CN107586727B

CN107586727B - Fermentation method of ganoderma lucidum mycelia

Info

Publication number: CN107586727B
Application number: CN201711013780.3A
Authority: CN
Inventors: 秦路宁
Original assignee: Hunan Heping Biotechnology Co Ltd
Current assignee: Hunan Heping Biotechnology Co Ltd
Priority date: 2017-10-26
Filing date: 2017-10-26
Publication date: 2020-03-13
Anticipated expiration: 2037-10-26
Also published as: CN107586727A

Abstract

The invention relates to a fermentation method of ganoderma lucidum mycelia, and belongs to the technical field of fungus culture. The fermentation method of the ganoderma lucidum mycelia provided by the invention comprises the following steps: 1) inoculating the activated ganoderma lucidum strain into a liquid fermentation culture medium, and culturing for 5-7 d at 23-26 ℃, wherein the ventilation volume is 0.6-1V/V.min, and the air pressure is 0.03-0.06 Mpa to obtain a fermentation strain; 2) inoculating the fermentation strain obtained in the step 1) into a solid fermentation culture medium for dark culture, wherein the dark culture time is 30-40 days. The fermentation method of the ganoderma lucidum mycelia combines the solid fermentation technology and the liquid fermentation technology, and has the advantages of large production scale, short production period, simple and convenient production process, high content of effective components and high product recovery rate.

Description

Fermentation method of ganoderma lucidum mycelia

Technical Field

The invention relates to the technical field of fungus culture, in particular to a fermentation method of ganoderma lucidum mycelia.

Background

The ganoderma lucidum polysaccharide has multiple physiological functions of resisting tumor, improving immunity, resisting aging, reducing blood fat and the like, has larger medicinal and health-care values, is commonly concerned by the current medical field and food industry, and the prepared medicament and health-care product are popular with people and have wide development prospects.

The extraction method of the effective components of the ganoderma comprises the following steps: the extraction of sporocarp and mycelium have different components and different contents, and the components cannot be mutually replaced. Wherein the mycelium extraction adopts a mycelium culture process comprising the following steps: liquid fermentation culture and solid fermentation culture, wherein the liquid fermentation is finally used for extracting effective components such as fungal polysaccharide, protein and the like in the bacterial pellet and the leachate concentrate, and has the advantages of higher purity or content of the extract, more application in the aspect of preparing high-efficiency medicaments, and the defects of high fermentation process requirement, high investment, complex extraction process and lower extraction rate. The solid fermentation has the advantages of low investment, simple and convenient process, high product recovery rate and the defects of low extraction content and long culture period, and can only be applied to the aspects of common-effect health-care medicines and health-care products. The prior art does not provide a high-efficiency fermentation method of ganoderma lucidum mycelia.

Disclosure of Invention

The invention aims to provide a fermentation method of ganoderma lucidum mycelia. The ganoderma lucidum fermentation method provided by the invention combines the solid fermentation technology with the liquid fermentation technology, and has the advantages of large production scale, short production period, simple and convenient production process, high content of effective components and high product recovery rate.

The invention provides a comprehensive fermentation method of ganoderma lucidum mycelia, which comprises the following steps:

1) inoculating the activated ganoderma lucidum strain into a liquid fermentation culture medium, and culturing for 5-7 d at 23-26 ℃, wherein the ventilation volume is 0.6-1V/V.min, and the air pressure is 0.03-0.06 Mpa to obtain a fermentation strain;

the liquid fermentation medium comprises the following components in percentage by mass: 0.5-1% of glucose, 0.1-0.2% of soybean meal, 0.1-0.2% of corn flour, 0.1-0.2% of wheat flour, 0.1-0.3% of yeast extract, 0.003-0.005% of magnesium sulfate, 0.08-0.1% of monopotassium phosphate, 0.001% of vitamin B complex, 0.03-0.05% of defoaming agent and the balance of water;

2) inoculating the fermentation strain obtained in the step 1) into a solid fermentation culture medium for dark culture, wherein the dark culture time is 30-40 days.

Preferably, the inoculation amount of the ganoderma lucidum strain activated in the step 1) in the liquid fermentation culture medium is 0.1-0.2%.

Preferably, the biomass of the fermentation strain obtained in the step 1) is (2.00 +/-0.50) multiplied by 10^-2g/mL, the average diameter of the bacteria balls is 1.00 +/-0.10 mm, the density of the bacteria balls is 120.00 +/-2.00 bacteria balls/mL, and the pH value is 5-6.5.

Preferably, the solid fermentation medium of step 2) is a bag solid fermentation medium.

Preferably, the inoculation amount of the zymophyte in the step 2) is 50-100 mL/kg.

Preferably, the carbon-nitrogen ratio in the solid fermentation medium is (20-28): 1, the water content is 42-50%, and the pH value of the culture medium before sterilization is 7-8.

Preferably, the solid fermentation medium comprises the following components in percentage by mass: 5-10% of rice, 10-15% of wheat, 35-45% of corn flour, 5-8% of corncobs, 5-10% of bean pulp, 15-25% of bran, 1-2% of calcium hydroxide and 1-2% of light calcium carbonate.

Preferably, the humidity of the dark culture in the step 2) is 60-65%; the dark culture comprises the following 4 stages:

1 st to 9 th, the dark cultureThe temperature of the culture is 25-26 ℃, and CO is₂The concentration is 1000-1500 ppm;

10 th to 15 th days, the temperature of dark culture is 23 ℃ to 24 ℃, and CO is added₂The concentration is 1500-2000 ppm;

16 th to 30 th days, the temperature of dark culture is 24 ℃ to 25 ℃, and CO is added₂The concentration is 2000-3000 ppm;

and (30) finishing the culture, wherein the temperature of the dark culture is 25-26 ℃, and the temperature of the CO is CO₂The concentration is 1500-2000 ppm.

Preferably, the step 2) dark culture further comprises bag removal, slicing and baking processes.

The invention provides a comprehensive fermentation method of ganoderma lucidum mycelia. The method for fermenting the ganoderma lucidum mycelia combines the solid fermentation technology and the liquid fermentation technology, integrates the advantages of solid fermentation and liquid fermentation, and has the advantages of large production scale, short production period, simple and convenient production process, low pollution rate, high content of effective components and high product recovery rate. Is beneficial to the high-efficiency extraction of the subsequent effective components and the recovery of the product. Compared with the existing solid fermentation, the fermentation method has the advantages of large production scale, short production period and low pollution rate, and compared with liquid fermentation culture, the fermentation method has the advantages of simple production process, high content of effective components and high product recovery rate. The extraction rate of the ganoderma lucidum volatile oil of the mycelium mixture cultured by the comprehensive fermentation method is up to 0.03%, the extraction rate of the ganoderma lucidum mycolic acid is up to 3.2%, the purity of the acid containing solid product acid is up to more than 25%, the extraction rate of the ganoderma lucidum polysaccharide is up to 10.1%, and the purity of the polysaccharide is up to more than 45%.

Detailed Description

The invention firstly carries out the preparation of the activated ganoderma lucidum strain. In the invention, the activation times of the strains are preferably 2 times, a primary strain and a secondary strain are obtained by culturing in an activation culture medium, and the secondary strain is selected as the activated ganoderma lucidum strain for liquid fermentation culture. In the present invention, the primary strain activation medium is preferably a fumaragus potato medium well known to those skilled in the art, and the secondary strain activation medium preferably includes glucose per liter of water: 10-20 g, peptone: 2-4 g, yeast extract: 1-2 g, potassium dihydrogen phosphate: 1-2 g, magnesium sulfate heptahydrate: 0.75-1.5 g and vitamin B complex: 0.25 to 0.5 g. The source of the ganoderma lucidum strain is not specially limited, the conventional commercial ganoderma lucidum strain known by the technical personnel in the field can be adopted, and the ganoderma lucidum strain which is from China general microbiological culture Collection center and has the strain number of CGMCC5.743 is preferably selected.

The invention preferably selects hypha with the growth speed of 5.5-6.5 mm/d as a primary strain. In the process of selecting the first-level strains, the invention preferably selects strains with neat colony edges, white color and strong strength. The primary strain preparation of the invention can screen out the original strain with weak growth and ensure the activity of the strain. The condition for culturing the primary strain is preferably 22-28 ℃ dark culture for 5-8 days, and more preferably 23 ℃ dark culture for 7 days.

After the primary strain is obtained, hyphae with 1.5-2.5 mm of the edge of a colony of the primary strain are preferably selected and placed in a secondary strain activation culture medium for culture, the condition of activation culture is preferably constant-temperature shaking culture for 5-7 days, and the biomass is 1.5-2.5 multiplied by 10^-2g/mL of the strain is used as a secondary strain. Before the constant temperature shaking culture, the first class strain is preferably activated and cultured in the second classAnd standing the culture medium until hypha blocks germinate, wherein the standing condition is preferably dark condition, the standing is performed for 28-32 h at 23-25 ℃, and the standing is more preferably performed for 30h at 24 ℃. In the invention, the temperature of the shaking culture is preferably 23-26 ℃, and more preferably 24 ℃. The shaking culture is preferably carried out by adopting a constant-temperature shaking table, and the rotating speed of the shaking table is preferably 140-160 r/min, and more preferably 150 r/min. In the invention, the second activation culture can ensure the homology and the synchronism of the strains. In the invention, the strains obtained after the second activation are preferably uniform in size and distribution, do not sink, have full villi on the spherical surface, and have obviously lighter and transparent culture medium color than the sterilized culture medium. In the existing ganoderma lucidum liquid fermentation technology, secondary metabolites are easy to generate, the method can ensure the homology and the synchronism of strains strictly through twice strain activation and screening, meanwhile, the inoculation amount is ensured to be 0.1-0.2% according to the method, the liquid fermentation time can be greatly shortened, the activity of the fermentation product is ensured to reach the peak value synchronously, and the subsequent solid fermentation is facilitated.

After the activated ganoderma lucidum strain is obtained, the activated ganoderma lucidum strain is inoculated in a liquid fermentation culture medium and cultured for 5-7 d at the temperature of 23-26 ℃, the ventilation volume is 0.6-1V/V.min, and the air pressure is 0.03-0.06 Mpa, so that the liquid fermentation strain is obtained. In the invention, the liquid fermentation medium comprises the following components in percentage by mass: 0.5-1% of glucose, 0.1-0.2% of soybean meal, 0.1-0.2% of corn flour, 0.1-0.2% of wheat flour, 0.1-0.3% of yeast extract, 0.003-0.005% of magnesium sulfate, 0.08-0.1% of monopotassium phosphate, 0.001% of vitamin B complex, 0.03-0.05% of defoaming agent and the balance of water. In the invention, the glucose and the corn flour mainly provide a carbon source of a culture medium, the soybean meal, the wheat flour and the yeast extract mainly provide a nitrogen source of the culture medium, the monopotassium phosphate mainly controls the pH value in the culture medium, the potassium ions provide a metabolic environment, and the magnesium ions in the magnesium sulfate are activators of various enzymes. In the culture medium of the invention, the yeast extract is preferably Angel yeast LM800, the glucose is preferably edible grade, the chemicals such as magnesium sulfate, urea and the like are preferably analytical pure grade, the sources of the other components are not specially limited, and the conventional market of the components, which is well known to those skilled in the art, is adoptedAnd (5) selling the product. In the invention, the inoculation amount of the ganoderma lucidum strain activated in the step 1) in the liquid fermentation culture medium is 0.1-0.2%. In the present invention, the biomass of the obtained liquid fermentation strain was (2.00. + -. 0.50). times.10^-2g/mL, the average diameter of the bacteria balls is 1.00 +/-0.10 mm, the density of the bacteria balls is 120.00 +/-2.00 bacteria balls/mL, and the pH value is 5-6.5. In the invention, the culture temperature of the liquid fermentation culture is 23-26 ℃, and more preferably 24 ℃. In the invention, the time of the liquid fermentation culture is 5-7 d, and more preferably 6 d. In the present invention, the aeration rate in the liquid fermentation culture is 0.6 to 1V/V.min, more preferably 0.8V/V.min. The ventilation of the invention preferably adopts a sterile air system to treat air, the main principle is that clean air is compressed by an air compressor, specifically, high-pressure gas is stored by an air storage tank, the air is dehumidified, dedusted and cooled to 22-24 ℃ by a freeze dryer, microorganisms such as bacteria and the like are filtered by a double-layer 0.1 mu m microporous membrane, and finally the air is introduced into a culture device. In the invention, the air pressure of the liquid fermentation culture is 0.03-0.06 MPa, and more preferably 0.05 MPa. In the invention, the gas introduced in the fermentation process is preferably cold dry sterile air.

The specific operation process of the liquid fermentation culture is not particularly limited in the invention, and a liquid fermentation method well known to those skilled in the art can be adopted. The fermentation is carried out by adopting the following method: in the present invention, the fermentation apparatus for liquid fermentation culture particularly preferably includes: a steam sterilization system, a cold and hot circulating water system, an aseptic air system and an aseptic pipeline inoculation system. In the invention, the steam sterilization system is used for sterilizing the fermentation device, and adopts steam introduction type sterilization, and the steam pressure is preferably 0.12 MPa. The fermentation device is preferably a fermentation tank, the material is preferably SUS304 stainless steel, the volume is preferably 800L, the fermentation tank comprises an interlayer, and the interlayer is used for controlling the fermentation temperature through circulating water at constant temperature. The present invention is not particularly limited in the sterilization method of the fermentation medium, and a sterilization method known to those skilled in the art may be used. Specifically, when the water temperature is increased to 75 ℃, raw materials except the defoaming agent are preferably dissolved and stirred by hot water, the water temperature in a fermentation tank is increased to 90 ℃, the raw materials and the defoaming agent are added, sterilization is carried out for 45min at 121 ℃, after the sterilization is finished, cold water of a cold and hot water circulating system is used for carrying out cooling treatment through an interlayer, and inoculation is carried out after the culture medium is cooled to 24 ℃. In the liquid fermentation process, the pressure, temperature, smell, ventilation and other data of the fermentation tank are preferably observed every 2h and recorded. In the invention, preferably, two sampling detections are performed in the fermentation culture process, and the detection is to determine whether the bacterial liquid is contaminated, and the detection method is preferably as follows: judging whether the bacteria liquid has peculiar smell or not by odor, judging whether the bacteria liquid has color change turbidity or not by naked eyes, judging whether the bacteria liquid has bacterial infection or not by gram staining microscopy, and detecting whether the bacteria liquid has bacterial and other fungal pollution or not by flat plate scribing.

After the fermentation strain is obtained, the fermentation strain is inoculated in a solid fermentation culture medium through a sterile pipeline for dark culture. The strain inoculation process is preferably carried out by adopting an aseptic pipeline inoculation system, specifically, the inoculation pipeline is sterilized by adopting high-pressure steam, the sterilization condition is preferably 121 ℃, the sterilization time is 45min, and the inoculation is carried out after the pipeline is cooled. In the invention, the dark culture time is 30-40 d; in the invention, the carbon-nitrogen ratio in the solid fermentation medium is (20-28): 1, the water content is 42-50%, and the pH value of the culture medium before sterilization is 7-8. In the present invention, the dark culture time is preferably 35d, and the carbon-nitrogen ratio in the solid medium is preferably 25: the water content is preferably 48%, the water content is set to ensure the thoroughness of sterilization, the tightness of fungus bags and the requirement of hyphae on water, and the pH value of the culture medium before sterilization is preferably 7.5. In the invention, the setting of the carbon-nitrogen ratio in the solid fermentation medium can ensure the growth of the ganoderma lucidum mycelia and can ensure the normal growth of the mycelia on the premise of lowest raw material cost. In the present invention, the solid fermentation medium of step 2) is preferably a bag solid fermentation medium, i.e. solid culture of ganoderma lucidum mycelia is performed in a bag. The material of the fungus bag is not limited in the invention, and PP (polypropylene) fungus bags known to those skilled in the art can be adopted, and the specification is preferably 36cm multiplied by 15cm multiplied by 0.5 mm. The fungus bag preferably contains 2.5 +/-0.5 jin of culture medium per bag, and the height of the fungus bag is preferably 17.5-18 cm. In the present invention, the sterilization conditions after the fungus is bagged are preferably 123 ℃ for 200 min.

In the present invention, the inoculum size of the fermentation strain is 50 to 100mL/kg, and more preferably 60 mL/kg. The method of inoculating the fermentation culture of the present invention is not particularly limited, and the inoculation may be carried out by using an inoculating device known to those skilled in the art. Specifically, the method selects an inoculation gun with the length of 17.5cm and four rows of eight liquid outlet holes for inoculation, more preferably, the method controls the single output of the strains by using a preset counter and an electromagnetic valve, and specifically, the strains are inoculated in a sterile environment, the inoculation gun is inserted into the liquid outlet holes during inoculation, and a switch is stepped to spray the strains, so that the strains are uniformly sprayed to the surface, the middle and the bottom of the material. In the invention, the solid fermentation medium preferably comprises the following components in percentage by mass: 5-10% of rice, 10-15% of wheat, 35-45% of corn flour, 5-8% of corncobs, 5-10% of bean pulp, 15-25% of bran, 1-2% of calcium hydroxide and 1-2% of light calcium carbonate. In the invention, the solid fermentation medium preferably further comprises 0.1-0.2% by mass of urea. In the invention, the grain size of the corn flour is preferably 0.6-1.0 mm, the grain size of the soybean meal is preferably 0.8-1.2 mm, the grain size of the rice is preferably 2.0-2.5 mm, and the grain size of the wheat is preferably 2.4-2.8 mm. In the invention, the grain diameters of the corn flour, the bean pulp, the rice and the wheat can ensure the integral physical structure of the fungus bag and balance the air permeability and the sterilization thoroughness of the fungus bag. In the present invention, the wheat is preferably soaked in water at 25 ℃ for 36 hours before use, and the rice is preferably soaked in water at 25 ℃ for 12 hours.

In the invention, the humidity of dark culture in the step 2) is 60-65%; the dark culture comprises the following 4 stages:

1 st to 9 th days, the temperature of dark culture is 25 to 26 ℃, and CO is₂The concentration is 1000-1500 ppm;

16 th to 30 th days, the temperature of the dark culture is 24 to 25 DEG℃，CO₂The concentration is 2000-3000 ppm;

In the invention, the dark culture also comprises bag removal, slicing and baking processes to obtain the flaky mushroom blocks. In the invention, the ganoderma lucidum mycelia cultured by the fungus bag solid fermentation medium are preferably subjected to bag removal, the mycelium subjected to bag removal is preferably sliced, in the invention, the slicing process is preferably carried out by a self-made slicing machine, the thickness of the slices is preferably 1-1.5 cm, and in the invention, baking trays with holes are preferably adopted for subpackaging. In the present invention, baking is preferably carried out in a baking room having a volume of preferably 360m³The baking is preferably carried out by adopting steam pipeline space heating, the preferred power is 2.2kw, and the air volume is 9000m³The air induced by the axial flow fan is subjected to auxiliary heat dehumidification through the steam pipeline cooling fins, the preferred power is 0.55kw, and the air volume is 3000-4000 m³The drying temperature is preferably between 70 and 75 ℃. The baking method of the invention can ensure that: the total steam flow is 1.25-1.5 tons/time, the finished product amount in single drying is 1.8 tons, the baking time is less than 22 hours, and the moisture of the baked mushroom blocks is not 7 percent. After the flaky mushroom blocks are obtained, the subsequent processing process of the flaky mushroom blocks is not specially limited, and the ganoderma lucidum powder is obtained by adopting a conventional processing mode of technicians in the field, such as crushing; or with supercritical CO₂Fluid extracting the powder to obtain Ganoderma volatile oil, Ganoderma polysaccharide and ganoderic acid.

The fermentation method of ganoderma lucidum mycelia according to the present invention will be further described in detail with reference to the following specific examples, and the technical solutions of the present invention include, but are not limited to, the following examples.

Example 1

Inoculating Ganoderma original strain to multiple plate culture dishes containing culture medium, and culturing.

The specific preparation method of the ganoderma lucidum primary activated strain comprises the following steps:

the original strain of Ganoderma is derived from China general microbiological culture Collection center, and has a strain number of CGMCC 5.743.

Transferring the original strain of the ganoderma lucidum to a constant-temperature incubator at 24 ℃ from the preservation condition of 4 ℃, recovering the activity of the biological enzyme, and placing the strain in an ultra-clean workbench for standby after 24 hours.

The ganoderma lucidum primary activation strain adopts a fumaric potato culture medium, and the specific preparation method is as follows:

(1) 200g of potatoes are cleaned, peeled, sprout eyes are removed, and the potatoes are sliced to be 0.2-0.3 cm thick;

(2) placing the obtained potatoes in hot water, boiling the potatoes for 3-5 minutes by using strong fire until the potatoes are softened, and then boiling the potatoes for 10 minutes by using slow fire to fully disperse the nutrients of the potatoes in the water;

(3) filtering with 6-8 layers of gauze, and keeping the leaching solution;

(4) adding 1g of yeast extract, 2g of peptone, 1g of monopotassium phosphate, 0.5g of magnesium sulfate heptahydrate, 1g of vitamin B complex, 20g of glucose and 20g of agar powder into the leachate obtained in the step (3), and adding water to a constant volume of 1000 mL;

(5) and (5) subpackaging the mixed solution obtained in the step (4) into a plurality of culture dishes with the specification of 10cm, wherein the thickness of the culture medium is about 5 mm.

Bundling the culture dish with 5 pieces of kraft paper and rubber band, placing in a vertical autoclave, sterilizing at 121 deg.C for 45min, and preventing the culture dish from tilting. And cooling and placing in a superclean bench for standby.

Picking the original strains of the lucid ganoderma in a culture dish in a superclean workbench. Specifically, an original strain block with the length and the width of 5mm is cut by an inoculation hook, placed in the center of a culture dish, inoculated with 15-20 culture dishes, sealed by a sealing film, marked, and cultured in the dark at 23 ℃ for 7 d. And (5) inverting the culture dishes after the hyphae germinate, pricking the sealing film with a sterile inoculation needle, pricking 20 holes in each culture dish, and continuing culturing.

And after the bacterial colony grows to about 1.5cm of the edge of the culture dish, selecting bacterial hyphae of the strain with the growth speed of 5.5-6.5 mm/d, tidy edges of the bacterial colony and pure white and strong hyphae as a first-level activating strain of the lucid ganoderma.

And inoculating a fungus block of hypha with the edge of 1.5-2.5 mm of the colony of the primary strain in a secondary activated culture medium for culture.

The specific preparation method of the ganoderma lucidum secondary activation strain comprises the following steps:

the ganoderma lucidum secondary activation culture medium comprises the following components: glucose: 20g, peptone: 3g, yeast extract: 1g, potassium dihydrogen phosphate: 2g, magnesium sulfate heptahydrate: 1g, vitamin B complex: 0.5 g.

Dissolving the above materials in 75 deg.C water, and making the volume to 1000 mL.

The mixture is subpackaged in a plurality of 500mL straight triangular bottles, each triangular bottle contains 250mL, a 2.5cm magnetic stirrer is placed in each triangular bottle, the triangular bottle is tightly covered by a self-made cotton plug, the triangular bottle mouth is wrapped by kraft paper, and the bottle mouth is tightly tied by a rope.

The self-made cotton plug adopts non-absorbent cotton with longer fibers, and the specific manufacturing method is as follows: cutting cotton blocks matched with the size of the opening of the triangular flask, wrapping the cotton blocks by using a first web of a left hand, pressing one side of the cotton blocks by using a thumb of a right hand, ensuring that the other side of the cotton blocks is smooth and U-shaped, cutting medical gauze blocks matched with the size of the cotton blocks, wrapping the cotton blocks, pressing the cotton blocks, and fastening gauze at the U-shaped opening of the cotton blocks by using ropes to prevent deformation.

The subpackaged triangular bottles are placed in a vertical high-pressure sterilization pot and sterilized for 45 minutes at the temperature of 121 ℃. And cooling and placing in a superclean bench for standby.

Placing the first-stage activated strain of the ganoderma lucidum in an ultraclean workbench, selecting hypha blocks with the edge of 1.5-2.5 mm of the colony of the first-stage activated strain for inoculation, wherein the inoculation number of each triangular flask is 6 hypha blocks. The mycelium block was prepared using a 6.0mm diameter punch. The strain block floats on the surface of the culture medium.

And (3) after inoculation, placing the triangular flask in a constant-temperature incubator at 24 ℃ under a dark condition, standing for 30h, transferring the triangular flask to a constant-temperature shaking table after hyphae germinate, carrying out dark culture at 24 ℃ for 7d at a rotating speed of 150r/min, and selecting strains with uniform size and distribution of fungus balls, no settlement, full fuzz on the spherical surface, and taking the strains which are obviously lighter and transparent after the culture medium is sterilized as second-stage activated strains of the lucid ganoderma.

Inoculating the second strain to fermentation tank with inoculum size of 0.1%, culturing at 24 deg.C, and ventilatingThe amount is 0.6-1V/V.min, the air pressure is 0.03-0.06 Mpa, and the biomass is 2.00 x 10 after culturing for 7 days^-2g/mL, the diameter of the pellet is 1.00mm, the density of the pellet is 122 pellets/mL, and the pH value is 6.2.

The specific preparation method of the ganoderma lucidum liquid fermentation strain comprises the following steps:

the ganoderma lucidum liquid fermentation culture medium comprises the following components in percentage by mass: 1% of glucose, 0.2% of soybean meal, 0.2% of corn flour, 0.2% of wheat flour, 0.1% of yeast extract, 0.004% of magnesium sulfate, 0.08% of monopotassium phosphate, 0.001% of vitamin B complex, 0.03% of defoaming agent and the balance of water.

The fermentation device is a fermentation tank made of SUS304 stainless steel and having a volume of 800L, and the fermentation tank comprises an interlayer which is used for controlling the fermentation temperature through circulating water at constant temperature.

Calculating the total amount of the required liquid strains according to the inoculation amount and the number of inoculated strain packages, adding equivalent water into a fermentation tank, introducing 0.12Mpa steam into an interlayer of the tank body to heat up, dissolving and stirring the raw materials except the defoaming agent by hot water when the water temperature rises to 75 ℃, and adding the raw materials and the defoaming agent when the water temperature in the fermentation tank rises to 90 ℃. When the water temperature rises to 121 ℃, the interlayer steam switch is closed, the steam switch in the tank is opened to directly introduce steam into the tank body, and the filter sterilization circuit switch is opened to enter a sterilization procedure. Sterilizing at 121 deg.C for 45 min. After sterilization, cold water of a cold and hot water circulating system is utilized to carry out cooling treatment through the interlayer, and inoculation is carried out after the culture medium is cooled to 24 ℃.

Placing the ganoderma lucidum secondary activated strain before inoculation on a magnetic stirrer, and stirring for 12h to break the strain balls, wherein the breaking purpose is multipoint germination, so that the germination time of ganoderma lucidum mycelia in a fermentation tank is shortened, and meanwhile, the inoculation pipeline is prevented from being blocked.

And during inoculation, the air inlet valve of the fermentation tank is closed, the pressure in the tank is reduced until the exhaust pipe slightly exhausts the gas, and the exhaust valve is closed. The flame ring is sleeved on the inoculation port to be ignited, and after the inoculation port is opened, the air inlet switch is opened slightly to enable the inoculation port to exhaust slightly air. Opening the bottle mouth of the triangular flask under the protection of the flame ring, and pouring into the tank. The flame ring was extinguished with a wet towel after the inoculation port cap was tightened. Opening the air inlet and outlet valve, regulating the pressure in the tank to 0.05Mpa, and introducing the air flow of 0.8V/V.min into the culture stage. A brass vertical check valve is added in front of the exhaust valve to prevent air from being sucked backwards.

In the ganoderma lucidum liquid fermentation process, data such as pressure, temperature, smell, ventilation capacity, presence or absence of color change turbidity and the like of the fermentation tank are observed every 2 hours and recorded. And 4d, sampling and detecting twice before the fermentation is finished and inoculation, wherein the detection is to judge whether the bacterial liquid is polluted, and the detection method adopts a detection method which is well known by a person skilled in the art and comprises the following steps: the presence or absence of bacterial infection is detected by gram staining microscope, and the presence or absence of bacterial and other fungal contamination is detected by plate streaking.

Selecting biomass as (2.00 +/-0.50) x 10^-2And g/mL, the average diameter of the bacteria balls is 1.00 +/-0.10 mm, the density of the bacteria balls is 120.00 +/-2.00, and the pH value is 5-6.5.

The method for measuring the biomass of the strain comprises the following steps: centrifuging part of the bacterial liquid at 5000r/min for 5min, washing repeatedly for 3 times, freeze drying at-45 deg.C under vacuum degree of 15Pa, and measuring mycelium biomass after 48 hr. The method for measuring the diameter of the bacterial ball comprises the following steps: the diameters of 10 cocci were randomly measured in the same sample by means of a microscope eyepiece micrometer, and the arithmetic mean thereof was taken as the mean diameter of the cocci. The method for measuring the density of the bacteria balls comprises the following steps: accurately sucking 1mL of fermentation liquid by a pipette, counting in a culture dish, and calculating the number of the bacterial balls in each milliliter.

Inoculating the ganoderma lucidum fermentation strain into a solid fermentation culture medium through an aseptic inoculation pipeline, transferring the solid fermentation culture medium into a culture room for dark culture, and growing hypha over a fungus bag to obtain a comprehensive fermentation product.

The high-pressure resistant silicone tube with the specification of 10 multiplied by 20mm inside and outside diameters of the sterile inoculation pipeline is connected with a stainless steel inoculation gun at the inoculation end. The length of the inoculation gun is 17.5cm, and four rows of eight liquid outlet holes are uniformly distributed. The inoculation gun is connected with a 20cm stainless steel tubular jacket, and the other end of the inoculation gun is provided with a ball valve switch which is connected with a drainage pipeline and an exhaust pipeline. The bottom discharge port of the fermentation tank is connected with a cross joint which is divided into (1) a tank discharge port, (2) a sampling port, (3) an inoculation pipe port and (4) a water inlet pipe port or a steam pipe port.

The inoculation pipeline sterilization mode is as follows:

after fermentation of the ganoderma lucidum liquid strain is completed, the inoculation pipeline is connected to the pipe orifice, the pipe orifice is connected with the water inlet pipe, the water pipe and the inoculation pipeline valve are opened, the inoculation pipeline is washed by tap water for 20min, and after washing is completed, the water pipe at the pipe orifice is replaced by a steam pipeline for connection.

And opening a steam valve, introducing high-pressure steam into the inoculation pipe, opening a ball valve switch of the inoculation gun jacket at the other end, and discharging the steam. Sterilizing at 121 deg.C under 0.12Mpa for 45min, and cooling to below 25 deg.C.

The purpose of the flushing is to flush residual bacteria balls and nutrient substances in the pipeline and the inoculation gun and prevent the growth of mixed bacteria.

The preparation of the lucid ganoderma solid fermentation culture medium comprises the following steps:

the lucid ganoderma solid fermentation culture medium comprises the following components in percentage by mass: 10% of rice, 15% of wheat, 40% of corn flour, 8% of corncob, 5% of bean pulp, 20% of bran, 1% of calcium hydroxide and 1% of light calcium carbonate. The carbon-nitrogen ratio of the culture substrate is 25: 1.

the setting of the carbon-nitrogen ratio in the solid fermentation culture medium can ensure the growth of ganoderma lucidum mycelia and can ensure the normal growth of the mycelia on the premise of the lowest raw material cost.

Before use, all raw materials need to be pretreated, including crushing and soaking. The grain size of the corn flour is 0.6-1.0 mm, the grain size of the soybean meal is 0.8-1.2 mm, the grain size of the rice is 2.0-2.5 mm, and the grain size of the wheat is 2.4-2.8 mm. The wheat is soaked in water of 25 deg.C for 36 hr before use, and the rice and corn cob are soaked in water of 25 deg.C for 12 hr before use.

The grain diameters of the corn flour, the bean pulp, the rice and the wheat can ensure the integral physical structure of the fungus bag, and balance the air permeability of the fungus bag and the thoroughness of sterilization. Wheat, rice, corncob are in order to adjust the gas permeability of fungus package, and the granularity that sets up is great, leads to the water absorption capacity big and difficult water absorption, soaks the purpose and prevents thereby the raw materials is done the core and is leaded to the sterilization not thorough.

Pouring the components of the culture medium into a container with the thickness of 10.4m³The stirring kettle is 1000 kg/kettle, water is added after uniform stirring, the water content in the matrix before packaging is ensured to be 48%, and the pH value is 7.5. The mixture is evenly stirred and then is conveyed into an automatic packing machine, the height of the fungus bags is ensured to be 17cm, and 2.4 jin of culture medium is packed in each bag. And punching twice in the packaging process of the automatic packaging machine, wherein punching is performed at the center of the fungus bag matrix for the second time, and the depth is 15.5 cm.

The setting of water content can guarantee the thoroughness of sterilization, the elasticity of fungus package and the requirement of hypha to moisture.

The solid fermentation is carried out in a high-temperature-resistant fungus bag, and the fungus bag can be a PP (polypropylene) fungus bag well known to those skilled in the art, and the specification is 36cm multiplied by 15cm multiplied by 0.5 mm.

After the fungus bags are manufactured, the fungus bags are placed in matched high-temperature resistant plastic baskets with 12 bags in each basket, the plastic baskets are placed in a sterilization vehicle, and the sterilization vehicle is pushed into a high-pressure steam sterilization cabinet for sterilization.

The sterilization process comprises vacuumizing for 2 times, heating and keeping temperature for 1 to 105 deg.C for 30min, heating and keeping temperature for 2 to 115 deg.C for 30min, sterilizing for 123 deg.C for 200min, and stewing and exhausting for 30 min. After sterilization, the bags were cooled to below 25 ℃ for inoculation.

The inoculation mode is as follows:

and conveying the cooled fungus bags to an inoculation chamber with cleanliness less than 10 ten thousand grade for inoculation, wherein the principle of taking a gun with one hand and taking a cover with the other hand is adopted during inoculation, the inoculation operation flow comprises the steps of opening the cover ①, inserting a ② inoculation gun into a fungus bag material hole until a tail disc clamps a lantern ring, stepping a pedal switch with ③ feet to spray out the strains, staying for ④ for 1-2 seconds to spray out the strains, simultaneously opening the next fungus bag cover, immediately covering the cover after the fungus bags are extracted by the ⑤ inoculation gun, sequentially inoculating according to a ②③④⑤ flow, and conveying the inoculated fungus bags on a conveyor belt to be discharged from the inoculation chamber.

The inoculation amount of the ganoderma lucidum liquid strain is 60 mL/kg. The length of the inoculation gun is 17.5cm, four rows of eight liquid outlet holes are averagely arranged, and a counter and an electromagnetic valve are preset in the control equipment of the inoculation amount.

The specific mode of culturing the ganoderma lucidum mycelia is as follows:

after inoculation, the ganoderma lucidum fungus bags are transferred to a culture room for dark culture. The humidity of the dark culture is 60-65%; the dark culture comprises the following 4 stages:

the first stage is 1-9 days, belonging to hypha germination stage, the hypha self-heating quantity is low, the culture temperature needs to be provided to 25-26 ℃ by equipment, and CO is generated due to small hypha respiration quantity₂The concentration is 1000-1500 ppm;

the second stage is 10-15 days, which belongs to the first stage of hypha permanent planting, the hypha has the maximum self-heating quantity, the oxygen consumption begins to increase, the culture temperature needs to be reduced to 23-24 ℃, and CO is required₂The concentration is controlled to be 1500-2000 ppm;

the third stage is 16-30 days, belonging to the second stage of hypha permanent planting, wherein the hypha has large self-heating value and CO₂The highest accumulation concentration, the culture temperature is set at 24-25 ℃, and CO is added₂The concentration is 2000-3000 ppm;

the fourth stage is from 30d to the completion of culture, belongs to the post-maturation stage of hyphae, and has a hypha calorific value substantially equal to that of the first stage, a culture temperature of 25-26 ℃, and CO₂The accumulated concentration is 1500-2000 ppm higher than that in the first stage.

The invention adopts the solid fermentation and liquid fermentation combined technology to carry out fermentation culture on the ganoderma lucidum mycelia, the water content and the inoculation amount of a solid fermentation substrate are different from those of the traditional process, and the invention adopts a mode of reducing the water content of the solid culture medium and increasing the inoculation amount of liquid strains. The water content standard of the solid culture medium is established according to the proportion of each component, the granularity and the water absorption performance of the formula, on the premise of ensuring the sterilization thoroughness principle, the drier the culture medium is, the better the permeability of the liquid strain is, and on the premise that the principle standard of the liquid strain amount is that no water is accumulated at the bottom in the culture medium, the larger the strain amount is, the more the strain base number is, and the faster the hypha grows. The local water accumulation of the fungus bag leads to slow or difficult growth of local hypha growth, and meanwhile, the larger the liquid strain amount is, the higher the content of fungus polysaccharide extracted in the later stage is.

By contrast, on the specific premise of the formula of the solid fermentation raw material, the proportion of each component and the particle size, the solid fermentation substrate has the water content of 48 percent, the inoculation amount of 60ml/kg and the fastest growth vigor of hyphae.

According to the invention, the ganoderma lucidum mycelia are prepared by adopting a liquid fermentation mode, the extraction cost is lower when the single fermentation amount is larger, but on the premise of the same diameter ratio of the fermentation tank body, the fermentation equipment is larger, the requirements of the fermentation equipment process and the matched equipment are higher, the requirements of the operation process and the fermentation process are severer, the propagation times are increased, the total fermentation period is longer, the risk coefficient is larger, the required early investment is higher, and the requirements of the extraction process and the equipment are higher. The method adopts the 800L fermentation tank to prepare the liquid fermentation strain, is not directly used for extraction, but is propagated into the solid fermentation substrate, and has the advantages of low early investment, low pollution rate, strong risk resistance and relatively simple and convenient production process.

Example 2

Processing treatment of ganoderma lucidum fermentation products:

adopting the mature ganoderma lucidum mycelium fermentation product obtained in the embodiment 1, slicing and drying to obtain flaky fungus blocks; crushing the flaky fungus blocks to obtain ganoderma lucidum fungus powder; by supercritical CO₂Fluid extracting the powder to obtain Ganoderma volatile oil, Ganoderma polysaccharide and ganoderic acid.

Slicing and drying the fermented product of the mature ganoderma lucidum mycelia, and specifically comprises the following steps:

and (3) carrying out manual cover taking, ring taking and bag removing treatment on the ganoderma lucidum fungus bags subjected to solid fermentation culture, and mechanically slicing fermentation products subjected to bag removing, wherein the thickness of each slice of the sliced ganoderma lucidum fungus bags is 1-1.5 cm.

The slicing machine is a self-made slicing machine, the inspiration comes from a cutting machine of a paper mill, a fixed knife handle capable of being closed by 90 degrees is arranged, 13 stainless steel blades with the maximum thickness of 3mm are welded under the handle, and the distance between the blades is 1.25 cm. A stainless steel cylindrical fixing clamping groove which is 17cm long and 5cm in diameter is formed in the table top below the blade, and 13 transverse hollow stripes are arranged in the clamping groove. The space between the hollow stripes in the upper semicircle of the clamping groove is 3.35mm, the fungus dregs can be prevented from remaining in the blade gap when the knife blade of the switch is lifted, the space between the stripes in the lower semicircle clamping groove is 8mm, and the slice-shaped fungus block can pass through the clamping groove after the slicing is finished. A tabletop capable of placing a baking pan is arranged below the clamping groove, the height of the tabletop from the clamping groove is 20cm, and the fungus blocks passing through the clamping groove directly fall into the baking pan. One end of the clamping groove is connected with an arc-shaped feeding cylinder which is 50cm long and has the same diameter, and the tail end of the cylinder is 15 degrees to the table top, so that fungus bags can be conveniently put into the cylinder. The feed cylinder, the clamping groove and the blade can be opened and closed or disassembled, so that the slicing is convenient to clean after being finished.

The drying efficiency of the drying pan with holes is improved, the aperture is 3mm, and the density of the drying pan holes is 25/dm². The baking pans are made of stainless steel materials with the specification of 80 multiplied by 50 multiplied by 2cm and the thickness of 1mm, 3 ganoderma lucidum fungus bags are placed in each baking pan, and 3 multiplied by 14 pieces are placed in the baking pans.

The drying is carried out in a drying room with the volume of 360m³And 4 temperature probes and humidity probes are respectively arranged on the upper part, the lower part, the front part and the rear part of the drying room. The rear end of the drying room is provided with a power of 0.55kw and an air volume of 3000-4000 m³The centrifugal fan is provided with a circulation channel and an exhaust channel, multiple rows of moisture in the exhaust channel are normally opened in the early stage of baking, the circulation channel is opened more in the later stage, the exhaust channel is opened less, the baking time can be shortened, and the energy can be saved. Baking house steam conduit space heating, outside the baking house was arranged in to steam conduit drainage air discharge valve door, diameter 25mm iron pipe to the griddle lower extreme is arranged in to the coiled tubing form, griddle sets up 8 layers, and the individual layer is high 30cm, and width 1.6m, every layer sets up 4 coiled pipe heating. In addition, the power of 2 baking rooms at the front end of each drying room is 2.2kw, and the air quantity is 9000m³The/h axial flow fan induces air to assist heat and dehumidify.

The drying process is divided into 3 steps: heating, dehumidifying and preserving heat. After the baking pan is fully filled in the drying room, opening a coil steam valve to heat the drying room, properly reducing the steam quantity of the coil after the temperature is raised to 70 ℃, opening a radiating fin steam valve, opening an axial flow fan and a centrifugal machine, and inducing air and discharging moisture. And when the average humidity of the space is lower than 5%, entering the heat preservation stage, setting the circulating air per hour for starting for 45min by the centrifugal fan, starting the external air for 15min, and finishing baking after heat preservation for 6 h. When the baking temperature is lower than 65 ℃ for a long time, the mould can be bred in a high-temperature, high-humidity and high-nutrition environment, and the polysaccharide substance structure can be damaged when the baking temperature is higher than 80 ℃. When the temperature is raised, the steam pressure is ensured to be sufficient, and the temperature-raising time is not more than 3 hours; during baking, the amount of steam entering a baking room is strictly controlled, and the baking temperature is between 70 and 75 ℃.

The slice baking method can ensure that: the total amount of steam for single baking is 1.25-1.5 tons/time, the amount of finished products for single baking is 1.8 tons, the baking time is less than 22 hours, and the moisture of the baked fungus blocks is not higher than 7%.

After baking, subpackaging, weighing to 50 jin/bag, sealing by a sewing machine, packaging by a packaging bag with a film inner bag, and storing in a cool and ventilated and dry place for later use after packaging.

The mycelium mixture cultured by the comprehensive fermentation method is determined that the extraction rate of the ganoderma lucidum volatile oil is up to 0.03%, the extraction rate of the ganoderma lucidum mycolic acid is up to 3.2%, the purity of the acid containing solid product is up to more than 25%, the extraction rate of the ganoderma lucidum polysaccharide is up to 10.1% according to different varieties, and the purity of the polysaccharide is up to more than 45%.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A comprehensive fermentation method of ganoderma lucidum mycelia comprises the following steps:

2) inoculating the fermentation strain obtained in the step 1) into a solid fermentation culture medium for dark culture, wherein the dark culture time is 30-40 d;

the inoculation amount of the fermentation strain in the step 2) is 50-100 mL/kg;

the carbon-nitrogen ratio in the solid fermentation medium is (20-28): 1, the water content is 42-50%, and the pH value of the culture medium before sterilization is 7-8;

the solid fermentation medium comprises the following components in percentage by mass: 5-10% of rice, 10-15% of wheat, 35-45% of corn flour, 5-8% of corncobs, 5-10% of bean pulp, 15-25% of bran, 1-2% of calcium hydroxide and 1-2% of light calcium carbonate;

the humidity of dark culture in the step 2) is 60-65%; the dark culture comprises the following 4 stages:

2. The fermentation method according to claim 1, wherein the inoculation amount of the activated ganoderma lucidum strain in the liquid fermentation medium in the step 1) is 0.1-0.2%.

3. The fermentation method according to claim 1, wherein the biomass of the fermentation strain obtained in step 1) is (2.00 ± 0.50) × 10^-2g/mL, the average diameter of the bacteria balls is 1.00 +/-0.10 mm, the density of the bacteria balls is 120.00 +/-2.00 bacteria balls/mL, and the pH value is 5-6.5.

4. The fermentation process of claim 1, wherein the solid fermentation medium of step 2) is a bag-in-bag solid fermentation medium.

5. The fermentation method of claim 1, wherein the step 2) further comprises bag removal, slicing and baking processes after dark culture.