WO2024250176A1 - Mycélium de champignon filamenteux et procédé de préparation de mycélium - Google Patents

Mycélium de champignon filamenteux et procédé de préparation de mycélium Download PDF

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Publication number
WO2024250176A1
WO2024250176A1 PCT/CN2023/098665 CN2023098665W WO2024250176A1 WO 2024250176 A1 WO2024250176 A1 WO 2024250176A1 CN 2023098665 W CN2023098665 W CN 2023098665W WO 2024250176 A1 WO2024250176 A1 WO 2024250176A1
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Prior art keywords
mycelium
seawater
culture medium
liquid culture
filamentous fungi
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PCT/CN2023/098665
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English (en)
Chinese (zh)
Inventor
陈劲初
陈炎鍊
林珊
杨碧华
陈彦博
林明义
江玲慧
李宗儒
朱心彤
林子淳
陈婉屏
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Grape King Bio Ltd
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Grape King Bio Ltd
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Priority to PCT/CN2023/098665 priority Critical patent/WO2024250176A1/fr
Priority to CN202380013215.4A priority patent/CN119585413A/zh
Publication of WO2024250176A1 publication Critical patent/WO2024250176A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/77—Fusarium

Definitions

  • the present invention relates to a method for preparing mycelium of a fungus, and in particular to a method for preparing mycelium of a filamentous fungus.
  • a flexitarian diet refers to consuming a large amount of non-animal foods such as plant-based, fungal and/or algae foods, and consuming fish, poultry, eggs and dairy products in moderation, but consuming a small amount of red meat and processed meat, or a vegan diet at least one day a week.
  • the flexitarian population accounts for more than 40% of the total population.
  • beans In addition to being eaten directly, beans can also be processed into soy milk, dried tofu, bean curd skin, tofu and vegetarian meat products.
  • soy milk dried tofu
  • bean curd skin tofu
  • vegetarian meat products need to be further extruded, which causes a lot of carbon emissions.
  • filamentous fungi contains high protein, high dietary fiber and low fat, which can improve the intestinal flora and is a common material for health foods.
  • filamentous fungi have a fibrous taste and can be cultivated in large quantities in a fermentation tank through a fermentation step, but do not have the above-mentioned disadvantages of bean cultivation. Therefore, they can be used in the food industry as an excellent protein source for vegan, vegetarian and flexible vegetarian diets.
  • the filamentous fungi used in the food industry are terrestrial, so fresh water is required for the fermentation step. In areas with insufficient and/or uneven rainfall, fresh water resources are very precious, and the cultivation of filamentous fungi will have a negative impact on the environment.
  • one aspect of the present invention is to provide a method for producing mycelium of filamentous fungi, which comprises adding seawater to a culture medium to promote the growth of mycelium of filamentous fungi and increase its dry weight yield and/or protein yield. Therefore, mycelium of filamentous fungi can be used as a source of excellent vegetarian protein and solve the problems of consumption of land resources, fresh water resources and/or carbon emissions.
  • Another aspect of the present invention is to provide a mycelium of a filamentous fungus, which is prepared by the above-mentioned production method, wherein the mycelium has a higher content of protein.
  • a method for producing mycelium of a filamentous fungus is proposed.
  • a solid culture step is performed on a first mycelium of Fusarium venenatum at 15°C to 30°C using a solid culture medium for 3 to 5 days to obtain a second mycelium.
  • a liquid culture step is performed on the second mycelium at 15°C to 30°C, pH 5 to pH 8, and an oscillation rate of 10rpm to 100rpm using a first liquid culture medium for 2 to 7 days to obtain a third mycelium.
  • the third mycelium is fermented for 2 to 7 days using the second liquid culture medium at 15°C to 30°C, pH 5 to pH 8, and a rotation speed of 10 rpm to 100 rpm to obtain a fermented product.
  • the solid culture medium, the first liquid culture medium, and/or the second liquid culture medium are added with seawater or diluted seawater.
  • the fermented product is subjected to a solid-liquid separation step to obtain mycelium.
  • Fusarium spp. is deposited in the American Type Culture Collection (ATCC) with the deposit number being ATCC 20334.
  • the first liquid culture medium and the second liquid culture medium may selectively contain cereals, beans, inorganic salts, sugars, yeast extracts and/or malt extracts.
  • the diluted seawater is obtained by diluting seawater with fresh water, and the concentration of the diluted seawater may be, for example, greater than 0 volume percentage (vol%) and less than 100 volume percentage.
  • the fermentation step is performed in a fermentation tank.
  • gas can be selectively introduced into the fermentation tank, wherein the gas system is selected from a group consisting of air, oxygen, carbon dioxide, helium, or any combination thereof.
  • the manufacturing method may optionally include a drying step and/or a grinding step for the fermented product to obtain a dried mycelium product.
  • the seawater is subjected to a sedimentation step and a seawater filtration step.
  • the volume ratio of the first liquid culture medium to the second liquid culture medium may be, for example, 1:100 to 1:200.
  • the dry weight yield of the mycelium can be, for example, 1.00 weight percentage (wt%) to 1.50 weight percentage.
  • a mycelium of a filamentous fungus which is obtained by the above-mentioned production method, wherein the protein yield of the mycelium of Fusarium aureum can be, for example, greater than 45 weight percent and less than or equal to 55 weight percent.
  • the method for producing mycelium of filamentous fungi of the present invention is to add seawater to at least one of the solid culture medium, the first liquid culture medium and the second liquid culture medium of the filamentous fungi, thereby promoting the growth of the mycelium of the filamentous fungi and increasing its dry weight yield and/or protein yield. Therefore, the mycelium of the filamentous fungi can be used as a source of excellent vegetarian protein and solve the problems of consumption of land resources, fresh water resources and/or carbon emissions.
  • FIG. 1 shows the wet weight yield of mycelium of Fusarium aureum obtained by adding first and second liquid culture media with different concentrations of salt water or seawater according to one embodiment of the present invention.
  • FIG. 2 shows the dry weight yield of mycelium of Fusarium aureum obtained by adding first and second liquid culture media with different concentrations of salt water or seawater according to one embodiment of the present invention.
  • FIG. 3 shows the protein yield of mycelia of Fusarium aureum obtained by adding first and second liquid culture media with different concentrations of salt water or seawater according to one embodiment of the present invention.
  • the present invention provides a mycelium of a filamentous fungus and a method for producing the same, which may include but is not limited to adding seawater to a culture medium of the filamentous fungus, thereby promoting the growth of the mycelium of the filamentous fungus and increasing its dry weight yield and/or protein yield. Therefore, the mycelium of the filamentous fungus can be used as a source of excellent vegetarian protein and solve the problems of land resource and fresh water resource consumption and/or carbon emissions.
  • the vegetarian protein can be derived from plants, algae, blue-green algae and/or fungi.
  • the above-mentioned plants can include but are not limited to nuts (such as almonds, pistachios, pumpkin seeds), grains (such as rice, wheat, corn) and/or beans (such as peanuts, peas, soybeans, red beans, mung beans, pinto beans, chickpeas and/or emperor beans).
  • the above-mentioned algae can include but are not limited to Chlorella, red algae and/or kelp.
  • the above-mentioned blue-green algae can include but are not limited to Arthrospira maxima and/or Arthrospira platensis.
  • the above-mentioned fungi can include but are not limited to the fruiting bodies, mycelium and/or mycelium of filamentous fungi of large fungi.
  • the aforementioned vegetarian protein can be, for example, mycelium of filamentous fungi.
  • filamentous fungi described herein also known as molds, refer to fungi that can produce mycelium with long chains and/or branches but do not produce large fruiting bodies.
  • Filamentous fungi can be any edible species.
  • filamentous fungi may include but are not limited to Fusarium, Aspergillus and/or Neurospora.
  • filamentous fungi may include but are not limited to Fusarium venenatum, Aspergillus oryzae and/or Neurospora intermedia.
  • Fusarium aureum is also known as golden Fusarium or Fusarium solani, and is a fungus widely found in the soil.
  • Fusarium aureum has a high content of protein and can be mass-produced by a fermentation step, so it is often used in the manufacture of food compositions.
  • the strain of Fusarium aureum is not particularly limited, and may be any strain that can be mass-produced by a fermentation step, is edible and non-pathogenic.
  • Fusarium aureum may include but is not limited to strain A3/5 deposited in the American Type Culture Collection (ATCC) with a deposit number of ATCC20334, which can be purchased from ATCC.
  • ATCC American Type Culture Collection
  • the seawater referred to herein refers to salt water from the ocean, which may include but is not limited to 30 g/L to 40 g/L of sea salt and active substances.
  • the active substance refers to a substance that can promote the growth of mycelium of filamentous fungi.
  • the source of the above seawater is not particularly limited and can be taken from any ocean.
  • the seawater is shallow (or called the true light layer) seawater off the coast of Keelung, Taiwan province, China.
  • the above shallow seawater refers to seawater 50m to 200m below sea level, which has a moderate concentration of active substances, is conducive to the growth of mycelium of filamentous fungi, and there are fewer solids in the seawater, which can reduce the cost of the precipitation step and the seawater filtration step.
  • the seawater may be selectively subjected to a sedimentation step and a seawater filtration step to remove solids (e.g., marine debris, sand, biological debris, and/or humus) in the seawater.
  • the seawater filtration step may include, but is not limited to, filtering and/or centrifuging the seawater.
  • the seawater excludes evaporation, crystallization, and ion exchange membrane electrodialysis to retain active substances.
  • the diluted seawater is obtained by mixing fresh water with seawater.
  • the concentration of seawater in the diluted seawater may be, for example, greater than 0 volume percentage (vol%) and less than 100 volume percentage, or 50 volume percentage to 90 volume percentage, or 60 volume percentage to 80 volume percentage.
  • the water described herein includes an aqueous solution with water molecules ( H2O ) as a solvent.
  • water can be divided into fresh water and salt water.
  • Salt water is water with a salt content greater than or equal to 0.5 g/L, and salt water includes the above-mentioned seawater.
  • Fresh water is water with a salt content less than 0.5 g/L.
  • the chloride content of fresh water is less than or equal to 250 mg/L.
  • the sulfate content of fresh water is less than or equal to 250 mg/L.
  • the source of fresh water is not particularly limited, and may include but is not limited to underground fresh water, surface fresh water, biological water, desalinated seawater and/or sewage treated with purified water.
  • surface fresh water may include lake water, river water and/or glacier water.
  • Surface fresh water may come from precipitation such as rain, snow, snow grains and/or hail.
  • Fresh water may be further treated with water purification, wherein the method of water purification is not particularly limited.
  • water purification may include but is not limited to gelation, precipitation, filtration and disinfection to remove impurities and/or pathogens in fresh water, thereby obtaining tap water.
  • fresh water is primary water, secondary water or pure water obtained by further treatment with reverse osmosis, ion exchange resin and/or activated carbon.
  • the aforementioned fresh water, groundwater, tap water, primary water, secondary water and pure water may be used interchangeably.
  • fresh water is equivalent to no seawater or salt water added, in other words, the content of seawater or salt water is 0 volume percent.
  • seawater can promote the growth of mycelium of filamentous fungi compared to adding fresh water.
  • adding salt water to the culture medium such as sea salt prepared from sea salt reprocessed from seawater (e.g., evaporation, crystallization, and ion exchange membrane electrodialysis)
  • sea salt prepared from sea salt reprocessed from seawater
  • ion exchange membrane electrodialysis can inhibit the growth of mycelium of filamentous fungi compared to adding fresh water, which means that the active substances contained in seawater play an important role in increasing the dry weight yield and/or protein yield of mycelium of Fusarium aureum.
  • the mycelium of Fusarium aureum can be obtained by a multi-step process, wherein the multi-step process may include but is not limited to a solid culture step, a liquid culture step, and a fermentation step.
  • the solid culture step is to perform a solid culture step using the first mycelium of Fusarium aureum cultured on a solid state to obtain the second mycelium.
  • the temperature of the solid state culture step is not particularly limited, and may be, for example, 15° C. to 30° C. If the temperature of the solid state culture step is too high or too low, the growth activity of the second mycelium will be reduced or even lost.
  • the duration of the solid state culture step depends on the manufacturing efficiency and the growth activity of the mycelium. In some embodiments, the duration of the solid state culture step may be, for example, 3 to 5 days, otherwise the second mycelium with better growth activity cannot be obtained.
  • the liquid culture step is to use the first liquid culture medium to carry out a liquid culture step on the second mycelium to obtain the third mycelium.
  • the liquid culture step can be carried out at a conventional suitable temperature, which can be, for example, 15°C to 30°C. If the temperature of the liquid culture step is too high or too low, the growth activity of the third mycelium will be reduced or even lost. There is no particular restriction on the time for the liquid culture step, which can be determined according to the manufacturing efficiency and the growth activity of the mycelium.
  • the time of the liquid culture step can be, for example, 2 days to 7 days, otherwise the third mycelium with better growth activity cannot be obtained, or the wet weight yield, dry weight yield and/or protein yield of the third mycelium are not improved under the premise of increased time cost.
  • the shaking rate of the liquid culture step can be, for example, 10 rpm to 100 rpm to increase the ventilation rate of the first liquid culture medium, thereby promoting the growth of the third mycelium.
  • the fermentation step may be performed on the third mycelium using the second liquid culture medium to obtain a fermentate.
  • the temperature and time of the fermentation step may be the same as those of the liquid culture step, but the production scale is different.
  • the fermentation step is performed in a fermentation tank, and a stirring process may be selectively combined, wherein the stirring process has a rotation speed of 10rpm to 100rpm, or 10rpm to 40rpm.
  • the aforementioned fermentation tank is a ton-level fermentation tank.
  • the volume ratio of the first liquid culture medium and the second liquid culture medium may be, for example, 1:10 to 1:200, or 1:150 to 1:170, or 1:155 to 1:165.
  • the fermentation step may be performed in a fermentation tank.
  • gas may be selectively introduced into the fermentation tank, wherein the gas may be, for example, selected from the group consisting of air, oxygen, carbon dioxide, helium, or any combination thereof.
  • the aeration rate of the fermentation tank may be, for example, 0.01 volume of air per unit volume of culture medium per minute (VVM) to 1.5 VVM.
  • the tank pressure of the fermentation tank may be, for example, 0.1 kg/cm 2 to 1.0 kg/cm 2 , or 0.1 kg/cm 2 to 0.5 kg/cm 2 , so as to promote the growth of the third mycelium.
  • the types of the above-mentioned solid culture medium, the first liquid culture medium and the second liquid culture medium are not particularly limited.
  • the solid culture medium may be, for example, malt extract agar culture medium, Sabouraud's agar culture medium and/or a modified formula thereof.
  • the solid culture medium may preferably be 325 malt extract agar culture medium, which comprises 20.0 g/L malt extract, 20.0 g/L glucose, 1.0 g/L peptone, 20.0 g/L agar and a balanced amount of fresh water, seawater and/or diluted seawater.
  • the first liquid culture medium may include but is not limited to cereals, beans, inorganic salts, sugars, yeast extracts and/or malt extracts to provide sufficient nitrogen sources and/or carbon sources for the second mycelium, but may be adjusted as required.
  • the first liquid culture medium may include but is not limited to sugars, organic acid salts and inorganic salts.
  • the pH value of the first liquid culture medium may be, for example, pH 5 to pH 8, otherwise the growth activity of the third mycelium is poor.
  • the types of cereals may include but are not limited to barley, wheat, rye, oats, rice, millet, sorghum and/or millet.
  • rice may include but are not limited to white rice, germ rice and/or brown rice.
  • rice may include but are not limited to glutinous rice, indica rice and/or glutinous rice.
  • beans may include but are not limited to red beans, pinto beans, emperor beans, soybeans, mung beans and/or macadamia beans.
  • inorganic salts may include but are not limited to potassium nitrate (KNO 3 ), magnesium sulfate (MgSO 4 ), calcium chloride (CaCl 2 ), ammonium dihydrogen phosphate (NH 4 H 2 PO 4 ) and/or potassium dihydrogen phosphate (KH 2 PO 4 ).
  • sugars may include but are not limited to monosaccharides, disaccharides, oligosaccharides and/or polysaccharides, wherein monosaccharides may include but are not limited to fructose, glucose, ribose, galactose and/or mannose, disaccharides may include but are not limited to sucrose, lactose, maltose and/or trehalose, oligosaccharides may include but are not limited to fructooligosaccharides, maltooligosaccharides, isomaltooligosaccharides, galacto-oligosaccharides and/or soybean oligosaccharides, and polysaccharides may include but are not limited to inulin, starch, glycogen, oat ⁇ -glucan and/or chitin.
  • the organic acid salt may include but is not limited to citrate (eg, sodium citrate), acetate, malate, tartrate and/or quinate.
  • the formula of the second liquid culture medium may be the same as or different from the formula of the first liquid culture medium, and may be adjusted as needed, and the pH value may be, for example, pH 5 to pH 8, such as pH 5 to pH 6.
  • seawater and/or diluted seawater are added to at least one of the solid culture medium, the first liquid culture medium, and the second liquid culture medium.
  • seawater and/or diluted seawater are added to the solid culture medium, the first liquid culture medium, and the second liquid culture medium.
  • seawater and/or diluted seawater are added to the first liquid culture medium and the second liquid culture medium, and the solid culture medium is prepared with fresh water.
  • seawater and/or diluted seawater are added to the solid culture medium and the first liquid culture medium, and the second liquid culture medium is prepared with fresh water.
  • seawater and/or diluted seawater are added to the solid culture medium and the second liquid culture medium, and the first liquid culture medium is prepared with fresh water.
  • seawater and/or diluted seawater are added to the second liquid culture medium, and the solid culture medium and the first liquid culture medium are prepared with fresh water.
  • seawater and/or diluted seawater are added to the first liquid culture medium, and the solid culture medium and the second liquid culture medium are prepared with fresh water.
  • seawater and/or diluted seawater are added to the solid culture medium, and the first liquid culture medium and the second liquid culture medium are prepared with fresh water.
  • the fermented product is subjected to a solid-liquid separation step to obtain mycelium.
  • the solid-liquid separation step may include but is not limited to centrifugation and/or filtration.
  • bacterial sludge can be obtained.
  • mycelium and bacterial sludge can be used alternately.
  • the growth of mycelium of filamentous fungi can be promoted by adding seawater and/or diluting the culture medium with seawater. "Promoting the growth of mycelium of filamentous fungi" described herein may include but is not limited to increasing the wet weight yield, dry weight yield and protein yield of mycelium.
  • the wet weight yield, dry weight yield and protein yield described herein are calculated based on the weight of the fermented product as 100 weight percent (weight percentage, wt%).
  • the dry weight yield of mycelium is 1.00 weight percent to 1.50 weight percent.
  • the protein yield of mycelium is greater than 45 weight percent and less than or equal to 55 weight percent.
  • the mycelium may be further subjected to a drying step and/or a grinding step to obtain a dried product of the mycelium of Fusarium aureum.
  • the method of the drying step is not particularly limited, and only a portion or all of the moisture of the mycelium needs to be removed, such as freeze drying, vacuum drying, or spray drying.
  • the dried product obtained after the mycelium of Fusarium aureum is freeze-dried is a freeze-dried powder.
  • freeze-dried powder and dried products can be used alternately.
  • the grinding step can be performed using conventional methods, such as mechanical grinding, drum grinding, or pneumatic grinding.
  • the fermented product, the mycelium obtained by the solid-liquid separation step of the fermented product, and the dried product obtained by the drying step and/or grinding step of the mycelium can all be applied to food compositions or pharmaceutical compositions.
  • the mycelium can be used to prepare a vegetarian meat product with a fibrous taste without an extrusion step.
  • the dried mycelium can be used as a raw material for making high-protein powder.
  • the dosage form of the food composition or pharmaceutical composition may be, for example, tablets, capsules, granules, pills, tablets, powders, emulsifiers, liquid suspensions, dispersants or solvents.
  • the food composition or pharmaceutical composition may selectively add pharmaceutically and/or food-acceptable additives, wherein the additives may include but are not limited to carriers, excipients, diluents, adjuvants, solvents, dispersants, coatings, antibacterial agents and/or antifungal agents.
  • the additives may include but are not limited to carriers, excipients, diluents, adjuvants, solvents, dispersants, coatings, antibacterial agents and/or antifungal agents.
  • the carrier may include but is not limited to lactose, corn starch and/or a lubricant.
  • the above-mentioned lubricant may include but is not limited to magnesium stearate and/or sodium stearyl fumarate.
  • the diluent may include but is not limited to lactose and/or dry corn starch.
  • the dosage form when the dosage form is a liquid suspension or an emulsifier, the emulsifier or suspending agent used to dissolve or suspend the fermentation product, mycelium and/or the dried product may be an oily substance.
  • the food composition or the pharmaceutical composition may selectively add sweeteners, flavoring agents and/or pigments.
  • the first mycelium of Fusarium aureum (hereinafter referred to as FV) with the collection number of ATCC20334 was purchased from the American Type Culture Collection (ATCC).
  • the first mycelium of FV was inoculated on a solid culture medium and subjected to a solid culture step at 25°C for 3 to 5 days to obtain a second mycelium.
  • the solid culture medium is 325 malt extract agar medium, which contains 20.0 g/L malt extract, 20.0 g/L glucose, 1.0 g/L peptone, 20.0 g/L agar and the balance amount of pure water.
  • the second mycelium is inoculated into the first liquid culture medium, and a liquid culture step is performed for 2 days at 25°C, pH 7, a rotation speed of 10 rpm to 100 rpm, and an aeration rate of 0.01 VVM to 1.5 VVM to obtain the third mycelium.
  • the first liquid culture medium comprises 1.0 weight percent glucose, 0.3 weight percent sodium citrate, 0.3 weight percent potassium nitrate (KNO 3 ), 0.1 weight percent diammonium phosphate (NH 4 H 2 PO 4 ), 0.1 weight percent calcium chloride (CaCl 2 ) and 20 volume percent seawater (seawater is diluted with secondary water to a volume percent concentration of 20 volume percent).
  • the seawater is taken from 50m to 60m below the sea level off the coast of Keelung, Taiwan province, China. Upon analysis, the seawater contained 3.64 ppm zinc, 0.71 ppm manganese, 1.09 ppm copper, 912.8 ppm calcium, 12.85 ppm iron, 11998 ppm phosphorus, 4406 ppm potassium, 13927 ppm sodium, and no nitrite was detected.
  • the third mycelium is placed in a fermentation tank, and a fermentation step is performed for 2 days at 25° C., pH 7, a rotation speed of 10 rpm to 100 rpm, and an aeration rate of 0.01 VVM to 1.5 VVM using a second liquid culture medium to obtain a fermentation product.
  • the second liquid culture medium comprises 1.0 weight percent glucose, 0.3 weight percent sodium citrate, 0.3 weight percent potassium nitrate (KNO 3 ), 0.1 weight percent diammonium phosphate (NH 4 H 2 PO 4 ), 0.1 weight percent calcium chloride (CaCl 2 ) and 20 volume percent seawater.
  • the fermentation product is centrifuged to obtain a pellet (also called mycelium).
  • the methods for producing the fermented products of Preparation Examples 2 to 5 are the same as those of Preparation Example 1, except that 40 volume percent, 60 volume percent, 80 volume percent and 100 volume percent of seawater are added to the first liquid culture medium and the second liquid culture medium (hereinafter collectively referred to as the first and second liquid culture media) of Preparation Examples 2 to 5, respectively.
  • the manufacturing methods of the fermented products of Comparative Examples 1 to 7 are the same as those of Example 1, except that the first and second liquid culture media of Comparative Examples 1 and 2 are added with secondary water (equivalent to 0 volume percent of seawater or sea salt water), and the first and second liquid culture media of Comparative Examples 3 to 7 are added with 20 volume percent, 40 volume percent, 60 volume percent, 80 volume percent and 100 volume percent of sea salt water, respectively.
  • the 100 volume percent sea salt water contains sea salt with a weight volume concentration of 35 g/L.
  • the percentage of the weight of the bacterial sludge and the weight of the fermented product was calculated to obtain the wet weight yield of the fermented product (unit: weight percentage).
  • the mycelium is dehydrated to obtain a dried product.
  • the weight of the dried product is measured, and the percentage of the weight of the dried product to the weight of the fermented product is calculated to obtain the dry weight yield of the mycelium (unit: weight percentage).
  • the percentage of the weight of the protein in the dried product to the weight of the fermentation liquid is tested to obtain the protein yield of the mycelium (unit: weight percentage).
  • the protein weight is tested using the Kjeldahl nitrogen determination method, which is briefly described below.
  • sulfuric acid is added to the dried product and heated to decompose the protein in the dried product into ammonia. Since ammonia will further react with sulfuric acid to form ammonium sulfate, a strong base can be added to decompose the ammonium sulfate into ammonia, and then the ammonia can be distilled into a standard acid solution by distillation.
  • the degree of neutralization of the standard acid solution by ammonia can be tested by acid-base titration to further obtain the nitrogen content in the dried product. Then, the nitrogen content is multiplied by the nitrogen factor (the dried powder here is classified as a general food, and the nitrogen factor is 6.25) to obtain the protein weight in the sample.
  • the nitrogen factor the dried powder here is classified as a general food, and the nitrogen factor is 6.25) to obtain the protein weight in the sample.
  • FIG. 1 illustrates the wet weight yield of mycelia of FV obtained by adding the first and second liquid culture media with different concentrations of salt water or seawater according to one embodiment of the present invention, wherein the horizontal axis indicates that the first and second liquid culture media are added with seawater or salt water, the vertical axis indicates the wet weight yield (unit: weight percentage), and the bars 110, 120, 130, 140, 150, and 160 respectively indicate that the concentrations of salt water or seawater are 0 volume percent, 20 volume percent, 40 volume percent, 60 volume percent, 80 volume percent, and 100 volume percent, that is, the bars from left to right are Preparation Comparative Example 2 to Preparation Comparative Example 7, Preparation Comparative Example 1, and Preparation Example 1 to Preparation Example 5, and "*" indicates that there is a statistically significant difference (p ⁇ 0.05) from Preparation
  • the wet weight yield of the mycelium obtained in Preparation Comparative Example 2 using the first and second liquid culture media with 0 volume percent sea salt water added was 5.3 weight percent.
  • the wet weight yield of the mycelium obtained in Preparation Comparative Example 3 to Preparation Comparative Example 7 using the first and second liquid culture media with 20 volume percent to 100 volume percent sea salt water added decreased with the increase of sea salt water, wherein the wet weight yield of the mycelium obtained in Preparation Comparative Example 7 using the first and second liquid culture media with 100 volume percent sea salt water added was only 4.4 weight percent.
  • FIG. 2 shows the dry weight yield of mycelium of Fusarium aureum (FV) obtained by adding the first and second liquid culture media with different concentrations of salt water or seawater according to one embodiment of the present invention, wherein the horizontal axis indicates whether the first and second liquid culture media are added with seawater or salt water, and the vertical axis indicates the dry weight yield (unit: weight percentage), ...
  • Bar 260 represents that the concentration of sea salt water or sea water is 0 volume percent, 20 volume percent, 40 volume percent, 60 volume percent, 80 volume percent and 100 volume percent, respectively, that is, the vertical bars from left to right are Preparation Comparative Example 2 to Preparation Comparative Example 7, Preparation Comparative Example 1 and Preparation Example 1 to Preparation Example 5, respectively, and "*" and "**” indicate that there are statistically significant differences between Preparation Comparative Example 3 to Preparation Comparative Example 7 and Preparation Comparative Example 2 or between Preparation Example 1 to Preparation Example 5 and Preparation Comparative Example 1 (p ⁇ 0.05, p ⁇ 0.01, respectively).
  • the dry weight yield of the mycelium obtained in Preparation Comparative Example 3 to Preparation Comparative Example 7 decreased with the increase of sea salt water, wherein the dry weight yield of the mycelium obtained in Preparation Comparative Example 6 and Preparation Comparative Example 7 using the first and second liquid culture media with 80 volume percent of sea salt water and 100 volume percent of sea salt water was 0.85 weight percent and 0.73 weight percent, respectively, which was significantly lower than Preparation Comparative Example 2 (the first and second liquid culture media with 0 volume percent of sea salt water added).
  • the dry weight yield of the mycelium of Preparation Examples 1 to 5 obtained by adding 20 volume percent to 100 volume percent of seawater will increase with the increase of seawater.
  • the dry weight yields of the mycelium of Preparation Examples 3 and 4 obtained by adding 60 volume percent of seawater and 80 volume percent of seawater were 1.25 weight percent and 1.23 weight percent, respectively, which were significantly higher than that of Preparation Comparative Example 1 (the first and second liquid culture media of 0 volume percent seawater).
  • seawater inhibits the growth of FV mycelium, but seawater promotes the growth of FV mycelium, which means that the active substances contained in seawater can promote the growth of FV mycelium, and seawater and seawater cannot be equivalently replaced.
  • FIG. 3 shows the protein yield of the mycelium of Fusarium aureum (FV) obtained by using the first and second liquid culture media added with different concentrations of sea salt water or seawater according to one embodiment of the present invention, wherein the horizontal axis represents the components of the first and second liquid culture media, the vertical axis represents the protein yield (unit: weight percentage), and the bars 310, 320, 330, 340, 350, and 360 represent that the concentrations of sea salt water or seawater are 0 volume percent, 20 volume percent, 40 volume percent, 60 volume percent, 80 volume percent, and 100 volume percent, respectively, that is, the bars from left to right are Preparation Comparative Examples 2 to Preparation Comparative Examples 7, Preparation Comparative Example 1, and Preparation Examples 1 to Preparation Examples 5, and "*" indicates that there are statistically significant differences between Preparation Examples 1 to Preparpar
  • the protein yield of the mycelia obtained in Comparative Example 3 to Comparative Example 7 using the first and second liquid culture media with 20 volume percent to 100 volume percent of sea salt water added did not change significantly compared to Comparative Example 2 (the first and second liquid culture media with 0 volume percent of sea salt water added), and even decreased with the increase of sea salt water.
  • the protein yield of the mycelium of Preparation Examples 1 to Preparation Examples 5 obtained by adding 20 volume percent to 100 volume percent of seawater to the first and second liquid culture media increased with the increase of seawater salt water, wherein the protein yield of the mycelium of Preparation Example 5 obtained by adding 80 volume percent of seawater salt water to the first and second liquid culture media was 52 weight percent, which was significantly higher than that of Preparation Comparative Example 1 (the first and second liquid culture media with 0 volume percent of seawater added).
  • At least one of the solid culture step, liquid culture step and fermentation step is carried out by adding seawater and/or diluted seawater to the culture medium, which can also increase the dry weight yield and/or protein yield of the mycelium of the filamentous fungi.
  • adding seawater and/or diluted seawater to other conventional culture mediums for at least one of the solid culture step, liquid culture step and fermentation step can also increase the dry weight yield and/or protein yield of the mycelium of the filamentous fungi.
  • the mycelium of filamentous fungi and the method for producing the same have the advantage that the mycelium of filamentous fungi can be prepared using seawater, which can not only promote the growth of mycelium of filamentous fungi and increase the dry weight yield and/or protein yield of mycelium, but also reduce the consumption of land resources and fresh water resources and/or carbon emissions. Therefore, the mycelium of filamentous fungi can be used as a source of excellent vegetarian protein and can solve the problem of food shortage when fresh water resources are scarce.

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Abstract

L'invention concerne un procédé de préparation d'un mycélium d'un champignon filamenteux, comprenant : l'ajout d'eau de mer à un milieu de culture de Fusarium venenatum, pour ainsi favoriser la croissance d'un mycélium de Fusarium venenatum, et améliorer le rendement en poids sec et le rendement protéique. L'invention permet au mycélium de Fusarium venenatum d'être utilisé comme source d'excellentes protéines végétales, et de résoudre les problèmes liés à la consommation de ressources terrestres et de ressources en eau douce et/ou à l'émission de carbone.
PCT/CN2023/098665 2023-06-06 2023-06-06 Mycélium de champignon filamenteux et procédé de préparation de mycélium Pending WO2024250176A1 (fr)

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CN202380013215.4A CN119585413A (zh) 2023-06-06 2023-06-06 丝状真菌的菌丝体及其制造方法

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CN1315501A (zh) * 2000-03-24 2001-10-03 食品工业发展研究所 一种利用固态营养基质悬浮液培养丝状真菌的方法
CN101273120A (zh) * 2005-10-05 2008-09-24 朝日啤酒株式会社 生产丝状真菌培养产物的方法
CN108239607A (zh) * 2018-04-10 2018-07-03 邹丹丹 一种培养、收集紫菜腐霉菌菌丝的方法
WO2019046480A1 (fr) * 2017-08-30 2019-03-07 Sustainable Bioproducts, Inc. Composition comestible à champignons filamenteux et système de bioréacteur pour sa culture
CN112226373A (zh) * 2020-12-07 2021-01-15 中国科学院天津工业生物技术研究所 一株产蛋白的菌株及其应用
CN115038779A (zh) * 2019-12-10 2022-09-09 芬德集团公司 用于在发酵培养基中培养丝状真菌的方法
CN115820438A (zh) * 2023-01-06 2023-03-21 中国科学院天津工业生物技术研究所 一株高产蛋白菌株及应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1315501A (zh) * 2000-03-24 2001-10-03 食品工业发展研究所 一种利用固态营养基质悬浮液培养丝状真菌的方法
CN101273120A (zh) * 2005-10-05 2008-09-24 朝日啤酒株式会社 生产丝状真菌培养产物的方法
WO2019046480A1 (fr) * 2017-08-30 2019-03-07 Sustainable Bioproducts, Inc. Composition comestible à champignons filamenteux et système de bioréacteur pour sa culture
CN108239607A (zh) * 2018-04-10 2018-07-03 邹丹丹 一种培养、收集紫菜腐霉菌菌丝的方法
CN115038779A (zh) * 2019-12-10 2022-09-09 芬德集团公司 用于在发酵培养基中培养丝状真菌的方法
CN112226373A (zh) * 2020-12-07 2021-01-15 中国科学院天津工业生物技术研究所 一株产蛋白的菌株及其应用
CN115820438A (zh) * 2023-01-06 2023-03-21 中国科学院天津工业生物技术研究所 一株高产蛋白菌株及应用

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