CN114532155A

CN114532155A - Preparation method of tremella liquid strain

Info

Publication number: CN114532155A
Application number: CN202210325602.9A
Authority: CN
Inventors: 黄勇云; 陈祥珍; 雷德赐; 黄暖云
Original assignee: Fujian Cloud Biotechnology Development Co ltd
Current assignee: Fujian Cloud Biotechnology Development Co ltd
Priority date: 2022-03-30
Filing date: 2022-03-30
Publication date: 2022-05-27
Anticipated expiration: 2042-03-30
Also published as: CN114532155B

Abstract

The invention discloses a preparation method of a tremella liquid strain, which comprises the following steps: a. selecting a tremella protospecies with a diameter of 5-7 cm from a sporocarp as a starting strain for producing a liquid strain; b. taking the tremella breeder seeds selected in the step a, eradicating old hyphae on the surfaces of sporocarp and a substrate by using an inoculating knife under aseptic conditions, uniformly mixing the tremella and the grifola frondosa hyphae by using a special tremella mixing machine, and carrying out constant-temperature recovery culture for 1-2 days at 19-23 ℃; c. and (c) adding a liquid culture medium into the fermentation tank, sterilizing, inoculating the tremella fuciformis strain obtained in the step b, and culturing at a constant temperature of 20-22 ℃ for 5-6 days to obtain the tremella fuciformis liquid strain. The liquid strain prepared by the method does not need artificial mixed culture, the yield is more than 97%, the yield of the produced product is high, the liquid strain is the only liquid strain which meets the commercial use requirement at present, and the method is suitable for bottle cultivation and bag cultivation.

Description

Preparation method of tremella liquid strain

Technical Field

The invention belongs to the technical field of artificial cultivation of tremella, and particularly relates to a preparation method of a liquid strain of tremella, which is suitable for bottle cultivation and bag cultivation of tremella.

Background

White fungus (Tremella fuciformis (berk))Tremella fuciformisBerk.) also known as Tremella, is a very precious edible and medicinal fungus with the name "crown of fungus". In terms of nutrition, tremella is low in energy and fat, but rich in protein, polysaccharide and dietary fiber. It is rich in minerals, trace elements and vitamins. Recent researches show that the tremella polyphenol has high antioxidant activity. The natural polysaccharide extracted from the body of the human body is considered to contain main active ingredients of nutrition and health of the human body, has various biological activities of enhancing immunity, reducing blood sugar, reducing blood fat, resisting aging, resisting ulcer, resisting thrombosis and the like, and is an ideal raw material of health-care food and medicines. Human beings have been cultivated by manpower consciously from the last 40 th century, and the cultivation history is nearly 80 years.

At present, the manual cultivation of white fungus adopts solid strains which comprise white fungus and ash fungus(s) ((R))Annulohypoxylon stygium) Two kinds of hypha. The cinerea belongs to ascomycetes, degrades lignocellulose in a substrate into soluble small molecular substances, and continuously transmits the soluble small molecular substances to the tremella fuciformis, so that the tremella fuciformis is used for growth and development of hypha and sporocarp of the tremella fuciformis. Tremella hyphae only grow in the area 1 cm around its inoculation point, while Gracilaria hyphae grows throughout the cultivation substrate. Before inoculation, the solid strains need to be artificially mixed, the white fungus and the grifola frondosa hyphae are uniformly mixed, and the strains inoculated in each cultivation bottle/bag are ensured to contain the white fungus and the grifola frondosa hyphae. Due to the characteristic of the tremella fuciformis strain, the solid strain inoculation process can only be carried out manually, time and labor are consumed, the uniformity of mixed strains is poor, large-scale mechanical operation cannot be carried out, and the problem of 'neck clamping' of industrial and large-scale cultivation of tremella fuciformis is solved.

The liquid white fungus strain is produced by adopting a fermentation tank, the strain is stirred and produced, white fungus hyphae are uniformly distributed at each corner of the strain, and the fact that the strain inoculated in each cultivation bottle/bag contains white fungus and ash fungus hyphae in equal proportion is ensured. The strain is uniform, can realize large-scale mechanical production, and is an ideal choice for tremella factory-like and large-scale cultivation. Liquid strains of the tremella are explored by predecessors, the prepared strains are unstable, the yield of the produced tremella is far lower than the level of solid strains, the yield of the produced tremella does not exceed 50%, and the tremella cannot be applied to production.

Disclosure of Invention

The invention provides a preparation method of liquid tremella fuciformis strains suitable for large-scale cultivation, and solves the problems that existing solid tremella fuciformis strains can only be mixed manually, are time-consuming and labor-consuming, are poor in uniformity, cannot be operated in a large-scale mechanized mode and the like.

In order to realize the purpose, the following technical scheme is adopted: the preparation method of the white fungus liquid strain comprises the following steps:

a. selecting a tremella protospecies with a fruiting body growing to a diameter of 5-7 cm or a tremella production species cultured for 8-9 days as a starting strain for liquid strain production.

b. Taking the tremella protospecies selected in the step a, eradicating old hyphae on the surfaces of the sporocarp and the substrate by using an inoculating knife under aseptic conditions, uniformly mixing the tremella and the cinerea hyphae by using a special tremella mixing machine for the rest strains, and performing constant-temperature recovery culture for 1-2 days at 19-23 ℃;

c. and (c) adding a liquid culture medium into a fermentation tank, sterilizing, inoculating the tremella fuciformis strain obtained in the step b, and culturing at a constant temperature of 20-22 ℃ for 5-6 days to obtain the tremella fuciformis liquid strain.

The tremella breeder seed selected in the step a needs to meet the following conditions: the gray fungus hypha has fast material intake, even growth, fast gelatinization of white hair mass, early ear emergence, round shape of fruiting body, good extension of ear, and no pollution.

In the mixed planting of the white fungus and the ash-like fungus in the step b, a special inoculation machine is adopted to firstly scatter the hard block matrix of the basal part of the fruit body, then other positions of a strain bottle are uniformly mixed, the strain is mixed through the head of the rotary inoculation machine, and a seed spoon is used for assisting in mixing the strain on the upper interface and the strain on the lower interface when necessary. Mixing each bottle of stock seeds for 2-3 minutes.

The liquid culture medium comprises the following components: 1000 ml of culture medium comprises 40-60 g of bran fine powder, 4-5 g of glucose, 6-9 g of peptone, 2-3 g of magnesium sulfate, 4-6 g of monopotassium phosphate, 2-3 g of vegetable oil and the balance of water.

The preparation steps of the liquid culture medium are as follows: adding water according to the formula amount into the fine bran powder according to the proportion, boiling for 30-40 min, filtering by two layers of gauze, adding glucose, peptone, magnesium sulfate, potassium dihydrogen phosphate and vegetable oil into the filtrate, stirring uniformly, and sterilizing for 20-40 min at the pressure of 0.11 MPa and the temperature of 121 ℃.

Furthermore, the fine bran powder is particles with the particle size of less than 75 mu m after being subjected to superfine grinding.

Further, the specific operation steps of the superfine grinding are as follows: drying fresh bran until the water content is lower than 6%, repeatedly crushing for 10-20 times by a shearing crusher, crushing by a high-speed dispersion crusher, and sieving by a standard sieve of 200 meshes after crushing for one time; pulverizing the fine powder which does not pass through the sieve by using a high-speed dispersion pulverizer and sieving the fine powder by using a 200-mesh standard sieve, and repeating the steps, wherein the particle size of the fine particles of the final bran is less than 75 mu m. The culture medium is added with bran fine powder: 1) the fine bran powder provides nutrition for the growth of tremella and cinerea; 2) the tremella hyphae can grow rapidly only by being tightly combined with the grifola frondosa, and the shear force formed by compressed air in the liquid fermentation process easily causes the separation of the free tremella and the grifola frondosa hyphae, so that the growth of the tremella is slow; after the mixed hypha is fixedly planted on the bran, the tremella and the incense ashes are fixed in the bran and cannot be influenced by air shearing force.

In the step c, the fermentation tank is a special air-lift fermentation tank for edible fungi with the inner cavity volume of 50L, and the conditions for culturing the white fungus liquid strain fermentation tank are as follows: the culture temperature is 20-22 ℃, the ventilation rate L/min and the dissolved oxygen amount are 50-90%, high-speed air is sprayed out by an air nozzle and dispersed in liquid in a bubble mode, liquid circulation in a fermentation tank is formed through the gas-liquid density difference, and the mixing of the tremella and the grifola frondosa hyphae is promoted.

In the step c, the amount of the liquid culture medium added into the fermentation tank is 65-70% of the volume of the inner cavity of the fermentation tank.

And c, inoculating 400-500 g of solid strains into 35L of liquid culture medium according to the inoculation amount of the mixed strains in the step c.

Further, the inoculation of the liquid culture medium comprises the following specific steps: sterilizing the peripheral surface of the tremella protospecies bottle by using 75% ethanol, and further sterilizing the bottle mouth of the protospecies bottle by using flame of an alcohol lamp; pouring some absolute ethyl alcohol into the groove on the outer ring of the inoculation port of the fermentation tank, and igniting to form a flame ring; inoculating the solid strain in the stock culture bottle into a liquid culture medium by using an inoculating shovel.

The technical effects obtained by the invention are as follows:

(1) the invention provides a preparation method of a white fungus liquid strain, which comprises the steps of spraying high-speed air by using an air nozzle, dispersing the air in liquid in a bubble mode, forming liquid circulation in a fermentation tank through a gas-liquid density difference, promoting the white fungus and the incense ash fungus hypha to be mixed, wherein the obtained strain does not need manual stirring, and the white fungus and the incense ash fungus hypha are uniformly distributed.

(2) Compared with the existing solid strains, the cost of materials used for inoculating each bottle of cultivation bottle with the liquid strains obtained by the invention is reduced by 12%, the labor cost is reduced by about 70%, and the inoculation speed is increased to 3 times.

(3) The liquid strain obtained by the invention can germinate and fix within 1-2 days, and the speed is equivalent to that of the existing solid strain; the hypha grows over the cultivation bottle in 8-9 days, and the bottle-filling time is 1-2 days faster than that of the existing solid strain; the formation of the auricular base begins in 25-26 days, and the auricular yielding time is 1-2 days faster than that of the existing solid strain; the entity maturation time of the inoculated culture bottle is 43-45 days, and the culture period of the tremella is equivalent to that of solid strains.

(4) The tremella sporophore pedicel produced by the liquid strain obtained by the invention is very small, and the average dry weight of the tremella sporophore pedicel produced by the solid strain is 50-70% of that of the tremella sporophore produced by the solid strain; the average dry weight of the produced tremella sporophore (pedicle removed) is equal to or slightly higher than the average dry weight of tremella produced by the solid strain.

Drawings

FIG. 1 is a photograph of liquid seed culture prepared in example 3;

FIG. 2 is a photomicrograph (600X) of the hyphae of the liquid spawn prepared in example 3, containing both the hyphae of Botrytis cinerea and the hyphae of Tremella fuciformis;

FIG. 3 is a photograph (B) of a liquid seed culture prepared in example 3 inoculated into a culture flask, with a solid seed culture (A) as a control;

FIG. 4 is a photograph (B) of the liquid seed culture prepared in example 3 inoculated with mycelia overgrowing the culture flask, and the solid seed culture (A) was used as a control;

FIG. 5 is a photograph (B) of the liquid seed culture inoculated with the primordia prepared in example 3, and the solid seed culture (A) is a control;

FIG. 6 shows a photograph (B) of the inoculated liquid seed culture prepared in example 3, when the fruit body matured, and the solid seed culture (A) was used as a control.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof, and all simple modifications which are within the spirit of the invention are intended to be within the scope of the invention as claimed.

Example 1: a preparation method of a white fungus liquid strain adopts stock seeds as starting strains for producing the white fungus liquid strain, and comprises the following specific steps:

selecting a strain which is fast in material intake, uniform in growth, fast in gelatinization and early in earring of the cinerea mycelia, round in shape of fruiting body leaves, good in earring stretching and free of pollution from a tremella protospecies with the diameter of fruiting body being 5-7 cm. Removing old hyphae on the surface of the sporocarp and the substrate by using an inoculating knife under aseptic conditions, scattering the hard substrate on the basal part of the sporocarp by using a special inoculating machine, uniformly mixing other positions of a strain bottle, mixing strains by rotating the head of the inoculating machine, and using an inoculating spoon to assist in mixing the strains on the upper interface and the lower interface if necessary. Mix 3 minutes per bottle stock. The mixed strains are restored and cultured for 1 day in a constant temperature incubator at 22 ℃.

Drying fresh bran to water content of 6%, repeatedly crushing by a shearing crusher for 15 times, crushing by a high-speed dispersion crusher, and sieving by a standard sieve of 200 meshes after crushing for one time; pulverizing the fine powder which does not pass through the sieve by using a high-speed dispersion pulverizer and sieving the fine powder by using a 200-mesh standard sieve, and repeating the steps, wherein the particle size of the fine particles of the final bran is less than 75 mu m.

The liquid strain formula is as follows: 1000 ml of medium contained 50 g of bran fines, 5g of glucose, 8 g of peptone, 3 g of magnesium sulfate and 5g of monopotassium phosphate. The liquid culture medium is prepared by adding water into 5% bran fine powder, boiling for 35 min, filtering with two layers of gauze, adding glucose 0.5%, peptone 0.8%, magnesium sulfate 0.3%, potassium dihydrogen phosphate 0.5% and vegetable oil 0.3%, stirring, and sterilizing at 121 deg.C under 0.11 MPa for 30 min.

When the stability of the sterilized liquid medium is lowered to below 25 ℃, inoculation can be started; sterilizing the peripheral surface of the tremella protospecies bottle by using 75% ethanol, and further sterilizing the bottle mouth of the protospecies bottle by using flame of an alcohol lamp; pouring some absolute ethyl alcohol into the groove on the outer ring of the inoculation port of the fermentation tank, and igniting to form a flame ring; inoculating the solid strain in the stock bottle into the liquid culture medium by using an inoculating shovel.

After the liquid culture medium is inoculated, culturing for 6 days at a constant temperature of 22 ℃, and keeping the ventilation volume of 100L/min and the dissolved oxygen volume of 50-90% in the culture process to obtain the tremella liquid strain.

Example 2: a preparation method of liquid white fungus strains adopts cultivated species as starting strains for producing the liquid white fungus strains, and comprises the following specific steps:

the production method comprises the step of selecting the strain which is fast in material taking, uniform in growth vigor, strong in white hairballs, large in quantity, uniformly distributed on the surface of the strain and free of pollution from the production strains of the white fungus cultured for 8-9 days. The culture medium in the production seeds is uniformly mixed by using a special inoculating machine for tremella under aseptic conditions, an inoculating spoon is used for assisting in mixing the strains on the upper interface and the lower interface when necessary, and each bottle of production seeds is mixed for 2 minutes. The mixed strains are restored and cultured for 1 day in a constant temperature incubator at 22 ℃.

Example 3: the 10 batches of liquid strains prepared in the example 1 and the effect of the liquid strains on cultivating tremella are as follows:

the liquid strain is flocculent and light yellow (figure 1), and is not layered obviously after standing for 30 minutes; microscopically, the mycelial pellets contained white fungus hyphae and gray fungus hyphae intertwined with each other (fig. 2).

Control solid strain: the preparation method of the tremella fuciformis solid strain (cultivar) comprises the following steps of preparing a culture material formula from 63-73% of wood chips of Betulaceae, 35-25% of bran, 2% of gypsum powder and a material-water ratio of 1: 1.5. And (3) uniformly stirring the compost, bottling, and sterilizing for 2 hours in autoclave sterilization at 121 ℃, wherein the material height is 7 cm-10 cm. 3-5 g of tremella protospecies particles are taken by an inoculating spoon and inoculated to the upper surface of the culture material, and the strain is dispersed to the whole upper surface by shaking the strain bottle. And (3) culturing in a strain room with the temperature of 20-22 ℃, the light resistance and the fresh air for 7-12 days, wherein the incense ash hypha grows strongly, is uniformly distributed, secretes melanin, and a plurality of white hair lumps appear on the surface of the culture medium, so that the cultivated species is obtained.

The shape difference of the inoculated part of the culture bottle inoculated by the liquid strain and the control solid strain is obvious (figure 3); on the 2 nd day of inoculation of the liquid strains, hyphae begin to germinate, and the germination speed is equivalent to that of the control solid strains; on the 9 th day of inoculation, hyphae overgrow the whole fungus bottle (fig. 4), 1 day faster than the control solid strain; on day 25 of inoculation, the formation of an ear base (fig. 5) began at the inoculation site, and the ear emergence time was 1 day faster than that of the control solid strain; on day 45 of inoculation, the fruiting bodies matured (FIG. 6), at a time comparable to that of the solid seed.

Randomly picking 20 bottles of cultivation bottles inoculated with the liquid strains and the control liquid strains respectively, and counting the weight of mature sporocarp. The average dry weight of pedicel of the white fungus fruiting body produced by the liquid strain is 2.2 g, which is 54% of that of the contrast solid strain; the average dry weight of the fruiting body (pedicle removed) of Tremella fuciformis produced was 31.0 g, which is 116% of the average dry weight of Tremella fuciformis produced by the control solid strain (Table 1).

The 10 batches of the strain were prepared from 12 months 4 to 12 months 16 days in 2021, and inoculated into culture bottles on the same day. The inoculation amount of each batch of strains is different from 4336-41152 bottles, 228738 bottles are inoculated together, wherein 4903 culture bottles cannot produce ears (bad products), 45 culture bottles produce abnormal fruit bodies (defective products), the yield is 97.84% (table 2), and the requirement of large-scale culture of tremella is met.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims

1. A preparation method of a white fungus liquid strain is characterized by comprising the following steps:

selecting a tremella protospecies with a fruiting body growing to a diameter of 5-7 cm or a tremella production species cultured for 8-9 days as a starting strain for liquid strain production;

taking the starting strain selected in the step a), removing old hyphae on the surfaces of the sporocarp and the substrate by using an inoculating knife under the aseptic condition, uniformly mixing the rest strains with the grifola frondosa hyphae by using a special tremella mixing machine, and carrying out constant-temperature recovery culture for 1-2 days at the temperature of 19-23 ℃ to obtain a tremella strain;

adding a liquid culture medium into a fermentation tank, sterilizing, inoculating the tremella fuciformis strain obtained in the step b), and culturing at a constant temperature of 20-22 ℃ for 5-6 days to obtain a tremella fuciformis liquid strain;

the liquid culture medium comprises the following components: 1000 ml of culture medium comprises 40-60 g of bran fine powder, 4-5 g of glucose, 6-9 g of peptone, 2-3 g of magnesium sulfate, 4-6 g of monopotassium phosphate and the balance of water.

2. The method for preparing liquid spawn of tremella according to claim 1, wherein the method comprises the following steps: the preparation steps of the liquid culture medium are as follows: adding water according to the formula amount into the fine bran powder, boiling for 30-40 min, filtering with two layers of gauze, adding glucose, peptone, magnesium sulfate and potassium dihydrogen phosphate into the filtrate, stirring uniformly, and sterilizing at 121 ℃ for 20-40 min under the pressure of 0.11 MPa.

3. The method for preparing liquid strain of tremella according to claim 1 or 2, wherein: the fine bran powder is prepared by micronizing into particles with particle size less than 75 μm.

4. The method for preparing a liquid strain of tremella as claimed in claim 1, wherein the method comprises the following steps: the selected tremella breeder seeds meet the following conditions: the gray fungus hypha has fast material intake, even growth, fast gelatinization of white hair mass, early ear emergence, round shape of fruiting body, good extension of ear, and no pollution.

5. The method for preparing liquid spawn of tremella according to claim 1, wherein the method comprises the following steps: the selected tremella fuciformis production seeds meet the following conditions: the grifola frondosa mycelia are fast in material intake, uniform in growth, strong in white hair clusters and large in quantity, and are uniformly distributed on strains, so that no pollution is caused.

6. The method for preparing liquid spawn of tremella according to claim 1, wherein the method comprises the following steps: in the mixed planting of the starting strain and the grifola frondosa hypha, a special inoculation machine is adopted to scatter the hard substrate of the basal part of the sporocarp, other positions of a strain bottle are uniformly mixed, the strain is mixed through a head of the rotary inoculation machine, an inoculation spoon is used for assisting in mixing the strain on the upper interface and the strain on the lower interface when necessary, and each bottle of original seeds is mixed for 2-3 minutes.

7. The method for preparing liquid spawn of tremella according to claim 1, wherein the method comprises the following steps: the strain inoculation amount in the fermentation tank is that 400-500 g of tremella strain is inoculated in 35L of liquid culture medium.

8. The method for preparing liquid spawn of tremella according to claim 1, wherein the method comprises the following steps: the conditions of the culture in the fermenter were: the culture temperature is 20-22 ℃, the ventilation volume is L/min, and the dissolved oxygen is 50-90%.

9. The liquid strain of Tremella fuciformis produced by the method of any one of claims 1-8 is used in bottle cultivation and bag cultivation of Tremella fuciformis.