WO2019095437A1 - 一种液体静置培养食药用真菌的方法 - Google Patents

一种液体静置培养食药用真菌的方法 Download PDF

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WO2019095437A1
WO2019095437A1 PCT/CN2017/113782 CN2017113782W WO2019095437A1 WO 2019095437 A1 WO2019095437 A1 WO 2019095437A1 CN 2017113782 W CN2017113782 W CN 2017113782W WO 2019095437 A1 WO2019095437 A1 WO 2019095437A1
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incubator
medicinal
culture
liquid
unit
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French (fr)
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丁重阳
徐萌萌
王琼
栾斌
赵丽婷
刘高强
艾连中
顾正华
石贵阳
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江南大学
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    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
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    • C12M23/00—Constructional details, e.g. recesses, hinges
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    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/48—Holding appliances; Racks; Supports
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    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/16—Vibrating; Shaking; Tilting
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M31/00—Means for providing, directing, scattering or concentrating light
    • C12M31/10—Means for providing, directing, scattering or concentrating light by light emitting elements located inside the reactor, e.g. LED or OLED
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    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M37/00—Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination
    • C12M37/04—Seals
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C12M41/14—Incubators; Climatic chambers
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    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas
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    • 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, e.g. protozoa; 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

Definitions

  • the invention relates to a method for liquidly cultivating edible and medicinal fungi, and belongs to the technical field of biological fermentation.
  • the edible and medicinal fungi are delicious, rich in protein, vitamins, mineral elements and other biologically active ingredients. They have more medicinal value and better health care functions, and are well received by the masses.
  • mushrooms, white fungus, ganoderma lucidum, ash tree flowers and other medicinal fungi can effectively reduce blood lipids; Agaricus blazei can lower cholesterol, lower blood pressure; sputum, fungus, etc. can effectively prevent cerebral thrombosis; long-term consumption of Hericium erinaceus can effectively prevent each Stomach disease; Cordyceps sinensis and Cordyceps militaris contain not only anti-cancer but also some bacteria and pathogenic fungi.
  • the biosynthesis of the main bioactive substances is more active in the liquid static culture mode. It was found that the effect of oxygen on the fermentation of Ganoderma lucidum mycelium was found to be conducive to the formation of total ganoderic acid. When the polysaccharide was produced by fermentation of Ganoderma lucidum mycelium, it was found that the nutrient-limiting strip could increase the yield of polysaccharide.
  • the Chinese patent (CN200710030072.0) discloses a method for producing deep- and shallow-layer statically coupled fermented high-fiber coconut fruit, which shortens the fermentation cycle of high-fiber coconut fruit by 20%-40%, and can improve the utilization rate of equipment.
  • the Chinese patent (CN201510646553.9) is a combined liquid fermentation shallow tank assembly and a liquid fermentation apparatus using the same, the combined liquid fermentation shallow tank assembly comprises: a mother tank, a sub tank; the liquid fermentation shallow tank provided by the invention
  • the device is simple to operate, is conducive to the growth of filamentous fungal mycelium, can solve the problem of insufficient dissolved oxygen in the liquid fermentation process of mycelium, and can also improve the shallow layer. Cultivate space utilization and increase production efficiency.
  • the present invention provides a device for cultivating a medicinal medicinal fungus and a method for using the device to liquidly culture a medicinal medicinal fungus.
  • the device for liquid static culture of edible medicinal fungi comprises an incubator, an incubator unit, a ventilation device, a temperature and humidity control device; the incubator is provided with one or more relatively independent incubators
  • the incubator unit is in the form of a three-dimensional container having a polygonal, circular or elliptical cross section, and is provided with a vent, a liquid inlet, and a liquid discharge port.
  • the incubator unit can be fixed by a partition built into the incubator.
  • the partition is provided with a portion for fixing the incubator.
  • the material of the incubator may be a whole stainless steel or a stainless steel frame with transparent glass.
  • the material of the separator may be stainless steel.
  • the height of the incubator unit is generally no more than 20 cm, for example: 6 cm or 10 cm.
  • the ventilation device is used to exchange the incubator with outside air.
  • the ventilation device can be automatically turned on or off by a solenoid valve.
  • the temperature and humidity control device includes a temperature sensor and a dehumidification thermostat. It is used to provide the temperature required for the growth of the medicinal fungi and to keep the incubator environment dry.
  • the incubator unit is a food grade sterile bag.
  • the incubator unit is detachable, including an incubator body and an upper cover; the upper cover has two through holes, and the bottom of the incubator body has a liquid discharge port,
  • the cover plate is made of a transparent material, and the material of the incubator body is a food grade, heat resistant material such as PPSU (polyphenylsulfone), PP (polypropylene) or stainless steel.
  • PPSU polyphenylsulfone
  • PP polypropylene
  • a silicone gasket with good sealing properties such as a food grade polytetrafluoroethylene material or a food grade silicone mat, may be provided between the main body of the incubator and the upper cover.
  • the incubator unit is further provided with a cell trapping device for separating the mycelium from the culture medium without affecting the bacterial body to obtain nutrients from the culture medium, which may be food grade filtration.
  • a cell trapping device for separating the mycelium from the culture medium without affecting the bacterial body to obtain nutrients from the culture medium, which may be food grade filtration.
  • Membrane such as a food grade collagen casing.
  • the incubator unit may further be provided with a supporting device for supporting the cell trapping device, and the supporting device may be a net or at least two supporting strips that are crossed or not crossed.
  • the material of the support device may be made of stainless steel.
  • the incubator unit is cylindrical and detachable, and has a diameter of about 50 to 500 mm and a height of about 60 to 100 mm.
  • the device for cultivating a medicinal medicinal fungus further comprises an ultraviolet sterilizing device.
  • the ultraviolet disinfection device may be an ultraviolet lamp.
  • the device for culturing a medicinal medicinal fungus is further provided with a lighting device.
  • the illumination device can be an LED light.
  • the apparatus for cultivating a medicinal medicinal fungus further includes a rocking device capable of shaking the incubator unit within a certain range; the rocking device includes a power source and a transmission.
  • the power source may be a motor and the transmission is a gear transmission.
  • the device for culturing a medicinal medicinal fungus has a support foot at the bottom, the support foot being curved.
  • the incubator performs a slight rocking motion.
  • the invention also provides a culture container unit for cultivating edible medicinal fungi, which is detachable, including an incubator body and an upper cover; the upper cover has two through holes, and the bottom of the incubator body is drained
  • the upper cover is made of a transparent material, and the material of the incubator is made of food grade, heat resistant material such as PPSU (polyphenylsulfone), PP (polypropylene) or stainless steel.
  • a silicone gasket with good sealing properties such as a food grade polytetrafluoroethylene material or a food grade silicone mat, may be provided between the main body of the incubator and the upper cover.
  • a cell trapping device is also provided in the incubator unit, and the cell trapping device may be a food grade filter, such as a food grade collagen casing.
  • the incubator unit may further be provided with a supporting device for supporting the cell trapping device, and the supporting device may be a net or at least two supporting strips that are crossed or not intersected.
  • the material of the support device may be made
  • a sterile liquid medium is added through the inlet of the culture vessel unit, the fermented medicinal fungus seed fermentation liquid is connected, the culture vessel unit is placed on the partition plate, and the appropriate temperature is controlled to carry out the culture. ;
  • the culture solution can be collected from the liquid discharge port below the culture vessel unit, and the culture is opened. The container unit is taken out and dried for use.
  • the medicinal medicinal fungus includes, but is not limited to, Ganoderma lucidum, Antrodia camphorata, Cordyceps sinensis, Hericium erinaceus, Trametes versicolor, Golden ear, and the like.
  • the liquid fermentation medium is a liquid medium commonly used in the art.
  • the inoculation is inoculation of 0.5 mL of homogenized seed fermentation broth per 20 mL of fermentation broth.
  • the fermentation is aerobic fermentation, and gas exchange with the outside is required during the fermentation process.
  • the fermentation is a liquid static fermentation, but the incubator can be shaken left and right for 5-15 minutes by a rocking device periodically (every 12-48 hours), and the left and right shaking angle is greater than 0 degrees but not more than 3 degrees.
  • dark and light culture can be carried out in accordance with the requirements for the growth of medicinal fungi.
  • the device provided by the present invention is widely applicable to large-scale cultivation and laboratory scale culture of a medicinal fungus having a small amount of oxygen demand but sensitive to shear force, and satisfies research or actual production requirements.
  • the user can select the number of incubator units as needed. Because no matter what culture scale, each incubator unit always serves as a separate culture space, from small-scale cultivation to large-scale cultivation, there is no need to explore new conditions for scale-up culture.
  • the bacteria trapping device is arranged in the incubator unit, which does not affect the influence of the cells from the culture medium, and can realize rapid growth of the strain and ensure complete solid-liquid separation.
  • the obtained mycelium is relatively pure, and the growth speed is fast, and the mycelium also has a mushroom flavor, which can be directly eaten.
  • LED lighting device can realize dark culture and light culture, and can cultivate the strain to improve its biological activity and nutritional value.
  • Dehumidification thermostat and temperature sensor device can meet the temperature required for the growth of edible and medicinal fungi and accelerate their growth.
  • the liquid static culture device provided by the utility model can be reused many times, and the large-scale cultivation of edible and medicinal fungi can realize the demand of low-cost, high-nutrition value and high-activity edible and medicinal fungi products. It provides a basis for a wide range of applications in the fields of food, medicine, and cosmetics.
  • the obtained mycelium is relatively pure, the growth rate is fast, the mycelial biomass and the active substance content are increased, the biomass is increased by about 60% compared with the liquid fermentation, and the intracellular polysaccharide is increased by about 40-50%, and the extracellular polysaccharide is increased. Basically unchanged;
  • Mycelium also has mushroom flavor and can be eaten directly
  • the production process does not require expensive equipment, and the production equipment integrates devices to save space and save production costs.
  • the present invention uses a sterile liquid fermentation apparatus that can be repeatedly reused to carry out large-scale medicinal fungal liquid static culture, thereby realizing the demand for low-cost, high-nutrition, high-activity medicinal fungi. It provides a basis for its wide application in the fields of food, medicine and cosmetics.
  • Figure 1 is a front elevational view of an apparatus for cultivating a medicinal medicinal fungus in an embodiment of the present invention
  • Figure 2 is a side view of an apparatus for cultivating a medicinal medicinal fungus in an embodiment of the present invention
  • Figure 3 is a plan view of an apparatus for cultivating a medicinal medicinal fungus in an embodiment of the present invention
  • Figure 4 is a front elevational view of the incubator unit in one embodiment of the present invention.
  • Fig. 5 is a diagram showing the results of liquid static culture of Ganoderma lucidum.
  • Biomass determination The hyphae were taken out and dried at 60 ° C to constant weight.
  • Determination of extracellular polysaccharide collect a certain volume of culture solution from the discharge port of the culture vessel unit, add 4 times volume of 95% ethanol alcohol to precipitate overnight, centrifuge to remove the supernatant, wash the precipitate with 75% ethanol, and then use a certain volume of distilled water. The precipitate was dissolved, and the extracellular polysaccharide was measured by a phenol-sulfuric acid method.
  • Determination of intracellular polysaccharide accurately weigh about 1g of dry powder obtained from the sample, put it into a triangular flask, add 30mL of water, water bath at 90 °C for 1.5h, centrifuge, repeat the precipitation twice, centrifuge and combine three times to obtain the supernatant. Up to 100mL. Take a certain volume of extract, add 4 times volume of 95% ethanol, stand in a 4 °C refrigerator for overnight precipitation, centrifuge, remove the supernatant, wash the precipitate with 75% ethanol, then dissolve the precipitate with distilled water, dilute to 100mL, take a certain The intracellular polysaccharide content was determined by a phenol-sulfuric acid method after appropriately diluting the volume.
  • Ganoderma acid determination The mycelium powder was weighed and extracted with ethanol. The supernatant was obtained after centrifugation and dried under vacuum to remove ethanol and water. The residue was dissolved in water, and the dispersed aqueous phase was extracted twice with chloroform to obtain a chloroform layer. After adding 5% baking soda water, the mixture was mixed and extracted, and the aqueous layer was taken. The pH of the aqueous layer was adjusted to 3 with 2M hydrochloric acid, and ginsolic acid was extracted by adding chloroform. The chloroform phase was dried in vacuo and dissolved in absolute ethanol. The absorbance is measured at 245mn, and then the corresponding ginsolic acid content is calculated according to the standard curve. the amount.
  • HPLC detection chromatographic conditions the column was reversed C18 Huijie high performance liquid chromatography column (filler: hypersil ODS 5 ⁇ m, column length 150mm, diameter 4.6mm); mobile phase was 10mmol KH 2 PO 4 dissolved in methanol / double steaming Water (6:94); detection wavelength was 254 nm; column temperature was 45 ° C; flow rate was 0.80 m L/min; injection volume was 20 ⁇ L.
  • Liquid seed medium (g ⁇ L -1 ): potato 200, glucose 20, natural pH.
  • Liquid resting medium (g ⁇ L -1 ): glucose 20, tryptone 5, YNB (amino-free yeast) 5, KH 2 PO 4 4.5, MgSO 4 . 7H 2 O 2, pH 6.0.
  • the incubator unit is a cylinder with a radius of 50 mm and a height of 60 mm.
  • the cell retention device is located 25 mm from the bottom of the container inside the container.
  • the Ganoderma lucidum slant-preserved strain was inserted into a 250 mL sterile flask containing 80 mL of liquid seed medium, and cultured at 30 ° C, 150 rpm for 7 days, which was a seed solution, which was transferred to a new 250 mL sterilized rotor and glass beads.
  • the triangular flask is broken up to homogenize the seed solution.
  • 230 mL of liquid static medium was added to the culture vessel unit, 5.75 mL of liquid homogenate seed solution was inoculated, and cultured at 30 ° C in a constant temperature incubator, and shaken by a rocking device for 10 min every 24 h, and the left and right shaking angle was 2 degrees.
  • the static culture was completed for 6 days.
  • the culture solution is collected from the lower opening of the culture vessel unit, and the cells are left on the edible film to form a mushroom.
  • the culture results are shown in Fig. 5.
  • the ganoderma lucidum cells grow rapidly and well, and the strains are overgrown on the surface of the incubator with uniform texture, the mycelium is dense, the colonies are intact, and the color is normal.
  • the yield of biomass, polysaccharide and ganoderma lucidum was significantly higher than that of liquid oscillating fermentation.
  • 4.16g of bacteria and about 0.2L of fermentation broth were obtained.
  • the active substance ganoderma acid content was 3.72mg/100mg (dry weight), and the yield of extracellular polysaccharide was obtained. 0.53 g / L, intracellular polysaccharide content of 269.72 mg / g.
  • Liquid seed medium (g ⁇ L -1 ): potato 200, glucose 20, natural pH.
  • Liquid resting medium (g ⁇ L -1 ): glucose 20, tryptone 5, YNB (amino-free yeast) 5, KH 2 PO 4 4.5, MgSO 4 . 7H 2 O 2, pH 6.0.
  • the incubator unit is a cylinder with a radius of 50 mm and a height of 60 mm.
  • the cell retention device is located 25 mm from the bottom of the container inside the container.
  • the Ganoderma lucidum slant-preserved strain was inserted into a 250 mL sterile flask containing 80 mL of liquid seed medium, and cultured at 30 ° C, 150 rpm for 7 days, which was a seed solution, which was transferred to a new 250 mL sterilized rotor and glass beads.
  • the triangular flask is broken up to homogenize the seed solution.
  • 230 mL of liquid static medium was added to the culture vessel unit, 5.75 mL of the liquid homogenate seed solution was inoculated, and the culture was carried out at 30 ° C in a constant temperature incubator, and the culture was allowed to stand for 6 days.
  • the culture results are shown in Fig. 5.
  • the ganoderma lucidum cells grow rapidly and well, and the strains are overgrown on the surface of the incubator with uniform texture, the mycelium is dense, the colonies are intact, and the color is normal.
  • the yield of biomass, polysaccharide and ganoderma acid was significantly higher than that of liquid oscillating fermentation.
  • 3.70g of bacteria and about 0.2L of fermentation broth were obtained.
  • the active substance ganoderma acid content was 2.63mg/100mg (dry weight), and the yield of extracellular polysaccharide was 0.52. g/L, intracellular polysaccharide content of 254.72 mg / g.
  • Liquid seed medium (g ⁇ L -1 ): potato 200, glucose 20, pH is natural.
  • Liquid resting medium sucrose 20, peptone 20, KH 2 PO 4 1, MgSO 4 ⁇ 7H 2 O 0.5, pH is natural.
  • the incubator unit is a cylinder with a radius of 50 mm and a height of 60 mm.
  • the cell retention device is located 25 mm from the bottom of the container inside the container.
  • the triangular flask is broken up to homogenize the seed solution.
  • Add 230 mL of liquid static medium to a sterile circular culture vessel, inoculate 5.75 mL of liquid homogenate seed solution place it in a constant temperature incubator at 25 ° C for light culture, and shake it for about 15 min every 28 h by a rotating device. The angle of shaking was 3 degrees, and the stationary culture was finished for 12 days.
  • the culture solution was collected from the lower opening of the incubator, and the cells were left on the edible film to form a microplate.
  • Cordyceps sinensis grows rapidly and well, and the strain grows over the surface of the incubator and has a uniform texture. The mycelium is dense and the colony is intact and the color is normal. At the end of the culture, 3.53g of bacteria and about 0.2L of fermentation broth were obtained. The yield of active substance cordycepin, adenosine and adenine was significantly higher than that of liquid oscillating fermentation. The yield of cordycepin was 65mg/L, and the yield of adenosine was 8mg/L. The yield was 7 mg/L.
  • Liquid seed medium (g ⁇ L -1 ): potato 200, glucose 20, pH is natural.
  • Liquid resting medium sucrose 20, peptone 20, KH 2 PO 4 1, MgSO 4 ⁇ 7H 2 O 0.5, pH is natural.
  • the incubator unit is a cylinder with a radius of 50 mm and a height of 60 mm.
  • the cell retention device is located 25 mm from the bottom of the container inside the container.
  • the triangular flask is broken up to homogenize the seed solution.
  • Add 230 mL of liquid static medium to a sterile circular culture vessel, inoculate 5.75 mL of liquid homogenate seed solution place it in a constant temperature incubator at 25 ° C for dark culture, and shake it for about 15 min every 28 h by a rotating device. The angle of shaking was 3 degrees, and the stationary culture was finished for 12 days.
  • the culture solution was collected from the lower opening of the incubator, and the cells were left on the edible film to form a microplate.
  • Cordyceps sinensis grows rapidly and well, and the strain grows over the surface of the incubator and has a uniform texture. The mycelium is dense and the colony is intact and the color is normal.
  • 3.78 g of bacteria about 0.2 L of fermentation broth, the yield of cordycepin and the light culture decreased slightly, the production of adenosine decreased drastically, but the yield of adenine increased, the yield of cordycepin reached 58 mg/L, and the yield of adenosine reached 3.5.
  • Mg/L adenine production reached 9.8mg / L.
  • Liquid seed medium (g ⁇ L -1 ): potato 200, glucose 20, natural pH.
  • Liquid resting medium (g ⁇ L -1 ): glucose 20, tryptone 5, YNB (amino-free yeast) 5, KH 2 PO 4 4.5, MgSO 4 . 7H 2 O 2, pH 6.0.
  • the incubator unit is rectangular, detachable, with a length and width of 200 mm and a height of 100 mm.
  • the cell retention device is located 45 mm from the bottom of the container inside the container.
  • the Ganoderma lucidum slant-preserved strain was inserted into a 250 mL sterile flask containing 80 mL of liquid seed medium, and cultured at 30 ° C, 150 rpm for 7 days, which was a seed solution, which was transferred to a new 250 mL sterilized rotor and glass beads.
  • the triangular flask is broken up to homogenize the seed solution.
  • 2 L of liquid static medium was placed in the culture vessel unit, 50 mL of the liquid homogenate seed solution was inoculated, and the culture was carried out by placing it in a constant temperature incubator at 30 °C.
  • the culture solution is collected from the lower opening of the culture vessel unit, and the cells are left on the edible film to form a mushroom.
  • Ganoderma lucidum cells grow rapidly and well, and the strains grow over the surface of the incubator and have a uniform texture. The mycelium is dense and the colonies are intact and the color is normal. At the end of the culture, 35.7 g of the cells were obtained, and about 1.7 L of a fermentation broth was obtained, wherein the active substance ganoderic acid was 3.85 mg/100 mg (dry weight), the extracellular polysaccharide yield was 0.61 g/L, and the intracellular polysaccharide content was 280.72 mg/g.
  • the apparatus for cultivating a medicinal medicinal fungus comprises a cuboid shaped incubator 1.
  • the incubator 1 is internally provided with a plurality of partitions 17, on which the incubator unit 3 is placed, and the LEDs are mounted under the partitions 17. light.
  • the top of the incubator 1 is provided with an exhaust pipe 4 on which an electromagnetic valve 5 is mounted for realizing exchange of the incubator with outside air.
  • the outside of the bottom of the incubator 1 is provided with a power interface 8 and a motor 11, and the motor 11 is mounted with a frequency converter 9 and a rocking controller 10, which can be used to control the swing frequency and amplitude of the incubator 1, and the motor 11 passes through a coupling device (such as The shaft drive drives the gear transmission to swing left and right, and the limit gear on the large gear.
  • the swing frequency is set according to the strain, and the swing angle is controlled to not exceed 3 degrees.
  • a temperature sensor 6 and a dehumidification thermostat 7 are mounted on the side wall of the incubator 1 for controlling the temperature and humidity in the incubator 1 to make it suitable for growth of the culture.
  • the incubator unit 3 is of a detachable cylindrical shape, including an incubator body and an upper cover; the upper cover is provided with a liquid inlet 12 and a vent 13; the bottom of the incubator body has a liquid discharge port 16 and the upper cover is made of food Grade transparent material; a silicone gasket with good sealing property may be provided between the main body of the incubator and the upper cover plate for sealing; a bacteria trapping device-edible film 14 is arranged in the main body of the incubator, and the main body of the incubator is further provided A support device 15 supporting the edible film 14, the support device 15 being a thin stainless steel mesh having an aperture of 2-3 cm. The protruding liquid discharge port 16 at the bottom of the incubator unit 3 is caught in the hole of the partition plate 17.
  • the liquid discharge port 16 may be externally connected with a rubber tube or the like for discharging and collecting the culture liquid.
  • the edible film 14 can also be replaced with a food-grade collagen casing, allowing the cells to adhere and grow to form a micro-slice without separating the cells from the food-grade collagen casing.
  • the prepared liquid medium is first added to the main body of the incubator, the upper cover plate is covered, and the incubator unit is sterilized. After sterilization, the liquid level of the culture medium exceeds the bacterial body.
  • the height of the intercepting device is 0.1-0.5 cm. If the incubator is equipped with an ultraviolet lamp, turn on the UV lamp and kill the inside of the incubator for a while. Then, under aseptic conditions, the seeds of the medicinal fungi to be cultured are introduced from the liquid inlet on the upper cover and fall into the upper surface of the bacterial retention device.
  • the medicinal fungi are cultured at a suitable temperature, and the incubator is periodically shaken to promote air circulation and oxygen dissolution.
  • the LED light built into the incubator as a light source, or use a stainless steel frame with a clear glass incubator to take advantage of natural light.
  • the fermentation broth is collected from the liquid discharge port at the bottom of the incubator body, and the grown pieces are removed from the cell retention device. The fermentation broth and the plaque are further processed to obtain the corresponding product.

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Abstract

本发明公开了一种液体静置培养食药用真菌的方法，属于生物发酵技术领域。本发明采用可多次重复利用的无菌液体发酵装置进行食药用真菌液体静置培养，可以实现其低成本、高营养价值、高活性的食药用真菌产品需求，为其在食品、医药、化妆品等领域广泛应用提供基础。

Description

一种液体静置培养食药用真菌的方法 技术领域
本发明涉及一种液体静置培养食药用真菌的方法，属于生物发酵技术领域。
背景技术
食药用真菌味道鲜美，富含蛋白质、维生素、矿质元素等多种生物活性成分，具有较多的药用价值及较好的保健功能，深受广大群众喜爱。例如：香菇、银耳、灵芝、灰树花等食药用真菌均可有效降血脂；姬松茸可降低胆固醇、降血压；茯苓、木耳等可有效预防脑血栓；长期食用猴头菇可有效预防各种胃病；冬虫夏草和北冬虫夏草含不仅能抗癌、而且对一些细菌和致病真菌有抑制作用。
传统的食药用真菌常采用固体栽培方式进行培养，这种培养方式下，真菌的生长环境条件接近自然状态，但固体栽培存在培养周期较长、产量低、代谢产物生物活性不稳定、易染菌等的问题。相对而言，液体发酵培养周期短、易于扩大培养、其制品丰富。许多食药用真菌，例如牛樟芝、斑金钱菌等，很难采用人工固体栽培方式进行大规模长时间培养，但可以通过液体发酵达到大规模长时间培养的目的。此外，灵芝、灰树花、姬松茸等几十种食药用真菌也实现了液体发酵培养。因此，采用液体培养食药用真菌已经成为一种趋势。
但是，传统的液体发酵培养食药用真菌，设备要求高且复杂；且菌体主要以菌丝体的形式生长，发酵结束时固液分离较复杂且相对困难。对于液体静置培养食药用真菌，操作比较简单，也容易实现快速分离菌体和发酵液，但是现有的一些细胞培养装置或多或少存在一些缺陷，无法满足各种规模的培养需求或者无法达到快速固液分离的目的。在食药用真菌发酵过程中，氧和营养物对菌丝的生长以及次级代谢物的生产有重要的作用。液体静置培养是一种高效生产的方式，与传统培养方式相比，主要生物活性物质(多糖、灵芝酸等)的生物合成在液体静置培养方式下更为活跃。研究发现氧对灵芝菌丝体发酵的影响时，发现较低的氧浓度有利于总灵芝酸的生成；用灵芝菌丝体发酵生产多糖时，发现营养限制条可以提高多糖的产量。中国专利(CN200710030072.0)公开了一种深层与浅层静态偶联发酵高纤椰果的生产方法，使高纤椰果浅层发酵周期缩短20％-40％，且可提高设备生产利用率，有利益降低浅层发酵中的杂菌污染，应用于大规模生产中可产生巨大的经济利益。中国专利(CN201510646553.9)是一种组合式液态发酵浅槽组件及使用该浅槽的液态发酵装置，组合式液态发酵浅槽组件包括：母槽，子槽；该发明提供的液态发酵浅槽装置操作简单，有利于丝状真菌菌丝体生长，能够解决菌丝体液态发酵过程中溶氧不足的问题，同时也能够提高浅层 培养的空间利用率，提高生产效率。
但是，以上的装置无法实现：(1)多种食药用真菌同时大规模培养，装置的高效利用；(2)隔离培养，大幅降低污染几率；(3)能够使食药用真菌生物活性物质含量增加，生物活性增强；(4)完全彻底的固液分离。
发明内容
为了克服以上技术缺点，本发明提供一种用于培养食药用真菌的装置以及应用该装置液体静置培养食药用真菌的方法。
本发明提供的用于食药用真菌液体静置培养的装置，包括培养箱、培养器单元、通风装置、温度和湿度控制装置；所述培养箱设有1个或多个相对独立的培养器单元；所述培养器单元，形状是横截面为多边形、圆形或者椭圆形的立体的容器，开设有通气口、进液口、排液口。
所述培养器单元可以通过培养箱内置的隔板进行固定。所述隔板设置有用于固定培养器的部分。所述培养箱的材质可以为整体不锈钢，或者不锈钢框架搭配透明玻璃。所述隔板的材质可以为不锈钢。
所述培养器单元的高度一般不超过20cm，例如：6cm或者10cm。
所述通风装置用于实现培养箱与外界空气的交换。所述通风装置可以由电磁阀自动控制开启或关闭。
所述温度和湿度控制装置，包括温度传感器和除湿恒温器。用于提供食药用真菌生长所需的温度且保持培养箱环境干燥。
在本发明的一种实施方式中，所述培养器单元为食品级别无菌袋。
在本发明的一种实施方式中，所述培养器单元为可拆卸式，包括培养器主体和上盖板；上盖板开有两个通孔，培养器主体底部开有排液口，上盖板采用透明材料，培养器主体的材质为食品级、耐热材料例如PPSU(聚苯砜)、PP(聚丙烯)或者不锈钢。培养器主体和上盖板之间可设有密封性良好的硅胶垫，如食品级聚四氟乙烯材质或食品级硅胶垫。所述培养器单元内还设有菌体截留装置，所述菌体截留装置用于将菌丝体与培养基分隔开但不影响菌体从培养基中获取营养成分，可以是食品级别滤膜，例如食品级别胶原蛋白肠衣。所述培养器单元内还可以设有支撑所述菌体截留装置的支撑装置，所述支撑装置可以是网或者至少两个交叉或不交叉的支撑条。所述支撑装置的材质可以是不锈钢材质。
在本发明的一种实施方式中，所述培养器单元为圆柱形、可拆卸式的，直径50～500mm左右、高度60～100mm左右。
在本发明的一种实施方式中，所述用于培养食药用真菌的装置，还包括紫外线消毒装置。所述紫外线消毒装置可以是紫外灯。
在本发明的一种实施方式中，所述用于培养食药用真菌的装置内部还安装了照明装置。所述照明装置可以是LED灯。
在本发明的一种实施方式中，所述用于培养食药用真菌的装置，还包括能够使培养器单元在一定幅度内晃动的摇摆装置；所述摇摆装置包括动力源、传动装置。所述动力源可以是电机，所述传动装置是齿轮传动装置。
在本发明的一种实施方式中，所述用于培养食药用真菌的装置的底部有支撑脚，所述支撑脚为弧形。当向所述用于培养食药用真菌的装置施加一定的力时，培养箱做轻微的摇摆运动。
本发明还提供一种用于培养食药用真菌的培养容器单元，为可拆卸式，包括培养器主体和上盖板；上盖板开有两个通孔，培养器主体底部开有排液口，上盖板采用透明材料，培养器主体的材质为食品级、耐热材料例如PPSU(聚苯砜)、PP(聚丙烯)或者不锈钢。培养器主体和上盖板之间可设有密封性良好的硅胶垫，如食品级聚四氟乙烯材质或食品级硅胶垫。所述培养器单元内还设有菌体截留装置，所述菌体截留装置可以是食品级别滤膜，例如食品级别胶原蛋白肠衣。所述培养器单元内还可以设有支撑所述菌体截留装置的支撑装置，所述支撑装置可以是网，或者至少两个交叉或不交叉的支撑条。所述支撑装置的材质可以是不锈钢材质。
本发明应用所述装置液体静置培养食药用真菌的步骤如下：
(1)准备食药用真菌匀浆种子发酵液(发酵液通过玻璃珠和转子打散至匀浆状态)；
(2)准备高温灭菌液体培养基；
(3)准备高温灭菌培养容器单元；
(4)在无菌环境下，通过培养容器单元进液口加入灭菌液体培养基，接入食药用真菌种子发酵液，将培养容器单元放置在隔板上，控制合适的温度，进行培养；
(5)当菌种长满无菌培养箱表面，且菌落完整，菌丝体细密、健壮、色泽好，即培养结束，可将培养液从培养容器单元下面排液口进行收集备用，打开培养容器单元，取出菌片、烘干备用。
在本发明的一种实施方式中，所述的食药用真菌包括但不局限于灵芝、牛樟芝、冬虫夏草、猴头菇、变色栓菌、金耳等。
在本发明的一种实施方式中，所述液体发酵培养基为本领域常用液体培养基。
在本发明的一种实施方式中，所述接种是每20mL发酵液接种0.5mL匀浆种子发酵液。
在本发明的一种实施方式中，所述发酵为好氧发酵，发酵过程中需要与外界进行气体交换。
在本发明的一种实施方式中，所述发酵为液体静置发酵，但可以定期(每12-48h)通过摇摆装置对培养箱进行左右摇晃5-15min，左右晃动角度大于0度但不超过3度。
在本发明的一种实施方式中，可以依据食药用真菌生长的需求进行黑暗与光照培养。
有益效果：
本发明提供的用于培养食药用真菌的装置，具有以下优点：
(1)本发明提供的装置广泛适用于生长需氧量不大但对剪切力敏感的食药用真菌的大规模培养及实验室规模培养，满足研究或实际生产需求。使用者可根据需求，选择培养器单元的数量。因为不管在何种培养规模下，每一个培养器单元始终作为单独的培养空间，从小规模培养到大规模培养的过程，不需要摸索新的放大培养的条件。
(2)培养器单元内设有菌体截留装置，不影响菌体从培养基中获取影响，可以实现菌种快速生长，并且保证实现彻底的固液分离。所得菌丝体较为纯净，生长速度快，菌丝体也有菇类香味，可直接食用。
(3)LED灯照明装置可以实现黑暗培养与光照培养，可以针对性对菌种进行培养以提高其生物活性和营养价值。
(4)除湿恒温器和温度传感器装置能够满足食药用真菌生长所需温度，加速其生长。
(5)可利用大型克氏瓶或相应的培养容器，扩大培养面积，提高产量，适应生产需要。
(6)具有萌芽点多、菌丝覆盖面早的优点。
(7)本实用新型提供的液体静置培养装置可以多次重复利用，大规模培养食药用真菌，可以实现其低成本、高营养价值、高活性的食药用真菌产品需求，为其在食品、医药、化妆品等领域广泛应用提供基础。
(8)设置一个个独立的培养空间，可以在每一个独立空间培养不同的菌种，可以依据生产需求对多种食药用真菌进行培养，实现装置的高效利用。
(9)菌体截留装置采用食品级别胶原蛋白肠衣这种耐高温、透气性好而且可食用的材质时，无需将菌体与膜分离，可以一起烘干食用。
本发明提供的培养食药用真菌的方法，具有以下优点：
(1)所得菌丝体较为纯净，生长速度快，菌丝体生物量和活性物质含量均有提高，生物量相对液体发酵提高约60％，胞内多糖提高约40-50％，胞外多糖基本不变；
(2)菌丝体也有菇类香味，可直接食用；
(3)具有流动快、发菌迅速、菌丝覆盖面早的优点；
(4)减少了发酵结束时固液分离的操作步骤，工业生产时间减少，同时也降低了成本；
(5)生产过程不需要昂贵的设备，生产设备集成装置，节约场地，节约生产成本。
总的来说，本发明采用可多次重复利用的无菌液体发酵装置进行大规模食药用真菌液体静置培养，可以实现其低成本、高营养价值、高活性的食药用真菌需求，为其在食品、医药、化妆品等领域广泛应用提供基础。
附图说明
图1本发明一种实施方式中用于培养食药用真菌的装置的主视图；
图2本发明一种实施方式中用于培养食药用真菌的装置的侧视图；
图3本发明一种实施方式中用于培养食药用真菌的装置的俯视图；
图4本发明一种实施方式中培养器单元的主视图；
图1～4中，1：培养箱，2：培养器单元，3：LED灯，4：排气管，5：电磁阀，6：温度传感器，7：除湿恒温器，8：电源接口，9：变频器，10：摇摆控制器，11：电机，12：进液口，13：通气口，14：可食用膜，15：支撑装置，16：排液口，17：隔板。
图5灵芝液体静置培养结果图。
具体实施方式
生物量测定：菌丝片取出，于60℃烘干至恒重。
胞外多糖测定：从培养容器单元排液口处收集一定体积的培养液，加4倍体积95％乙醇醇沉过夜，离心后去上清，用75％乙醇洗涤沉淀后再用一定体积的蒸馏水将沉淀物溶解，采用苯酚-硫酸法对胞外多糖进行测定。
胞内多糖测定：精确称取样品得到的干粉约1g，放入三角瓶中，加入30mL水，90℃水浴1.5h，离心，沉淀重复上步两次，离心后合并三次所得上清，定容至100mL。取一定体积提取液，加入4倍体积95％乙醇，4℃冰箱静置沉淀过夜，离心，去除上清，用75％乙醇洗涤沉淀后再用蒸馏水将沉淀物溶解，定容到100mL，取一定体积适当稀释后用苯酚-硫酸法对胞内多糖含量进行测定。
灵芝酸测定：称取菌丝体粉末，加入乙醇提取。离心后获得上清液，真空干燥去除乙醇和水。用水溶解剩余物，并用氯仿萃取分散后的水相两次取氯仿层，加入5％的小苏打水后混匀并萃取，取水层。用2M盐酸将水层pH调节为3，加入氯仿萃取灵芝酸。取氯仿相真空干燥后，用无水乙醇溶解。在245mn测定吸光值，然后依据标准曲线计算对应的灵芝酸的含 量。
虫草素、腺嘌呤和腺苷测定：将发酵完毕的发酵液4,000r/min离心20min，收集上清液并稀释至一定浓度，经0.45μm滤膜过滤，用HPLC检测虫草素、腺嘌呤和腺苷的含量。HPLC检测色谱条件：色谱柱采用反相C18惠杰型高效液相色谱柱(填料：hypersil ODS 5μm，柱长150mm，管径4.6mm)；流动相为10mmol KH2PO4溶于甲醇/双蒸水(6:94)；检测波长为254nm；柱温45℃；流速为0.80m L/min；进样量为20μL。
实施例1
菌种：灵芝
液体种子培养基(g·L-1)：马铃薯200，葡萄糖20，自然pH。
液体静置培养基(g·L-1)：葡萄糖20，胰蛋白胨5，YNB(无氨基酵母)5，KH2PO44.5，MgSO4﹒7H2O 2，pH 6.0。
无菌培养容器规格：培养器单元为圆柱体，半径为50mm、高度60mm，菌体截留装置在容器内部距离容器底部25mm处。
灵芝斜面保藏菌种接入装有80mL液体种子培养基的250mL无菌三角瓶，30℃、150rpm培养7天，即为种子液，将其转入新的放有转子和玻璃珠的250mL无菌三角瓶打碎得匀浆种子液。在培养容器单元内加入230mL液体静置培养基，接种液体匀浆种子液5.75mL，放置恒温培养箱中30℃进行培养，每24h通过摇摆装置对其进行左右摇晃10min，其左右晃动角度2度，静置培养6天结束。从培养容器单元的下方开口处收集培养液，菌体留在可食用膜上形成菌片。
培养结果如图5所示，灵芝菌体生长迅速且良好，菌种长满培养箱表面且质地均匀，菌丝体密集且菌落完整，色泽正常。生物量、多糖和灵芝酸产量较液体振荡发酵有明显提高，培养结束得到菌体4.16g，约0.2L发酵液，其中活性物质灵芝酸含量达3.72mg/100mg(干重)，胞外多糖产量0.53g/L，胞内多糖含量269.72mg/g。
对照实施例1
菌种：灵芝
液体种子培养基(g·L-1)：马铃薯200，葡萄糖20，自然pH。
液体静置培养基(g·L-1)：葡萄糖20，胰蛋白胨5，YNB(无氨基酵母)5，KH2PO44.5，MgSO4﹒7H2O 2，pH 6.0。
无菌培养容器规格：培养器单元为圆柱体，半径为50mm、高度60mm，菌体截留装置在容器内部距离容器底部25mm处。
灵芝斜面保藏菌种接入装有80mL液体种子培养基的250mL无菌三角瓶，30℃、150rpm培养7天，即为种子液，将其转入新的放有转子和玻璃珠的250mL无菌三角瓶打碎得匀浆种子液。在培养容器单元中加入230mL液体静置培养基，接种液体匀浆种子液5.75mL，放置恒温培养箱中30℃进行培养，静置培养6天结束。
培养结果如图5所示，灵芝菌体生长迅速且良好，菌种长满培养箱表面且质地均匀，菌丝体密集且菌落完整，色泽正常。生物量、多糖和灵芝酸产量较液体振荡发酵有明显提高，培养结束得到菌体3.70g，约0.2L发酵液，其中活性物质灵芝酸含量2.63mg/100mg(干重)，胞外多糖产量0.52g/L，胞内多糖含量254.72mg/g。
实施例2
菌种：冬虫夏草
液体种子培养基(g·L-1)：马铃薯200，葡萄糖20，pH自然。
液体静置培养基(g·L-1)：蔗糖20，蛋白胨20，KH2PO41，MgSO4·7H2O 0.5，pH自然。
无菌培养容器规格：培养器单元为圆柱体，半径为50mm、高度60mm，菌体截留装置在容器内部距离容器底部25mm处。
冬虫夏草斜面保藏菌种接入装有80mL液体种子培养基的250mL无菌三角瓶，25℃、150rpm培养5天，即为种子液，将其转入新的放有转子和玻璃珠的250mL无菌三角瓶打碎得匀浆种子液。在无菌圆形培养容器上加入230mL液体静置培养基，接种液体匀浆种子液5.75mL，放置恒温培养箱中25℃进行光照培养，每48h通过转动装置对其进行左右摇晃15min，其左右晃动角度3度，静置培养12天结束。从培养器的下方开口处收集培养液，菌体留在可食用膜上形成菌片。
冬虫夏草菌体生长迅速且良好，菌种长满培养箱表面且质地均匀，菌丝体密集且菌落完整，色泽正常。培养结束得到菌体3.53g，约0.2L发酵液，其中活性物质虫草素、腺苷和腺嘌呤产量较液体振荡发酵有明显提高，虫草素产量65mg/L，腺苷产量8mg/L，腺嘌呤产量7mg/L。
对照实施例2
菌种：冬虫夏草
液体种子培养基(g·L-1)：马铃薯200，葡萄糖20，pH自然。
液体静置培养基(g·L-1)：蔗糖20，蛋白胨20，KH2PO41，MgSO4·7H2O 0.5，pH自然。
无菌培养容器规格：培养器单元为圆柱体，半径为50mm、高度60mm，菌体截留装置在容器内部距离容器底部25mm处。
冬虫夏草斜面保藏菌种接入装有80mL液体种子培养基的250mL无菌三角瓶，25℃、150rpm培养5天，即为种子液，将其转入新的放有转子和玻璃珠的250mL无菌三角瓶打碎得匀浆种子液。在无菌圆形培养容器上加入230mL液体静置培养基，接种液体匀浆种子液5.75mL，放置恒温培养箱中25℃进行黑暗培养，每48h通过转动装置对其进行左右摇晃15min，其左右晃动角度3度，静置培养12天结束。从培养器的下方开口处收集培养液，菌体留在可食用膜上形成菌片。
冬虫夏草菌体生长迅速且良好，菌种长满培养箱表面且质地均匀，菌丝体密集且菌落完整，色泽正常。培养结束得到菌体3.78g，约0.2L发酵液，虫草素、产量较光照培养稍有下降，腺苷产量大幅下降，但腺嘌呤产量上升，虫草素产量达58mg/L，腺苷产量达3.5mg/L，腺嘌呤产量达9.8mg/L。
实施例3
菌种：灵芝
液体种子培养基(g·L-1)：马铃薯200，葡萄糖20，自然pH。
液体静置培养基(g·L-1)：葡萄糖20，胰蛋白胨5，YNB(无氨基酵母)5，KH2PO44.5，MgSO4﹒7H2O 2，pH 6.0。
无菌培养容器规格：培养器单元为长方体、可拆卸式的，长和宽为200mm、高度100mm，菌体截留装置在容器内部距离容器底部45mm处。
灵芝斜面保藏菌种接入装有80mL液体种子培养基的250mL无菌三角瓶，30℃、150rpm培养7天，即为种子液，将其转入新的放有转子和玻璃珠的250mL无菌三角瓶打碎得匀浆种子液。在培养容器单元内加入2L液体静置培养基，接种液体匀浆种子液50mL，放置恒温培养箱中30℃进行培养。每12h通过摇摆装置对其进行左右摇晃8min，其左右晃动角度1度，静置培养8天结束。从培养容器单元的下方开口处收集培养液，菌体留在可食用膜上形成菌片。
灵芝菌体生长迅速且良好，菌种长满培养箱表面且质地均匀，菌丝体密集且菌落完整，色泽正常。培养结束得到菌体35.7g，得到约1.7L发酵液，其中活性物质灵芝酸达3.85mg/100mg(干重)，胞外多糖产量0.61g/L，胞内多糖含量280.72mg/g。
实施例4
如图1～4所示：
所述用于培养食药用真菌的装置包括一个长方体形的培养箱1。
培养箱1内部设有多个隔板17，隔板17上放置了培养器单元3，隔板17下方安装了LED 灯。
培养箱1的顶部设有排气管4，排气管4上安装有电磁阀5，用于实现培养箱与外界空气的交换。
培养箱1的底部外侧设有电源接口8和电机11，电机11上安装有变频器9、摇摆控制器10，可用于控制培养箱1的摇摆频率和幅度，电机11通过连轴装置(如连轴器)带动齿轮传动装置左右摆动，大齿轮上有限位开关。摇摆频率依据菌种不同进行设置，控制摇摆角度不超过3度。
培养箱1侧壁上安装了温度传感器6和除湿恒温器7，用于控制培养箱1内的温度和湿度，使适合培养物的生长。
培养器单元3为可拆卸式圆柱形，包括培养器主体和上盖板；上盖板开有进液口12、通气口13，培养器主体底部开有排液口16，上盖板采用食品级透明材料；培养器主体和上盖板之间可设有密封性良好的硅胶垫，用于密封；培养器主体内设有菌体截留装置-可食用膜14，培养器主体内还设有支撑所述可食用膜14的支撑装置15，所述支撑装置15是孔径2-3cm的薄的不锈钢网。培养器单元3底部的突出的排液口16卡入隔板17的孔洞中。排液口16可以外接橡胶管等用于排出、收集培养液。所述可食用膜14还可以替换为食品级别胶原蛋白肠衣，让菌体附着生长，形成菌片，无需将菌体从食品级别胶原蛋白肠衣上分离。
应用所述装置培养食药用真菌时，先向培养器主体中加入配制好的液体培养基，盖好上盖板，将培养器单元灭菌，灭菌后，培养基的液面超过菌体截留装置的高度0.1-0.5cm。如果培养箱配置了紫外灯，打开紫外灯，对培养箱内部消杀一段时间。然后，在无菌条件下，将待培养的食药用真菌的种子由上盖板上的进液口接入，落入菌体截留装置的上表面。在适宜的温度下培养食药用真菌，定期摇摆培养箱，促进空气流通、氧气的溶解。对于需要光照的食药用真菌，可打开培养箱内置的LED灯作为光源，或者选用不锈钢框架搭配透明玻璃的培养箱以利用自然光。培养结束后，将发酵液从培养器主体底部的排液口收集，长成一片的菌片可从菌体截留装置上取下。发酵液和菌片经进一步后加工，可得到相应的产品。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (18)

  1. 一种用于食药用真菌液体培养的装置，其特征在于，包括培养箱、培养器单元、通风装置、温度和湿度控制装置；所述培养箱内设有1个或多个相对独立的培养器单元；所述培养器单元是横截面为多边形、圆形或者椭圆形的立体的容器，开设有通气口、进液口、排液口。
  2. 根据权利要求1所述的一种用于食药用真菌液体培养的装置，其特征在于，所述培养器单元通过培养箱内置的隔板进行固定，所述隔板设置有用于固定培养器单元的部分。
  3. 根据权利要求1或2所述的一种用于食药用真菌液体培养的装置，其特征在于，所述培养器单元为食品级别无菌袋。
  4. 根据权利要求1或2所述的一种用于食药用真菌液体培养的装置，其特征在于，所述培养器单元为可拆卸式，包括培养器主体和上盖板；上盖板开有两个通孔，培养器主体底部开有排液口，上盖板采用透明材料。
  5. 根据权利要求4所述的一种用于食药用真菌液体培养的装置，其特征在于，所述培养器主体的材质为食品级、耐热材料。
  6. 根据权利要求4所述的一种用于食药用真菌液体培养的装置，其特征在于，培养器主体和上盖板之间设有密封性良好的硅胶垫。
  7. 根据权利要求4所述的一种用于食药用真菌液体培养的装置，其特征在于，所述培养器单元内还设有菌体截留装置，所述菌体截留装置用于将菌丝体与培养基分隔开但不影响菌体从培养基中获取营养成分。
  8. 根据权利要求7所述的一种用于食药用真菌液体培养的装置，其特征在于，所述菌体截留装置是食品级别胶原蛋白肠衣。
  9. 根据权利要求7所述的一种用于食药用真菌液体培养的装置，其特征在于，所述培养器单元内还设有支撑所述菌体截留装置的支撑装置，所述支撑装置是网，或者至少两个交叉或不交叉的支撑条。
  10. 根据权利要求7所述的一种用于食药用真菌液体培养的装置，其特征在于，培养箱内还设置了紫外线消毒装置和/或照明装置。
  11. 根据权利要求1所述的一种用于食药用真菌液体培养的装置，其特征在于，还包括能够使培养器单元在一定幅度内晃动的摇摆装置；所述摇摆装置包括动力源、传动装置。
  12. 根据权利要求1或12所述的一种用于食药用真菌液体培养的装置，其特征在于，所述培养箱的底部有支撑脚，所述支撑脚为弧形。
  13. 一种用于培养食药用真菌的培养容器单元，其特征在于，为可拆卸式，包括培养器主体和上盖板；上盖板开有两个通孔，培养器主体底部开有排液口；上盖板采用透明材料， 培养器主体和上盖板之间设有密封装置；所述培养器单元内还设有菌体截留装置，所述菌体截留装置用于将菌丝体与培养基分隔开但不影响菌体从培养基中获取营养成分。
  14. 根据权利要求13所述的一种用于培养食药用真菌的培养容器单元，其特征在于，所述培养器单元内还设有支撑所述菌体截留装置的支撑装置，所述支撑装置是网或者至少两个交叉或不交叉的支撑条。
  15. 一种应用权利要求1～12任一所述装置液体静置培养食药用真菌的方法，其特征在于，包括如下步骤：
    (1)准备食药用真菌匀浆种子发酵液；
    (2)准备高温灭菌液体培养基；
    (3)通过培养容器单元进液口加入灭菌液体培养基，高温灭菌；
    (4)在无菌环境下，接入食药用真菌种子发酵液，将培养容器单元放置在隔板上，控制合适的温度，进行发酵培养；
    (5)培养结束，将培养液从培养容器单元下面排液口进行收集备用，打开培养容器单元，取出菌片、烘干备用。
  16. 根据权利要求15所述的方法，其特征在于，所述的食药用真菌包括但不局限于灵芝、牛樟芝、冬虫夏草、猴头菇、变色栓菌、金耳等。
  17. 根据权利要求15所述的方法，其特征在于，定期通过摇摆装置对培养箱进行左右摇晃5-15min，左右晃动角度大于0度但不超过3度。
  18. 根据权利要求15所述的方法，其特征在于，依据食药用真菌生长的需求进行黑暗或光照培养。
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CN111979125A (zh) * 2020-08-28 2020-11-24 安徽惠恩生物科技股份有限公司 一种无血清细胞培养基
CN113215004A (zh) * 2021-06-11 2021-08-06 山东效峰生物科技股份有限公司 一种调控药食两用真菌混合培养过程中均衡生长方法
CN114480078A (zh) * 2022-02-23 2022-05-13 上海申亚动物保健品阜阳有限公司 一种饲料加工用秸秆发酵熟化装置及发酵方法
IT202200010058A1 (it) * 2022-05-16 2023-11-16 Massimo Ambrosi Impianto produzione in continuo di finta pelle da micelio
CN116004380A (zh) * 2022-06-24 2023-04-25 河南牧业经济学院 一种猪细小病毒疫苗研发自控温电磁培养箱
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