lentinus squarrosulus, Lentinus sajor-caju, Lentinus brumalis, Lentinus velutinus, Lentinus badius, Lentinus arcularius, Lentinus flexipes and Lentinus tuber-regium. It may be one or more selected from the group consisting of, but is not limited to, these.

[0023] In one specific example, the fungus may be cultured in, but is not limited to, PDA, YM, NU, YMG, LB, MYP, or MCM media. Preferably, the fungus may be cultured in PDA, YMG, or MCM media. More preferably, the fungus may be cultured in PDA media.

[0024] The above medium may be, but is not limited to, a liquid medium.

[0025] The above liquid medium may be, but is not limited to, PDB.

[0026] The above culture medium may include, but is not limited to, one or more selected from the group consisting of a carbon source, a nitrogen source, and an inorganic salt.

[0027] The carbon source is a food-grade carbon source that can be used industrially, and can be used without any particular limitation if it is generally used for fungal culture. For example, monosaccharides, disaccharides, polysaccharides with three or more sugars, alcohol, starch, and mixed sugars such as molasses generated as a by-product during sucrose production can be used. Preferably, the carbon source may be glucose, maltose, fructose, lactose, sucrose, starch, mannitol, sorbitol, glycerol, pyruvic acid, lactic acid, organic acid, starch hydrolysate, rice bran, molasses, sugarcane residue, corn steep liquor, or a mixture thereof, but is not limited thereto.

[0028] The above culture medium may contain 0.1% to 20% of a carbon source. Preferably, the carbon source may be a mixture of glucose, sugar, and starch.

[0029] The above glucose, sugar and starch may be included in the culture medium at 1 to 2% (w / v), 0.5 to 2% (w / v) and 0.5 to 2% (w / v), respectively. Preferably, the culture medium may include 1% glucose, 2% sugar and 0.5% starch.

[0030] The above nitrogen source is a food-grade nitrogen source that can be used industrially, and can be used without any particular restrictions if it is generally used for fungal culture. It may be, but is not limited to, soybean powder, yeast extract, soy peptone, ammonium chloride (NH4Cl), ammonium sulfate ((NH4)2SO4), potassium nitrate (KNO3), sodium nitrate (NaNO3), ammonium phosphate, ammonium carbonate, ammonium acetate, peptone, cornstiprica, casein hydrolysate, wheat bran, meat extract, amino acids, or mixtures thereof.

[0031] The above culture medium may contain 0.1% to 20% of a nitrogen source. Preferably, the nitrogen source may be soy peptone and yeast extract.

[0032] The above soy peptone and yeast extract may be included in the culture medium at a ratio of 0.5 to 1% (w / v), respectively. Preferably, the culture medium may include 1% soy peptone and 0.5% yeast extract.

[0033] The above inorganic salts are food grade and can be, but are not limited to, potassium phosphate monobasic, potassium phosphate dibasic, magnesium sulfate, manganese sulfate, sodium chloride, calcium carbonate, calcium chloride, iron chloride, iron sulfate or mixtures thereof.

[0034] The above culture medium may contain 0.005% to 10% of inorganic salts. Preferably, the inorganic salts may be potassium phosphate monobasic, potassium phosphate dibasic, and magnesium sulfate.

[0035] The above potassium phosphate monobasic, potassium phosphate dibasic, and magnesium sulfate may be included in the culture medium at 0.05 to 0.1% (w / v), respectively. Preferably, the culture medium may include 0.1% potassium phosphate monobasic, 0.05% potassium phosphate dibasic, and 0.05% magnesium sulfate.

[0036] The above culture medium may be manufactured to be edible.

[0037] In one specific example, the inoculation may be 0.5% to 5% (v / v) of the fungal pre-culture solution as the main culture inoculation amount. Preferably, the inoculation may be 1% (v / v) of the pre-culture solution as compared to the main culture solution.

[0038] In one specific example, the pH of the culture medium may be 4.0 to 9.0, for example, pH 4.5 to 9.0, pH 4.5 to 8.5, pH 4.5 to 8.0, pH 4.5 to 7.5, pH 5.0 to 7.5, pH 5.0 to 7.0, pH 5.0 to 6.5, pH 5.0 to 6.0 or pH 5.5.

[0039] The above culture can be adjusted to the pH of the culture by appropriately adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the culture. In addition, during the culture, foaming can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. In addition, to maintain the aerobic state of the culture, oxygen or an oxygen-containing gas can be injected into the culture, or to maintain anaerobic and microaerobic states, no gas can be injected or nitrogen, hydrogen, or carbon dioxide gas can be injected.

[0040] The above fungi can be cultured using a stirred bioreactor, and since the growth rate of the fungi or mycelia varies depending on the stirring speed, aeration amount, culture temperature, etc., it is very important to establish optimal culture conditions in the present invention.

[0041] Since the above stirring speed significantly differs depending on the shape and size of the stirring blade, the stirring speed must be set taking this into consideration. In one specific example, the cultivation is performed at a stirring speed of 10 to 900 rpm, for example, 10 to 850 rpm, 10 to 800 rpm, 10 to 750 rpm, 20 to 750 rpm, 20 to 700 rpm, 30 to 700 rpm, 30 to 650 rpm, 30 to 600 rpm, 40 to 600 rpm, 40 to 550 rpm, 40 to 500 rpm, 50 to 500 rpm, 50 to 450 rpm. It may be performed under conditions of 50 to 400 rpm, 60 to 400 rpm, 60 to 350 rpm, 60 to 300 rpm, 70 to 300 rpm, 70 to 250 rpm, 70 to 200 rpm, 80 to 200 rpm, 80 to 150 rpm, 90 to 150 rpm or 100 rpm. Preferably, the culturing may be performed at a stirring speed of 100 rpm.

[0042] In one specific example, the ventilation amount is 0.001 to 10.0 vvm, for example, 0.001 to 9.0 vvm, 0.001 to 8.0 vvm, 0.001 to 7.0 vvm, 0.005 to 7.0 vvm, 0.005 to 6.0 vvm, 0.005 to 5.0 vvm, 0.005 to 4.0 vvm, 0.005 to 3.0 vvm, 0.005 to 1.0 vvm, 0.01 to 1.0 vvm, 0.01 to 0.9 vvm, 0.05 to 0.8 vvm, 0.05 to 0.7 vvm, 0.1 to 0.5 vvm or 0.03 vvm. can

[0043] In one specific example, the culturing may be performed at 10 to 30°C, for example, 11 to 30°C, 12 to 30°C, 12 to 29°C, 13 to 29°C, 14 to 29°C, 14 to 28°C, 15 to 28°C, 16 to 28°C, 17 to 28°C, 17 to 27°C, 18 to 27°C, 19 to 27°C, 20 to 27°C, 20 to 26°C, 21 to 26°C, 22 to 26°C, 22 to 26°C, 23 to 26°C, 24 to 26°C or 25°C.

[0044] In one specific example, the culturing may be performed for 3 to 14 days, for example, 3 to 13 days, 3 to 12 days, 3 to 12 days, 3 to 11 days, 3 to 10 days, 4 to 10 days, 4 to 9 days, 5 to 9 days, 5 to 8 days, 6 to 8 days or 7 days.

[0045] In one specific example, the filtration of the fungus or mycelia may be, but is not limited to, filtration using a filter press. The filter plate of the filter press used to filter the fungus or mycelia may be, but is not limited to, a filter plate having a size of 600х600 (mm, mm). The filter plate of the filter press may vary depending on the filter press used.

[0046] The above filtration may use a filter plate of 200 to 20,000 mesh. Preferably, the above filtration may use a filter plate of 5,000 mesh.

[0047] The above filtration is performed under pressure conditions of 0.01 to 10.0 Mpa, for example, 0.01 to 9.0 Mpa, 0.05 to 9.0 Mpa, 0.05 to 8.0 Mpa, 0.05 to 7.0 Mpa, 0.05 to 6.0 Mpa, 0.05 to 5.0 Mpa, 0.1 to 5.0 Mpa, 0.1 to 4.0 Mpa, 0.1 to 3.0 Mpa, 0.1 to 2.0 Mpa, 0.1 to 1.0 Mpa, 0.2 to 1.0 Mpa, 0.3 to 1.0 Mpa, 0.3 to 0.9 Mpa, 0.3 to 0.8 Mpa, 0.3 to 0.7 Mpa, 0.4 to 0.7 Mpa, 0.5 to 0.7 It can be filtered at Mpa or 0.6 Mpa.

[0048] The mycelia recovered by the above filtration may contain 30 to 90% moisture.

[0049] The step of filtering the above fungus to recover the mycelia may additionally include a step of re-filtering the filter 1 to 5 times.

[0050] The step of recovering the mycelia may include a washing step.

[0051] The above washing may be performed by filtering out the fungus, recovering the mycelia, and re-filtering them with the washing solution, or by filtering the fungus together with the washing solution when filtering it.

[0052] In one specific example, the washing may be performed to remove the fragrant component of the mycelium.

[0053] The above cleaning solution may be, but is not limited to, water or water containing an emulsifier.

[0054] The above emulsifier may be, but is not limited to, glycerin fatty acid ester, sorbitan fatty acid ester, sucrose fatty acid ester, propylene glycol fatty acid ester, lecithin, soybean saponin, alginic acid, carrageenan, guar gum, carboxymethyl cellulose or sodium caseinate.

[0055] The washing may be performed 1 to 10 times using water containing the emulsifier. Preferably, the washing may be performed 3 to 5 times.

[0056] The above washing may use two or more filter plates. For example, the washing may use two, three, four, five, six, seven, eight, nine, or ten or more filter plates, but is not limited thereto. Preferably, the washing may use four to seven filter plates.

[0057] In one specific example, the washing may be performed in such a way that the amount of washing solution used varies depending on the number of filter plates.

[0058] The washing may be performed by placing the recovered mycelia in water at 5 to 2000% (w / v) relative to the amount of the recovered mycelia, stirring, and homogenizing. Preferably, the washing may be performed by placing the recovered mycelia in water at 20 to 200% (w / v) relative to the amount of the recovered mycelia, stirring, and homogenizing.

[0059] Another aspect provides a method for producing a meat substitute, comprising the step of producing mycelia by the above method.

[0060] Another aspect provides a meat substitute comprising mycelia prepared by the above method.

[0061] The above-mentioned meat substitute may be manufactured by grinding mycelia into various particle sizes, depending on the intended use. Preferably, the meat substitute may be added to food after being ground uniformly or unevenly into particles ranging from 1 μm to 10 cm. Alternatively, the meat substitute may be added to food after being torn into pieces.

[0062] The above substitute meat may be processed through extrusion molding.

[0063] The method for producing the above meat substitute may further include a step of mixing meat substitute broth into the produced mycelium.

[0064] In one specific example, the broth for the substitute meat may be manufactured by a method including the steps of heating burnt or cooked onions, peppers, green onions, garlic, and ginger with alcohol to evaporate the alcohol; boiling the vegetables with water and soy sauce to prepare a vegetable stock; and boiling the vegetables with star anise, cloves, and whole peppercorns, and then filtering the boiled vegetables to prepare a broth.

[0065] The above meat substitute broth may be used by manufacturing basic broth, beef broth, pork broth, chicken broth, or seafood broth depending on the purpose of use of the meat substitute.

[0066] The above beef broth may be prepared by boiling the above vegetables with additional radish and drainage.

[0067] The above pork broth may be prepared by boiling the above vegetables with additional pear and apple.

[0068] The above chicken stock may be prepared by boiling the above vegetables with additional carrots and potatoes.

[0069] The above seafood broth may be prepared by boiling the above vegetables with kelp and seaweed.

[0070] In one specific example, the broth may be manufactured by further including a step of storing it at a low temperature in a refrigerator for about a day before filtering.

[0071] In one specific example, the mixing of the mycelia and the meat substitute broth may be performed for about 24 to 30 hours.

[0072] The above substitute meat may be obtained by removing or drying the broth for the substitute meat or using it as its own.

[0073] The above drying is not particularly limited and can be performed using a known method, for example, natural drying, hot air drying, cold air drying, vacuum drying, or freeze drying can be used, but is not limited thereto.

[0074] Another aspect provides a processed meat substitute food comprising the above meat substitute.

[0075] Another aspect provides a feed composition comprising the mycelia or meat substitute.

[0076] The processed meat substitute food may be at least one selected from the group consisting of ham, sausage, bacon, dried and stored meat, seasoned meat, snacks, dumplings, fried foods, stir-fried foods, soy sauce, seasoning, powder mixes, bread, processed canned foods, and processed noodles, but is not limited thereto. Preferably, the processed meat substitute food may be bulgogi, dumplings, burger patties, kimbap, sausage, bacon, nuggets, beef jerky, meatballs, ham, ramen, hamburgers, fish cakes, fish cakes, steak, or fried meat, but is not limited thereto.

[0077] Ingredients that may be included in the above-mentioned processed meat substitute food are not particularly limited, except for the effective ingredients contained as essential ingredients, and various herbal extracts, food additives, or natural carbohydrates may be included as additional ingredients, as in conventional foods. In addition, the food additives may include, but are not limited to, conventional food additives in the art, such as sweeteners, flavoring agents, coloring agents, nutritional supplements, and stabilizers.

[0078] The sweetener may be a sugar substitute, monosaccharide or oligosaccharoid, such as, but not limited to, saccharin, cyclamate, monellin, thaumatin, curculin, miraculin, stevioside, phyllodulcin, glycyrrhizin, nitroaniline, dihydrochalcone, dulcin, susan, guanidine, oxime, oxathiazinone dioxide, aspartame, alitame, and the like. The monosaccharide may be, but is not limited to, galactose, fructose, glucose, sorbose, agatose, tagatose or xylose. The oligosaccharide may be, but is not limited to, sucrose, lactose, lactulose, maltose, isomaltose, maltulose, saccharose or trehalose. Other sweeteners that may be used include, but are not limited to, high fructose corn syrup.

[0079] The above flavoring agents may include, but are not limited to, monosodium glutamate, maltol, 5'-mononucleotides, such as inosine, and the like.

[0080] The above pigments are food colorings, cacao pigment, persimmon pigment, kaoliang pigment, laver pigment, lac pigment, marigold pigment, hibiscus pigment, berry pigment, saffron pigment, spirulina pigment, cyan nut pigment, turmeric pigment, annatto pigment, water-soluble annatto, alfalfa extract pigment, onion pigment, squid ink pigment, rosewood pigment, purple sweet potato pigment, purple corn pigment, purple yam pigment, red radish pigment, red cabbage pigment, pearl pigment, perilla pigment, gardenia red pigment, gardenia blue pigment, gardenia yellow pigment, caramel pigment, cochineal extract pigment, tamarind pigment, tomato pigment, paprika extract pigment, papiah pigment, grape juice pigment, grape skin pigment, pecan nut pigment, red yeast rice pigment, red yeast rice yellow pigment, red yeast rice, It may be, but is not limited to, safflower red pigment, safflower yellow pigment, black carrot extract pigment, chlorophyll, beet red, rutin, titanium dioxide, apo-8'-carotenal, β-carotene, carotene, carmine, Red No. 2, Red No. 3, Red No. 40, Red No. 102, Yellow No. 4, Yellow No. 5, Green No. 3, Blue No. 1 or Blue No. 2 extract pigment.

[0081] The above nutritional supplements may be, but are not limited to, fat-soluble vitamins consisting of retinol (vit A), calciferol (vit D), tocopherol (vit E), phytomenadione (vit K1), water-soluble vitamins consisting of thiamine (vit B1), riboflavin (vit B2), pyridoxine (vit B6), nicotinamide (niacin), pantothenic acid, biotin, folic acid, cyanocobalamin (vit B12), ascorbic acid (vit C), polyunsaturated fatty acids (PUFAs), etc.

[0082] The term "feed" in the present invention may refer to any natural or artificial diet, meal, etc., or ingredients of such meal, intended for or suitable for eating, ingesting, or digesting by an animal. The type of feed is not particularly limited, and any feed commonly used in the relevant technical field may be used.

[0083] The above feed composition may be a 20 to 90% concentrated solution or may be manufactured in the form of a powder or granules. The above feed composition may further include one or more of organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, and malic acid; phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, and polyphosphate (polyphosphate); and natural antioxidants such as polyphenol, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, and phytic acid. The above composition may be formulated in the form of a conventional feed and may include conventional feed ingredients together.

[0084] The above feed composition may be manufactured in the form of conventional feed compositions, such as powder and pellets, or in a liquid form, but is not limited thereto. The above feed composition may be manufactured into various forms of feed according to conventional methods known in the art.

[0085] The feed may further include grains such as ground or shredded wheat, oats, barley, corn and rice; plant-based protein feeds such as feeds mainly composed of rapeseed, soybeans and sunflower; animal-based protein feeds such as blood meal, meat meal, bone meal and fish meal; dry ingredients composed of sugars and dairy products such as various types of milk powder and whey powder, and may further include nutritional supplements, digestion and absorption enhancers, growth promoters and the like.

[0086] In one specific example, the feed may be, but is not limited to, a vegan feed.

[0087] The feed composition may be administered to animals alone or in combination with other feed additives in an edible carrier. Furthermore, the feed additives may be readily administered to animals as a top dressing, by mixing them directly into animal feed, or in an oral formulation separate from the feed. When the feed additives are administered separately from animal feed, they may be prepared as immediate-release or sustained-release formulations by combining them with food-grade edible carriers, as is well known in the art. Such edible carriers may be solid or liquid, such as cornstarch, lactose, sucrose, soybean flakes, peanut oil, olive oil, sesame oil, and propylene glycol. When a solid carrier is used, the feed additive may be in the form of a tablet, capsule, powder, troche, or saccharide tablet, or a top dressing in a microdispersible form. When a liquid carrier is used, the feed additive may be in the form of a gelatin soft capsule, or a syrup, suspension, emulsion, or solution.

[0088] Additionally, the feed may contain additives such as preservatives, stabilizers, wetting or emulsifying agents, and solution accelerators. The feed additives may be added to animal feed by injection, spraying, or mixing.

[0089] The feed or feed additive of the present invention can be applied to a number of animal diets including mammals, poultry, and fish.

[0090] As the mammals, it can be used for pigs, cows, sheep, goats, laboratory rodents, and laboratory rodents, as well as pets (e.g., dogs, cats), and as the poultry, it can be used for chickens, turkeys, ducks, geese, pheasants, and quail, and as the fish, it can be used for trout, but is not limited thereto.

[0091]

[0092] Fungal mycelia produced by a method according to the present invention have excellent cultivation efficiency and can be mass-produced, and can eliminate peculiar odors that may be generated during cultivation, so they have the effect of producing meat substitutes with excellent taste and texture, as well as processed meat substitute foods and feed compositions containing the same.

[0093]

[0094] Figure 1 is a graph showing the amount of cells according to the culture time.

[0095] Figure 2 shows the appearance of a meat substitute manufactured by marinating mycelia in meat substitute broth and then removing the moisture.

[0096] Figure 3 shows a stir-fried dish using a meat substitute containing mycelia.

[0097] Figure 4 shows a patty made using a meat substitute containing mycelia.

[0098] Figure 5 shows the appearance of frozen kimbap made using a substitute meat containing mycelia.

[0099]

[0100] Hereinafter, the present invention will be described in detail by way of examples to specifically explain the present invention.

[0101]

[0102] Example

[0103] Example 1. Confirmation of mycelial growth according to solid medium type

[0104] To determine a medium with excellent mycelial growth for meat substitute production, mycelial growth and density were measured using various media. The media used were PDA, YM, NU, YMG, LB, MYP, and MCM media, and were prepared according to the compositions shown in Table 1.

[0105]

[0106] Media composition PDAPotato 200 g, Dextrose 20 g, Agar 15 g / LYMYeast extract 3 g, Malt extract 3 g, Peptone 5 g, Dextrose 10 g, Agar 15 g / LNUPeptone 5 g, Beef extract 3 g, Agar 15 g / LYMGYeast extract 4 g, Malt extract 10 g, Glucose 4 g, Agar 15 g / LLBNaCl 10 g, Yeast extract 5 g, Tryptone 10 g, Agar 15 g / LMYPMalt extract 30 g, Yeast extract 2 g, Peptone 1 g, Agar 15 g / LMCMGlucose 20 g, Yeast extract 2 g, Peptone 3 g, MgSO4·7H2O 0.5 g, K2HPO4 1 g, KH2PO4 0.46 g, Agar 20 g / L

[0107] Each medium was sterilized after adjusting the pH to 6.5 before sterilization. For static culture, Lentinus edodes mycelia were cultured for 14 days in an incubator fixed at 25±1℃, and the mycelial diameter and mycelial density were measured to investigate growth. The results are shown in Table 2, and mycelial density was indicated as * for average, ** for good, and *** for excellent.

[0108]

[0109] BadgePDAYMNUYMGLBMYPMCMdiameter / mm76795470617167mycelial density****************

[0110] As shown in Table 2, the mycelia showed excellent growth properties in general on PDA, YM, YMG, and MCM media, and in particular, the mycelia showed the best growth properties when PDA and YM media were used.

[0111]

[0112] Example 2. Establishment of optimal medium composition for mycelial cultivation.

[0113] To determine the optimal medium composition for mycelial culture, a basic medium containing 2% glucose, 0.5% peptone, 0.1% potassium phosphate monobasic, 0.05% potassium phosphate dibasic, and 0.05% magnesium chloride was set. 4 L of the medium containing the above components was prepared in a 5 L incubator, sterilized at 121°C for 20 minutes, and then cooled to 24°C. 200 mL of Lentinus edodes mycelia cultured in PDB (Potato dextrose broth, Difco) for 10 days were inoculated into the cooled medium, and cultured for 10 days while maintaining the temperature at 24±1°C, the air supply at 1 vvm, and the pH at 5.5.

[0114] To determine the optimal medium composition, a medium was prepared by replacing the components of the basic medium with other components, and then an experiment was conducted to cultivate mycelia using the above method.

[0115]

[0116] Example 2.1. Selection of carbon source for mycelial cultivation

[0117] The type and concentration of carbon sources in the above-mentioned basic medium were varied to select an appropriate carbon source for mycelial culture. In this experiment, industrially available and food-grade carbon sources such as glucose, maltose, fructose, lactose, sucrose, and starch were used, and added at concentrations of 0.5% (w / v), 1% (w / v), or 2% (w / v), respectively. The results are shown in Tables 3 and 4.

[0118]

[0119] Carbon source Glucose Maltose Fructose Ratio 0.5% 1% 2% 0.5% 1% 2% 0.5% 1% 2% Cell mass (g / L) 4.9 5.9 5.24.34.7 5.35.25.44.4

[0120] Carbon source lactose sugar starch ratio 0.5% 1% 2% 0.5% 1% 2% 0.5% 1% 2% Cell mass (g / L) 3.3 3.4 2.9 5.0 5.7 6.3 5.8 5.8 5.7

[0121] As shown in Tables 3 and 4 above, when maltose, fructose, and lactose were used, generally low cell counts were observed. When 1% glucose or 2% sucrose was used, high cell counts were observed, and in the case of starch, excellent cell counts of 5.7 g / L or more were observed regardless of concentration.

[0122] Based on the above results, additional experiments were conducted to explore the use of carbon sources in combination. Starch was used at 0.5% concentration, as it produced similar cell masses regardless of the concentration used. Glucose was used at 1% and 2%, which produced relatively high cell masses. Sugar, which produced the highest cell masses, was used at concentrations of 0.5%, 1%, and 2%. The results are presented in Table 5.

[0123]

[0124] Sugar 0.5%Sugar 1%Sugar 2%Glucose (%)1%2%1%2%1%2%Bacterial mass (g / L)5.55.85.86.16.85.2

[0125] As shown in Table 5, a relatively higher cell mass was observed when carbon sources were used in combination than when used alone. Furthermore, the highest cell mass was observed when 2% sugar, 1% glucose, and 0.5% starch were included in the combination, and thus this mixed composition was used as the carbon source in subsequent experiments.

[0126]

[0127] Example 2.2. Selection of nitrogen source for mycelial cultivation

[0128] In addition to the carbon source, the type and concentration of nitrogen, which is one of the main components for increasing mycelial mass, were varied to select an appropriate nitrogen source for mycelial culture. In this experiment, industrially available food-grade nitrogen sources were used. Organic nitrogen sources such as soybean powder, yeast extract, and soy peptone, and inorganic nitrogen sources such as ammonium chloride (NH4Cl), ammonium sulfate ((NH4)2SO4), potassium nitrate (KNO3), and sodium nitrate (NaNO3) were used, and were added at 0.5% (w / v) or 1% (w / v), respectively. The results are shown in Tables 6 and 7.

[0129]

[0130] Nitrogen source (0.5%), soybean powder, soy peptone, yeast extract, ammonium chloride, ammonium sulfate, potassium nitrate, sodium nitrate, cell mass (g / L) 7.19.99.64.73.92.82.5

[0131] Nitrogen source (1%), soybean powder, soy peptone, yeast extract, ammonium chloride, ammonium sulfate, potassium nitrate, sodium nitrate, cell mass (g / L) 10.4, 13.3, 13.0, 1.7, 1.6, 1.3, 1.2

[0132] As shown in Tables 6 and 7, a generally low cell mass was observed when inorganic nitrogen sources were used. In the case of organic nitrogen sources, a higher cell mass was observed when 1% rather than 0.5% was used, and excellent cell mass was observed when soy peptone and yeast extract were used.

[0133] Based on the above results, additional experiments were conducted to explore the use of combined nitrogen sources. Soy peptone and yeast extract, which demonstrated the best results, were used, each mixed at a concentration of 0.5% or 1%. The results are presented in Table 8.

[0134]

[0135] Soy peptone 0.5%Soy peptone 1%Yeast extract 0.5%1%0.5%1%Bacterial mass (g / L) 11.814.116.113.7

[0136] As shown in Table 8, the highest cell mass was achieved when 1% soy peptone and 0.5% yeast extract were used, and the above mixed composition was used as a nitrogen source in subsequent experiments.

[0137]

[0138] Example 2.3. Selection of inorganic salts for mycelial cultivation

[0139] This study aimed to determine optimal culture conditions by varying the types of inorganic salts, which are important components that affect cell growth by increasing enzyme activity or aiding DNA synthesis. Industrially available food-grade inorganic salts were used in this experiment, and 0.1% (w / v) of potassium phosphate monobasic, potassium phosphate dibasic, magnesium sulfate, manganese sulfate, sodium chloride, calcium carbonate, and calcium chloride were added. The results are shown in Table 9.

[0140]

[0141] Inorganic salts (0.1%), potassium phosphate, potassium phosphate dibasic, magnesium sulfate, manganese sulfate, sodium chloride, calcium carbonate, calcium chloride, cell mass (g / L), 15.9, 15.2, 15.4, 9.0, 10.6, 5.8, 5.5

[0142] As shown in Table 9, high cell masses were observed when potassium phosphate monobasic, potassium phosphate dibasic, and magnesium sulfate were used. On the other hand, relatively low cell masses were observed when manganese sulfate, sodium chloride, calcium carbonate, and calcium chloride were used.

[0143] Based on the above results, additional experiments were conducted to investigate the use of inorganic salts in combination. Potassium phosphate monobasic, potassium phosphate dibasic, and magnesium sulfate were mixed at 0.05% (w / v) or 0.1% (w / v), and the results are shown in Table 10.

[0144] Monobasic potassium phosphate 0.05% Monobasic potassium phosphate 0.1% Monobasic potassium phosphate 0.05%0.1%0.05%0.1%0.05%0.1%0.05%0.1%Magnesium sulfate 0.05%0.05%0.1%0.1%0.05%0.05%0.1%0.1%Bacterial mass (g / L) 11.7 12.2 13.4 12.4 16.9 16.1 15.2 9.6

[0145] As shown in Table 10, the highest cell mass was achieved when using a combination of 0.1% potassium phosphate monobasic, 0.05% potassium phosphate dibasic, and 0.05% magnesium sulfate. Therefore, the above mixed composition was used as an inorganic raw material in subsequent experiments.

[0146]

[0147] Example 3. Establishment of optimal culture conditions for Lentinus edodes strains.

[0148] Based on the optimal carbon source, nitrogen source, and mineral salt composition selected in Example 2, a 50 L medium containing 1% glucose, 2% sugar, 0.5% starch, 1% soy peptone, 0.5% yeast extract, 0.1% potassium phosphate monobasic, 0.05% potassium phosphate dibasic, and 0.05% magnesium sulfate was prepared. The medium prepared with the above composition was sterilized in a 150 L incubator at 121°C for 20 minutes, and then inoculated with 500 mL of pre-cultured Lentinus edodes mycelia cultured in PDB and cultured for 10 days.

[0149] To determine the optimal culture conditions, an experiment was conducted to compare the cell mass by varying the inoculum amount, stirring speed, aeration amount, temperature, pH, and end point in the above culture method.

[0150]

[0151] Example 3.1. Selection of the optimal inoculum amount for mycelial cultivation

[0152] The inoculum dose has a significant effect on the growth of the cell mass. While a low inoculum dose may result in a high final cell mass, the initial cell growth rate may be low, resulting in a long incubation period. Conversely, a high inoculum dose may result in rapid initial cell growth but a low final cell mass. Therefore, an appropriate inoculum dose must be determined. Therefore, cultures were performed using 0.1%, 0.2%, 0.5%, 1%, 2%, 5%, or 10% (v / v) of the pre-culture solution as the main culture inoculum dose. The results are shown in Table 11.

[0153]

[0154] Inoculum amount (%)0.10.20.512510Culturing day 5 cell mass (g / L)1.32.33.13.65.26.76.7Culturing day 10 cell mass (g / L)13.415.217.317.016.616.316.3

[0155] As shown in Table 11, high inoculum doses initially resulted in high cell masses, but low final cell masses. Low inoculum doses initially resulted in low cell masses, but high cell masses later. Finally, the most suitable cell mass was confirmed to be 1% inoculum.

[0156]

[0157] Example 3.2. Selection of the optimal stirring speed for mycelial cultivation.

[0158] The agitation speed used in the incubator is a critical factor in fungal growth. Low agitation speeds result in poor mixing and oxygen supply to the culture medium, leading to low fungal growth. High agitation speeds, while ensuring smooth mixing and oxygen supply, impose shear stress on the mycelia, resulting in low fungal growth. Therefore, to investigate the effect of agitation speed, cultures were cultured at agitation speeds of 30, 50, 100, 150, or 200 rpm. The results are presented in Table 12.

[0159] Stirring speed (rpm) 30 50 100 150 200 Cell mass (g / L) 13.7 15.117.111.67.2

[0160] As shown in Table 12, the cell mass increased up to 100 rpm and then decreased at higher rpm. Therefore, the optimal stirring speed was selected as 100.

[0161]

[0162] Example 3.3. Selection of optimal aeration rate for mycelial cultivation

[0163] To investigate the growth of fungi according to the aeration rate, cultures were performed for 7 days at aeration rates of 0.05, 0.1, 0.2, 0.3, 0.5, 0.7, 1.0, 2, 3, or 4 vvm, and the fungal mass was then examined. The results are shown in Table 13.

[0164]

[0165] Aeration (vvm)0.050.10.20.30.51234Bacterial mass (g / L)16.216.416.316.416.416.214.414.314.0

[0166] As shown in Table 13, there was no significant difference from 0.05 vvm to 0.1 vvm, and although there was a slight decrease at higher aeration amounts, no significant difference was observed. Therefore, the optimal aeration amount was confirmed to be 0.05 to 0.7 vvm.

[0167]

[0168] Example 3.4. Selection of the optimal temperature for mycelial cultivation.

[0169] To investigate the growth of fungi according to the culture temperature, the culture was incubated for 10 days at 19, 20, 22, 24, 26, or 28℃. The results are shown in Table 14.

[0170]

[0171] Incubation temperature (℃) 18 20 22 24 26 28 Cell mass (g / L) 16.4 16.7 16.7 16.4 15.9 10.7

[0172] As shown in Table 14, a minimal decrease in cell mass was observed at low temperatures, but a significant decrease in cell mass was observed at 28°C. Therefore, it was confirmed that a culture temperature of up to 26°C is possible, and the optimal temperature was found to be 22°C.

[0173]

[0174] Example 3.5. Selection of the optimal pH for maintaining mycelial culture.

[0175] As the fungal cells proliferate, the pH continuously decreases, so the pH maintained during culture is important for the formation of continuous fungal cell growth. Therefore, to investigate the optimal pH maintained during culture, the culture was performed at 5.0, 5.3, 5.5, 5.7, 6.0, or 6.5. The results are shown in Table 15.

[0176]

[0177] Maintenance pH 5.0 5.3 5.5 5.7 6.0 6.5 Cell mass (g / L) 16.4 16.7 17.0 16.3 15.0 13.1

[0178] As shown in Table 15, the highest cell mass was observed when the maintained pH was 5.5. Therefore, subsequent cultures were conducted at an optimal maintained pH of 5.5.

[0179]

[0180] Example 3.6. Selection of the end point of cell culture

[0181] Generally, the pattern of fungal growth is divided into an early lag phase, a mid-exponential phase, a stationary phase, and a late death phase. To investigate the late exponential phase, which is believed to be advantageous for mushroom growth due to the highest fungal mass and mushroom flesh, the fungal mass and growth pattern according to the culture time were examined. The results are presented in Figure 1 and Table 16.

[0182]

[0183] Incubation time (day) 345678 Cell mass (g / L) 3.76.410.714.617.117.1

[0184] Figure 1 is a graph showing the amount of cells according to the culture time.

[0185] As shown in Figure 1 and Table 16, cell proliferation continued for up to 7 days, plateaued on the 8th day, and decreased with subsequent culture time. Therefore, the optimal culture termination time was selected as 7 days.

[0186]

[0187] Example 4. Recovery of mycelia from Lentinus edodes culture solution

[0188] The cultivation of Lentinus edodes mycelia was carried out under the optimal conditions selected in Examples 2 and 3 above. A 150 L microbiological culture medium was used as the bioreactor, and the culture components were dissolved in 120 L of the medium, and then autoclaved at 121°C for 20 minutes. After cooling the sterilized medium to 24°C, 1.2 L of the pre-culture solution of Lentinus edodes mycelia cultured in PDB for 10 days was inoculated. The medium inoculated with the pre-culture solution was cultured for 7 days under the conditions of 24±1°C, 100 rpm, and 0.3 vvm of air supply. After the cultivation was completed, the mycelia were recovered by filtration through a filter press with a filter plate size of 600 X 600 (mm, mm).

[0189] To determine the optimal conditions for mycelia recovery, an experiment was conducted to compare the amount of mycelia recovered by changing the pore size of the filter press filter cloth or the filter press filtration pressure.

[0190]

[0191] Example 4.1. Comparison of cell recovery amount according to filter press pore size

[0192] Filter press plates for mycelial filtration can utilize filter media with various pore sizes. Larger pore sizes result in faster filtration but lower mycelial recovery. Conversely, smaller pore sizes result in higher mycelial recovery but lower filtration speed, making filtration difficult. Therefore, selecting an appropriate pore size is crucial. To determine the appropriate pore size, filtration was performed using 200, 300, 800, 1340, 5000, or 12700 mesh filters. The results are shown in Table 17.

[0193]

[0194] Filter pore size 2003008001340500012700Filtration time (min) 151615171935Mycelia mass (kg) 1.92.12.52.72.92.9

[0195] As shown in Table 17, as pore size increased, filtration time increased, but the amount of recovered mycelia also increased. Considering the optimal amount of mycelia recovered and filtration time, the increase in filtration time did not differ significantly up to 5000 mesh, but a significant difference in mycelia amount was observed. Therefore, the use of a 5000 mesh filter was confirmed to be the most appropriate.

[0196]

[0197] Example 4.2. Comparison of cell recovery amount according to filter press filtration pressure

[0198] When mycelial culture is supplied to a filter press, the pressure of the filter press affects the filtration time, the amount of mycelia recovered through filtration, and the moisture content of the recovered mycelia. Therefore, the effects of filter press pressure on filtration time and the amount of mycelia recovered were investigated, and the results are shown in Table 18.

[0199]

[0200] Filtration pressure (MPa) 0.1 0.2 0.4 0.6 1.0 Filtration time (min) 16 17 17 18 25 Mycelial mass (kg) 1.9 2.5 2.9 3.0 2.2 Moisture content (%) 9 28 8 8 3 8 283

[0201] As shown in Table 18, filtration time increased as the pressure increased, and mycelial mass increased up to 0.6 MPa. The water content decreased up to a certain pressure, but after that, increasing pressure did not significantly affect the water content. Therefore, it was confirmed that 0.6 MPa was the most appropriate pressure for the filter press.

[0202]

[0203] Example 5. Removal of fragrance components and properties of filter cake according to mycelial washing

[0204] In general, fruiting bodies often do not have the unique mushroom odor, but the mycelia recovered after filtering the mycelia culture solution obtained by culturing the same mycelia through a filter press may be difficult to use as food due to the unique mushroom odor components. In the case of Lentinus edodes used in this example, the fruiting bodies have almost no odor, so there is no problem using them as food, but in the case of the mycelia culture solution, there were problems in manufacturing them as food due to the odor components generated as the mycelia proliferate. Therefore, experiments were conducted by changing the washing conditions of the mycelia to remove the odor components.

[0205] Mycelia were obtained under the conditions selected in Examples 2 to 4 above. A medium was prepared by dissolving the culture components in 120 L using a 150 L microbial culture incubator, and the medium was autoclaved at 121°C for 20 minutes. Afterwards, the sterilized medium was cooled to 24°C and inoculated with 1.2 L of the pre-culture solution cultured in PDB for 10 days. The medium inoculated with the pre-culture solution was cultured for 10 days under the conditions of 24±1°C, 100 rpm, and 0.3 vvm of air supply. After the culture was completed, the mycelia were recovered by filtration through a filter press with a filter plate size of 600 X 600 (mm, mm).

[0206]

[0207] Example 5.1. Investigation of removal of fragrance components and cake properties according to number of washes

[0208] Although washing can remove a certain amount of odor components, it can negatively affect the physical properties of the meat substitute. Therefore, to investigate the effect of washing, the mycelial culture was filtered through a filter press to collect the cake, then the cake was homogenized by adding it to water and stirring, and then filter press filtration was performed. The results are shown in Table 19, and the odor components and the degree of mycelial entanglement were indicated as ***** if very strong, **** if strong, *** if moderate, ** if weak, and * if very weak.

[0209]

[0210] Number of washes 0123510 Degree of fragrance ingredients ************** Degree of mycelial tangle *****************

[0211] As shown in Table 19, the aroma components decreased as the number of washes increased up to 5, but the difference was minimal for further washings. When washed more than 3 times, the mycelia became loosely entangled (in a fibrous form), resulting in properties unsuitable for manufacturing mushroom meat. Therefore, the optimal number of washes for the cake, obtained by recovering mycelia from the mycelia culture, was determined to be 3 to 5.

[0212]

[0213] Example 5.2. Investigation of the removal of fragrance components and cake properties by washing mycelia.

[0214] Example 5.2.1. Investigation of removal of fragrance components and cake properties according to washing liquid ratio

[0215] The ratio of water used for washing was 10, 20, 30, 50, 100, or 200% (w / v) based on the culture medium, and the degree of fragrance components and mycelial entanglement was investigated. The results are shown in Table 20, and the degree of fragrance components and mycelial entanglement was indicated as ***** for very strong, **** for strong, *** for average, ** for weak, and * for very weak.

[0216]

[0217] Washing liquid ratio (%) 10203050100200 Degree of fragrance ingredient******************** Degree of mycelial tangle********************

[0218] As shown in Table 20, when using a detergent solution of 20% or more, there was little difference according to the dilution ratio. Therefore, the appropriate dilution ratio for removing fragrance components was selected as 20% or more.

[0219]

[0220] Example 5.2.2. Investigation of bacterial counts when mycelial filtration and washing are performed separately and simultaneously.

[0221] In order to compare the case of using washing and filtration simultaneously with the case of performing washing and filtration separately, the amount of washing solution used was 90% of the culture solution, and washing was performed simultaneously with filtration, and after filtration, the mycelia were recovered and washed while adding 30% of the washing solution in a separate tank to loosen them, and then filtered. The results were compared three times. The results are shown in Table 20. If the fragrance component and the degree of mycelial entanglement were very strong, it was indicated as *****, if strong, ****, if average, ***, if weak, **, and if very weak, *. In order to measure the degree of contamination, 1 g of the recovered mycelia was extracted with sterile water and cultured on a bacterial medium to measure the number of bacteria.

[0222]

[0223] Filtration and washing method Simultaneous filtration and washing Washing method after filtration 3 times Process time (h) 0.53 Degree of fragrance components ****** Degree of mycelial entanglement **** Mycelial mass (kg) 2.3 2.1 Degree of contamination 335

[0224] As a result, as shown in Table 21 above, the degree of fragrance components showed similar results in both cases and the amount of mycelia decreased slightly, but when filtration and washing were performed simultaneously, the time required for recovery was not required, so the process time was reduced by about 6 times, and the time of exposure to the outside was reduced, so contamination by bacteria in the air was reduced by more than 10 times. In addition, when filtration and washing were performed simultaneously, the degree of mycelia entanglement was maintained to some extent, but when filtration and washing were performed separately, the mycelia became loose and the degree of mycelia entanglement was low, making it difficult to obtain the desired texture.

[0225] Therefore, when filtration and washing are performed simultaneously, the process time is shortened and there are fewer contamination factors than when filtration and washing are performed separately, so the subsequent process was performed by performing filtration and washing simultaneously.

[0226]

[0227] Example 5.2.3. Investigation of the washing solution ratio when filtration and washing are performed simultaneously.

[0228] The removal of fragrance components, the degree of mycelial entanglement, and the amount of mycelia were investigated by using the washing solution ratio of 0.5, 1, 2, 5, and 10 times that of the culture solution while performing filtration and washing simultaneously. The results are shown in Table 21, and the degree of fragrance components and mycelial entanglement was indicated as ***** if very strong, **** if strong, *** if average, ** if weak, and * if very weak.

[0229]

[0230] Washing liquid ratio 0.512510 Fragrance content degree************** Degree of mycelial entanglement************* Mycelial mass (kg) 2.42.42.22.12.1

[0231] As shown in Table 22 above, when the ratio of the washing solution used was more than 1 times that of the culture solution, the level of fragrance components decreased, the degree of mycelial entanglement was similar up to 2 times, and when more than 2 times the washing solution was used, the amount of mycelia produced decreased.

[0232] Therefore, considering the reduction in the degree of fragrance components, the degree of mycelial entanglement, and the amount of mycelia produced, it was advantageous to use the same amount of washing solution as the culture solution.

[0233]

[0234] Example 5.2.4. Investigation of the removal of flavor components and cake properties when mycelial filtration and washing are performed simultaneously.

[0235] The method of filtering mycelia to remove odor components and then adding a washing solution to wash them again has several problems, such as the time required for the work and the contamination factor. To improve this, it is advantageous to perform the filter press at once even if the amount of washing solution used is increased. Therefore, to apply this, the amount of culture solution introduced into each plate of the filter press was reduced compared to the existing method, so that the mycelia could be washed smoothly by the washing solution. Specifically, the number of filter plates, which occupy a volume of approximately 3 L per filter plate, and the amount of washing solution added after the culture solution filtration was completed were investigated. The filter plates used were 2, 3, 4, 5, 6, and 7, respectively, and the amount of washing solution was used in the same amount as the culture solution. The degree of odor components and entanglement of the mycelia were investigated. The results are shown in Table 21, and ***** was indicated for very strong odor components and the degree of mycelia entanglement, **** for strong, *** for moderate, ** for weak, and * for very weak.

[0236]

[0237] Number of filter plates 2345678 Degree of fragrance ingredients************************* Degree of mycelial tangle***********************

[0238] As shown in Table 23, the use of four to seven filter plates confirmed the removal of odor components, and allowed the recovery of a certain degree of entangled mycelia. Therefore, it was advantageous for the volume recovered on the filter plates to be 10% to 20% of the culture volume.

[0239]

[0240] Based on the above results, the number of filter plates used was set to 4, and the amount of washing solution used was 1, 3, 5, 10, or 20 times the volume of the culture solution, and the results were investigated. Table 22 shows the results, and ***** was used for very strong fragrance components and **** for strong, *** for average, ** for weak, and * for very weak.

[0241]

[0242] The ratio of the detergent is 1x3x5x10x20 times, the degree of fragrance ingredient is ************, and the degree of mycelial tangle is *****************

[0243] As shown in Table 24, the degree of mycelial entanglement was similar regardless of the amount of detergent used. However, superior effects were observed in removing fragrance components when using detergent five times or more. Therefore, it was confirmed that using detergent five times or more was advantageous.

[0244]

[0245] Example 5.2.5. Removal of fragrance components and investigation of cake properties by adding emulsifier during washing

[0246] In general, the fragrance components of mycelia are sometimes present in the culture medium, but sometimes they are bound to the inside or surface of the mycelial cells. If they are contained in the culture medium, the fragrance components often tend to be lipophilic, so they can be improved by simply removing the culture medium. However, if they are present inside or on the mycelial cells, it is advantageous to remove them by treating them with an emulsifier to elute them outside the mycelia. Therefore, the efficacy of glycerin fatty acid esters, sorbitan fatty acid esters, sucrose fatty acid esters, propylene glycol fatty acid esters, lecithin, soybean saponin, alginic acid, carrigina, guar gum, carboxymethyl cellulose, and sodium caseinate used as emulsifiers in removing fragrance components was investigated. The results are shown in Table 23, and the degree of fragrance component and mycelial entanglement is indicated as ***** if very strong, **** if strong, *** if moderate, ** if weak, and * if very weak.

[0247]

[0248] Emulsifier concentration (%) 10 20 30 50 100 200 Degree of fragrance ingredient ******************** Degree of mycelial entanglement ********************

[0249] As shown in Table 25, the addition of an emulsifier resulted in a slight loosening of the mycelial cake, but a significant reduction in odor components was observed compared to the absence of an emulsifier. Although the degree of mycelial entanglement decreased, this did not significantly affect mushroom meat production, indicating that odor components were effectively removed when washed with a washing solution containing an emulsifier.

[0250]

[0251] Example 6. Production of meat substitute using meat substitute broth and mycelia

[0252] When manufacturing meat substitutes using recovered mycelia, broth is crucial for achieving a cooking quality similar to animal meat. Therefore, to create broths suitable for mycelia, we prepared a basic broth, beef broth, pork broth, chicken broth, and seafood broth to produce meat substitutes.

[0253]

[0254] Example 6.1. Preparation of basic broth

[0255] Onions, peppers, green onions, garlic, and ginger were roasted or cooked, then alcohol was added and heated to evaporate the alcohol. Next, water and soy sauce were added and brought to a boil. Once boiling, star anise, cloves, and whole peppercorns were added and simmered for another 30 minutes. Afterwards, the broth was stored at low temperatures in the refrigerator for a day, then filtered through a cloth to collect the broth before use.

[0256]

[0257] Example 6.2. Preparation of beef broth

[0258] Onions, peppers, green onions, garlic, and ginger were roasted or cooked, then alcohol was added and heated to evaporate the alcohol. Next, water and soy sauce were added and brought to a boil. Once it began to boil, star anise, cloves, whole peppercorns, radish, and dried radish were added and boiled for another two hours. Afterwards, the broth was stored at low temperatures in the refrigerator for a day and then filtered through a filter paper before use.

[0259]

[0260] Example 6.3. Preparation of pork broth

[0261] Onions, peppers, green onions, garlic, and ginger were roasted or cooked, then alcohol was added and heated to evaporate the alcohol. Water and soy sauce were then added and brought to a boil. Once boiling, star anise, cloves, whole peppercorns, pears, and apples were added and simmered for two more hours. Afterwards, the broth was stored in the refrigerator for a day and then filtered through a filter paper bag before use.

[0262]

[0263] Example 6.4. Preparation of chicken stock

[0264] Onions, peppers, green onions, garlic, and ginger were roasted or cooked, then alcohol was added and heated to evaporate the alcohol. Water and soy sauce were then added and brought to a boil. Once boiling, star anise, cloves, whole peppercorns, carrots, and potatoes were added and simmered for another two hours. Afterwards, the broth was stored in the refrigerator for a day and then filtered through a filter paper bag before use.

[0265]

[0266] Example 6.5. Preparation of seafood broth

[0267] Onions, peppers, green onions, garlic, and ginger were roasted or cooked, then alcohol was added and heated to evaporate the alcohol. Next, water and soy sauce were added and brought to a boil. Once boiling, star anise, cloves, whole peppercorns, kelp, and seaweed were added and simmered for another two hours. Afterwards, the broth was stored in the refrigerator for a day and then filtered through a filter cloth before use.

[0268]

[0269] Example 6.6. Preparation of meat substitute containing mycelia

[0270] A meat substitute was prepared using the broth and mycelia of Examples 6.1 to 6.5. Specifically, the mycelia obtained under the above optimal conditions were soaked in cold broth, and then marinated in the broth for 24 to 30 hours while checking the level of odor. The broth was carefully removed from the marinated mycelia to ensure that the shape was not damaged, and a meat substitute was prepared. The meat substitute containing the mycelia prepared by the above method is shown in Fig. 2.

[0271]

[0272] Example 7. Manufacturing of food using meat substitutes

[0273] Example 7.1. Preparation of stir-fried dishes using meat substitutes

[0274] A stir-fry was prepared using the meat substitute containing mycelia prepared in Example 6. Specifically, the meat substitute was cut into the desired product shape. To season the cut meat substitute, smoky flavor powder, morel powder, minced garlic, sugar, salt, and MSG were added and mixed. The smoky flavor powder was used in an amount of 17% of the amount of the meat substitute, and the morel powder was used in an amount of 5% of the amount of the mycelia. The seasoned meat substitute was slowly cooked over low heat in a preheated pan with oil. After cooking, the meat substitute was removed from the pan, patted with paper towels to remove excess oil, and finished. The cooked meat substitute stir-fry is shown in Fig. 3.

[0275]

[0276] Example 7.2. Preparation of boiled dishes (dumplings) using meat substitutes

[0277] A boiled dish (dumpling) was prepared using the meat substitute containing mycelia prepared in Example 6. Specifically, the meat substitute containing mycelia was cut into the shape of the desired product. To prepare the filling for the dumplings, the cut meat substitute, vegetables, glass noodles, tofu, salt, and MSG were added and mixed. The dumpling filling was put into the dumpling skin, and the dumplings were made, then steamed. After the prepared dumplings were sufficiently cooked, they were taken out of the steamer and finished. The cooked dumplings are shown in Fig. 4.

[0278]

[0279] Example 7.3. Preparation of patties using meat substitutes

[0280] A boiled dish (dumpling) was prepared using the meat substitute containing mycelia prepared in Example 6. Specifically, the meat substitute containing mycelia was minced to form a patty shape. Smoke flavor powder, morel powder, minced garlic, sugar, salt, and MSG were added to the minced meat substitute, mixed, and then shaped into a circle. The seasoned meat substitute was slowly cooked over low heat in a preheated pan with oil. After cooking, the meat substitute was removed from the pan, patted with a paper towel to remove excess oil, and finished by applying barbecue sauce. The meat substitute patty prepared using the above method is shown in Fig. 5.

[0281]

[0282] Example 7.4. Preparation of kimbap using meat substitutes

[0283] Kimbap was prepared using the meat substitute containing mycelia prepared in Example 6. Specifically, the meat substitute containing mycelia was minced, and then smoke flavor powder, morel powder, minced garlic, sugar, salt, and MSG were added to the minced meat substitute and mixed. The seasoned meat substitute was slowly cooked over low heat in a preheated pan with oil. After cooking, the meat substitute was removed from the pan and patted dry with a paper towel. Along with the cooked meat substitute, carrots, burdock, pickled radish, spinach, and crab meat were prepared as kimbap filling ingredients, and then rice was spread widely on the seaweed and the prepared ingredients were placed on top. Afterwards, the seaweed was rolled into a circle to prevent the filling ingredients from falling out. The kimbap was stored at room temperature or frozen.

[0284]

[0285] Example 8. Preparation of feed using mycelia or meat substitutes

[0286] Vegan feed was manufactured using the mycelia or meat substitutes produced by the method of the above examples. Specifically, agricultural by-products such as crushed garlic bulbs, onion peels, defatted rice bran, or soybean meal, wheat flour, and mycelia or meat substitutes were ground and thoroughly mixed. Water was added to the mixture, kneaded to an appropriate consistency, and after kneading, the mixture was extruded into an appropriate size to manufacture a feed composition.

Claims

1. Step of pre-cultivating fungi; A step of inoculating the above pre-cultured fungus into a liquid culture medium and performing main cultivation; and A method for producing fungal mycelia, comprising: a step of filtering the above-mentioned cultured fungi to recover mycelia; 2. In claim 1, The above fungi are Agrocybe, Albatrellus, Amillaria, Agaricus, Bondarzewia, Cantharellus, Cerioporus, Climacodon, Cordyceps, Fistulina, Flammulina, Fomes, Fomitopsis, Fusarium, Grifola, Herecium, Hydnum, Hypomyces, Hypsizygus, Ischnoderma, Laetiporus, Laricifomes, A method for producing a fungal mycelium, wherein the fungal mycelium is at least one selected from edible species from the group consisting of the genera Lentinula, Lentinus, Lepista, Meripilus, Morchella, Ophiocordyceps, Panelus, Piptoporus, Pleurotus, Polyporus, Pycnoporellus, Rhizopus, Schizophyllum, Stropharia, Tuber, Tyromyces and Wolfiporia.

3. In claim 1, A method for producing fungal mycelia, wherein the liquid culture medium contains 0.1% to 20% of a carbon source, 0.1% to 20% of a nitrogen source, and 0.005% to 10% of an inorganic salt.

4. In claim 1, A method for producing fungal mycelia, wherein the liquid culture medium is manufactured to be edible.

5. In claim 1, A method for producing fungal mycelia, wherein the above main culture is cultured for 3 to 14 days under conditions of an aeration amount of 0.001 to 10 vvm, a culture temperature of 10 to 30°C, and a pH of 4 to 9.

6. In claim 1, A method for producing fungal mycelia, wherein the above filtration is performed using a filter press under pressure conditions of 0.01 to 10 MPa using 1 to 10 filter nets.

7. In claim 1, A method for producing fungal mycelia, wherein the above filtration includes washing of the mycelia.

8. In claim 1, A method for producing fungal mycelia, wherein the washing is performed by washing the recovered mycelia 2 to 10 times using 5 to 1000% (w / v) of water or water containing an emulsifier relative to the amount of mycelia.

9. A method for producing a meat substitute, comprising the step of producing mycelia by the method of claim 1.

10. A meat substitute comprising mycelia produced by the method of claim 9.

11. A processed meat substitute food comprising the meat substitute of claim 10.

12. A feed composition comprising mycelia manufactured by the method of claim 1.

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