BE1029710A1 - Liquid fermentation culture methods and active metabolites of Inonotus hispidus - Google Patents

Abstract

The invention provides a liquid fermentation culture method and active metabolites of Inonotus hispidus, wherein the liquid fermentation culture medium is maltose, yeast powder, potassium dihydrogen phosphate, magnesium sulfate and water; the culture strain is subjected to activation on the slant surface, plate culture, mycelium dispersion and fermentation culture in shake flask, and the obtained fermentation broth of Inonotus hispidus is subjected to filtration, ethanol precipitation, centrifugation and drying to obtain mycelia of Inonotus hispidus, flavonoids having activity to Scavenging free radicals in vitro and obtaining extracellular polysaccharides with activity to inhibit proliferation of tumor cells in vitro. The present invention adds wheat bran to adsorb and stabilize the dispersed mycelium and prevent the mycelium from growing on the walls, thus controlling the morphology, size and number of the mycelium balls during the fermentation process.

Description

Liquid fermentation culture methods and active metabolites of Inonotus hispidus

TECHNICAL AREA

The present invention relates to the field of fermentation technology, in particular

Liquid fermentation culture methods and active metabolites of Inonotus hispidus.

STATE OF THE ART

Inonotus hispidus is a large edible and medicinal mushroom of the genus Inonotus in the tribe of

Basidiomycota and the only species of fungus that matches the description of "Phellinus linteus" in ancient Chinese writings. The medicinal value of "Phellinus linteus" has long been mentioned in Chinese medical writings, from the Eastern Han Dynasty's "Sheng Nong's herbal classic" to the Ming Dynasty's "Compendium of Materia Medica" by Li Shizhen. It not only eliminates heat and detoxifies the body, but also activates blood circulation and relieves congestion. It is also effective for replenishing weakness and enlivening Qi, and can treat amenorrhea, hemoptysis, bleeding, Qi deficiency, and blood deficiency. In recent years, domestic and foreign research has found that the wild or artificially bred Inonotus hispidus has a wide range of biological activities, such as anti-aging, improving

Immunity, anti-virus, lowering blood lipids, activating blood circulation and dissolving

Blood stasis, which has an extremely high prospect of medical application. However, wild-growing Inonotus hispidus gradually declines with collection, resulting in a

resource shortage of Inonotus hispidus and high prices; while in the artificial cultivation of Inonotus hispidus, obtaining fruit bodies takes much time and consumes more resources, so the technology of the

Liquid fermentation attracting more attention. The current liquid

Fermentation voices are identified and isolated from wild strains, and the different strains show large genetic differences and vary in the choice and use of culture media, resulting in large differences in the type, content, and activity of fermentation metabolites. Therefore, the selection of highly viable Inonotus hispidus strains and the optimization of the

Fermentation conditions of crucial importance for the technology of

liquid fermentation.

Inonotus hispidus is a filamentous fungus and agar blocks are often used as the stock inoculum in the production of liquid fermentation. In this case, the mycelium appears in densely packed masses and grows continuously. Inside the masses is the nutrient and

Oxygen transfer is blocked, resulting in nutrient and oxygen starvation that

Mycelial growth and the synthesis of products are impaired and sometimes the

Caused autolysis of the mycelium inside the masses. Also, the mycelium tends to cling to the glass walls of the shake flask and grow upwards, resulting in limited utilization of the nutrients and a small amount of the active

Product is secreted into the liquid culture medium. The problem of agglomeration

Mycelial nodules and wall growth also occurs in the fermentation phase of this

Strain of Inonotus hispidus, resulting in long fermentation cycles, low

Product metabolic yields and low activity, which requires optimized control of fermentation conditions and inoculation procedures.

CONTENT OF THE PRESENT INVENTION

In view of the deficiencies of the prior art, the present invention aims to provide a liquid fermentation culture method and active metabolites of Inonotus hispidus

To make available.

A liquid fermentation culture method of Inonotus hispidus comprising the following

Steps:

Plate culture: inoculation of the activated Inonotus hispidus strain im

agar plate culture medium (PDA) for cultivation;

Preparation of the culture medium: Recipe of the carbon source culture medium:

Carbon Source 5-20 g/L, Yeast Extract Powder 5-20 g/L, Potassium Dihydrogen Phosphate 0.5-5 g/L,

Magnesium sulfate 0.1-2 g/L, Nitrogen source culture medium recipe: Maltose 5-20 g/L,

nitrogen source 5-20 g/L, potassium dihydrogen phosphate 0.5-5 g/L, magnesium sulfate 0.1-3 g/L;

Mycelial dispersion: Agar blocks obtained from the plate culture are used as stocks, in the above carbon source culture medium and/or

Nitrogen source culture medium is inoculated, cultured until mycelium balls are formed, and the liquid culture containing the mycelium balls is sheared and broken at high speed to finally obtain a mushroom suspension containing dispersed mycelium;

Inoculation of the mycelium and liquid fermentation culture: the concentration of the crushed

Mushroom suspension is adjusted according to the light absorption value, then the

Mushroom suspension is inoculated into the above culture medium, in which the wheat bran is added to obtain the inoculated broth for liquid fermentation culture.

Inonotus hispidus is selected by China General Microbiological Culture Collection Center, with number CGMCC3.15188.

Preferably, the activated Inonotus hispidus strain is obtained by

Malt juice culture medium is mixed with freeze-dried mushroom powder so that the freeze-dried mycelium is dissolved into a suspension state, and then the

mycelium suspension is transplanted into a solid culture medium, and the colonies which grow strongly are selected and inoculated into a new solid culture medium for cultivation, thereby obtaining a well-grown strain. The

Composition of the solid culture medium glucose 5-15 g/L, peptone 5-15 g/L,

Yeast extract powder 1-10 g/L and agar 10-30 g/L.

The amount of malt juice culture medium is sufficient when the freeze-dried

Mushroom powder can be dissolved.

In the step of plate culture, the culture conditions preferably include culturing at 22-28°C for 8 to 10 days under shielding from light.

Preferably, the carbon source in the step of preparing the culture medium is at least one of dextrose, cane sugar, maltose, lactose and soluble starch, the

nitrogen source is at least one of yeast extract powder, peptone, beef paste, soybean meal cake and urea.

In the step of preparing the culture medium, the formulation of the

carbon source culture medium carbon source 10-15g/L, yeast extract powder 10-15g/L,

Potassium dihydrogen phosphate 0.5-3 g/L, magnesium sulphate 0.1-1 g/L, the recipe of the

nitrogen source culture medium maltose 5-15 g/L, nitrogen source 5-15 g/L,

contains potassium dihydrogen phosphate 0.5-3 g/L and magnesium sulfate 0.1-1 g/L;

After the preparation of the culture medium, the culture medium is preferably a

Subjected to high pressure sterilization, cooled to room temperature and made available.

Preferably, the agar blocks in the step of mycelium dispersion are punched near the edge of the colonies in the plate culture with agar blocks

to obtain mycelium.

Preferably, the culture temperature in the mycelium dispersion step is 25-35°C.

In the step of mycelium dispersion, the one used in high speed shearing has

high-speed scissors a labor intensity of 5 to 6; where the

High speed shear has a working time of 60 to 120s.

Preferably, in the step of inoculating the mycelium and the liquid fermentation culture, the adjusted light absorption value of the mushroom suspension is 0.3 to 0.5, more preferably 0.35 to 0.5; if the light absorption value is too large, the original mushroom suspension before the

Vaccination to be diluted.

Preferably, in the step of inoculating the mycelium and the liquid fermentation culture, the amount of the fungal suspension inoculum is 0.5-1%.

Preferably, in the step of inoculating the mycelium and the liquid fermentation culture, the wheat bran used is crushed and sieved to a final grain size of 30 to 50 mesh. The wheat bran used is preferably washed three times and dried in order to remove soluble substances.

Preferably, in the step of inoculating the mycelium and the liquid fermentation culture, the amount of wheat bran added in the culture solution is 6-10 g/L, more preferably 8-10 g/L.

Preferably, the liquid fermentation culture is in the step of inoculating the

mycelium and the liquid fermentation culture that the obtained inoculated broth is further cultivated for 8-15 days.

The carbon content of microbial cells is quite high, generally reaching about 50% of the dry weight of the mycelium. It is one of the most important raw materials for the growth of mycelium and the main source of energy for microbial metabolism. The use of

Carbon by microorganisms is closely related to their own enzymatic systems and

related to metabolic processes. Although dextrose is widely used in fermentation production, as a fast-acting source of carbon, dextrose can

inhibit product formation, while slow-acting carbon sources such as lactose,

Cane sugar, maltose and starch are more suitable for product formation. Similar to

Carbon sources also play a role in the growth of nitrogen sources

Microorganisms that they form the cell structure and that are needed for cell growth

provide energy. There are mainly organic and inorganic sources of nitrogen, with the nature of the two differing greatly in the fermentation process. inorganic

Nitrogen sources are single-component, fast-acting nitrogen sources and are readily taken up by the mycelium. Organic nitrogen sources are complex and nutritious, containing proteins, peptides, free amino acids, etc., common organic ones

Sources of nitrogen are beef powder, peptone, powdered yeast, etc. Organic nitrogen sources cannot be ingested until they are hydrolyzed by the proteases secreted from microorganisms and further decompose and metabolize. 5 Therefore, the selection of suitable carbon and nitrogen sources is particularly important for the

Growth of mycelium and accumulation of metabolites.

The liquid strains of filamentous fungi are usually prepared by first activating the strain and then transferring it to a plate culture, after the colony has grown it is punched with a punch near the edge of the colony and a

agar block is selected for liquid culture. When the agar block is used as an inoculation seed, it has very few growth points, resulting in the mycelium surrounding the

Agar block grows outwards and forms large, dense balls of mycelium. Inside the

Nutrient and oxygen transfer is blocked, resulting in a nutrient and

lack of oxygen, which impairs the growth of the mycelium and the synthesis of products and sometimes causes the autolysis of the mycelium inside the sphere.

In addition, there are certain differences in growth between the agar blocks, making it difficult to precisely control the amount of inoculum and strain status. To the

To improve the growth point of the liquid strain, to facilitate inoculation and the

Accurately controlling the amount of inoculation can be done by physically crushing a large

number of small mycelia for vaccination.

Wheat bran is rich in crude protein, sugars (maltose, starch, etc.) and vitamins, so wheat bran juice is often added to microbial culture media as an organic nitrogen source; e.g. the bran in a selenium-rich mycelial growth medium for

Inonotus hispidus used as nitrogen source. In experiments it was found that the

Wheat bran during the fermentation process has a dispersing effect on the mycelium, which can reduce mycelium agglomeration, the formation of small more homogeneous

Mycelial balls facilitated and the contact area between the mycelium and the liquid

Culture medium enlarged. At the same time, due to the fibrous structure of the

Wheat bran will grow on the bran particles and fully utilize the nutrients of the liquid culture medium instead of sticking to the walls of the shake flask in large quantities.

The wheat bran itself consists of coarse fibers and has a loose structure with numerous

Wrinkles and small honeycomb pores on the surface. Therefore, the addition of

Wheat bran disperses the growth of the mycelium balls and increases the production yield of metabolites in the fermentation broth.

The present invention uses the culture medium for liquid fermentation of

Inonotus hispidus. Under the same culture conditions, the production yield of extracellular polysaccharides and flavonoids after optimization of carbon and

Nitrogen sources increased by 3.08x and 1.24x, respectively; after improving the

Inoculation procedure increases the production yield of mycelium, extracellular polysaccharides and flavonoids by 1.38-, 1.16- and 1.52-fold, respectively. After adding

Wheat bran is the production yield of mycelium, extracellular polysaccharides and

Flavonoids increased by 1.23, 1.23 and 1.13 fold, respectively. The culture medium is simple to prepare, easy to handle, inexpensive to produce and process, provides a high production yield of biomass and polysaccharides and other active ones

products and is easily cultivated on a large scale and commercially.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the free radical scavenging activity of the

Embodiments and the comparative examples obtained flavonoid components.

Fig. 2 shows the activity for inhibiting the proliferation of tumor cells (Hela) by the extracellular ones obtained from the working examples and the comparative examples

polysaccharide components.

Fig. 3 shows the activity for inhibiting the proliferation of tumor cells (HepG2) by the extracellular ones obtained from the working examples and the comparative examples

polysaccharides.

DETAILED DESCRIPTION

The present invention provides a method for the culture and fermentation of Inonotus hispidus, and the method for using the present invention will be explained below in connection with the working examples, but these

Exemplary embodiments are only normative and do not represent any limitation of the scope of the present invention

Details and the form of the technical solutions of the present invention are made without departing from the spirit and scope of the present invention, but these

Modifications and substitutions should still be made within the scope of the present

Invention covered.

The strains of Inonotus hispidus are provided by the China General Microbiological Culture

Collection Center, number CGMCC3.15188; the wheat bran is obtained from the

Lvwan Agricultural Products GmbH Shijiazhuang with article number 202202250002 and granular appearance, and pre-treatment is carried out before use:

Taking an appropriate amount of wheat bran, soaking in distilled water for a period of time, filtering under reduced pressure to obtain the filter cake,

Repeating for 3 times and drying the filter cake in an oven at 60°C, the completely dried wheat bran is crushed in a crusher, sieved and collected, the model of high-speed shear emulsifier is IKA

T10 Basic.

Unless otherwise stated, all raw materials of the present invention are commercially available and are not processed further; the experimental device is also commercially available.

The following working examples and comparative examples are based on their

Performance tested: 1. the mycelium yield after fermentation, the filtration is done under reduced pressure to recover the mycelium. After washing with distilled water three times, 10 mycelium balls (if the number is less than 10, all are selected) are randomly selected to make the

Measure diameter and then dried in the oven at 60°C and weighed. 2. the polysaccharide yield 5 mL of fermentation broth is taken, and anhydrous ethanol fourfold

Volume is added, then allowed to settle overnight. Centrifuge at 6000

rpm for 30 minutes and remove supernatant. The sedimentation is dissolved with 2 mL distilled water. The total sugar content is

phenol-sulfuric acid method. 2.0 mL of the solution is taken up, then 1 mL of a 5% phenol solution is added and shaken until steady, at the end 5 mL of concentrated sulfuric acid is added, shaken until steady and cooled in water bath for 15 min. The

Light absorbance is measured at 490 nm and total polysaccharide content is determined using the regression equation of the standard curve (Y=13.726X+0.0116, R2=0.9941).

3. Total content of flavonoids Take 1 ml of fermentation broth of Inonotus hispidus, add 3 ml of 95% ethanol, after sedimentation at 4°C for 24 hours, centrifuge for 15 min at 5000 rpm, sedimentation is removed and the supernatant is collected, and 1 mL of supernatant is added to a test tube, and in each tube, 0.3 mL of a 5% sodium nitrite solution (prepared and ready to use) and until uniform

State shaken, let stand for 6 min, then 0.3 mL of a 10%

Added aluminum nitrate solution and shaken until uniform,

Allow to stand for 6 min, then add 4.0 mL of 4% sodium hydroxide in each

Glass is added 42% ethanol so that the total volume is 10 mL,

Leave for 30 min, after which the light absorbance is measured at 510 nm. Under

Using gallic acid as a standard product, the total flavonoid content of the sample is determined according to the regression equation of the standard curve (Y=1.1321x, R°=0.9999), and the

Results are expressed as gallic acid equivalents in mg/mL. 4. Total content of polyphenols

Take the 0.5 mL sample and place it in the test tube, add distilled water to a total volume of 5.5 mL, shake until smooth

state, then 0.5mL of Folin-Ciocalteu color developer is added, reacting in dark environment for 10min after shaking to steady state, then 40mL of 7.5% sodium carbonate solution is added, reacting in dark environment for 1h after Shake until steady and the absorbance of light is measured at 765 nm. Using rutin as the standard product, the

The total polyphenol content of the sample is determined according to the regression equation of the standard curve (Y=10.674x, R2=0.9997) and the results are expressed as rutin equivalent in mg/mL. 5. Free radical scavenging activity

To a 96 well plate is added 20 µl of different sample solutions and 180 µl of a 150 µmol/L DPPH working solution. The blank and positive controls are distilled water and 0.4 mg/mL VC. Vibrate and mix evenly and the light absorbance value is measured at 517 nm. VC gradient concentrations of 12.5, 25, 50, 100, 200, and 400 µg/mL are used to calculate DPPH clearance using the following formula:

DPPH clearance (%) = (A0-A1)/A0 x 100, AO - light absorbance value of blank control; A1 -

light absorption value of the sample.

6. Activity to inhibit proliferation of tumor cells

Cytotoxicity analysis is performed using the CCK8 colorimetric assay.

Logarithmic growth phase human cervical cancer cells Hela and human liver cancer cells HepG2 are digested with 0.25% trypsin for 1-2 min, after

At the end of the digestion, a cell suspension with a cell density of 5x104 cells/ml is prepared. 100 µl of cell-containing broth is added to 96-well cell culture plates and an equal amount of cell-free broth is added to the blank control group. In each group, 3 parallel compound wells are made and cultured in a CO2 incubator. When the cell density is more than 80%, the original culture solution is carefully discarded, and 100 µL of the polysaccharide sample with a concentration of 0.1 mg/mL is added, and culturing for another 12 h. Then, the liquid is slowly aspirated and 10 µL of CCK-8 is added to each well and cultured for 4 h. The OD value of each well is measured at 450 nm with an enzyme marker and the cell survival rate is calculated using the following formula: Cell inhibition rate (%) = [OD(O dose) - OD(dose)/[OD(O dose) - OD (Void) x 100.

Example 1 Fermentation culture with 20 g/L maltose as carbon source

Using a sterile pipette to take 0.3 mL of a suitable malt juice culture medium and drop it into a vial of freeze-dried mushroom powder and gently vibrate to dissolve the freeze-dried mycelium into a suspension.

Transplant 0.2 mL of mycelial suspension into a solid culture medium containing the

Composition of the solid culture medium glucose 10 g/L, peptone 10 g/L,

Yeast extract powder 5 g/L and agar 20 g/L. The colonies that grow strongly are selected, and part of the colonies are inoculated into a new solid culture medium for cultivation, this step is repeated three times to obtain colonies that grow well, and keep them in the slant area, then becomes the activated stem from the

Slant surface inoculated with an inoculation ring in an agar plate culture medium (PDA),

Cultivate for 8-10 days at 27°C protected from light; the

Carbon source base medium is used in the ratio of yeast extract powder 10g/L,

Potassium dihydrogen phosphate 1 g/L and magnesium sulfate 0.5 g/L, and 20 g/L

Maltose are added as a carbon source. The prepared culture solution is poured into 250 mL Erlenmeyer flasks, 150 mL culture medium per flask, sterilize for 20 min at 121°C in autoclave and cool to room temperature. in an ultra-clean bench, it is punched near the edge of the colonies with a 0.5 cm diameter punch to obtain agar blocks with mycelium. The agar blocks are after

Randomly selected as strains and inoculated into 150 mL of liquid culture medium for 14

days the inoculated 250 mL shake flask is cultured at 30°C and 120 rpm in a shaker. After fermentation, the test is carried out using the performance test method. After fermentation, the obtained fermentation broth is filtered under reduced pressure to obtain mycelium and filtrate, then a certain amount of anhydrous ethanol is added to the filtrate, the mixture is thoroughly stirred and left to stand for a period of time, then centrifugation is carried out, the sedimentation obtained is the metabolite - extracellular polysaccharide, and the centrifugal solution obtained is concentrated and dried to obtain the metabolite - flavonoids; the activity of scavenging free radicals of flavonoids and the activity of inhibiting the proliferation of

Tumor cells from extracellular polysaccharides in the separated filtrate are each tested.

Example 2 Fermentation culture with 10 g/L maltose as carbon source

In the carbon source base medium, 10 g/L of maltose is added as a carbon source. All other operations are the same as in embodiment 1.

Embodiment 3 Fermentation culture with 20 g/L yeast extract powder as a nitrogen source the nitrogen source basal medium is used in the ratio of maltose 10 g/L,

Potassium dihydrogen phosphate 1 g/L and magnesium sulfate 0.5 g/L, and 20 g/L

Yeast extract powders are added as a nitrogen source. All other operations are the same as in embodiment 1.

Example 4 Fermentation culture with 10 g/l yeast extract powder as nitrogen source

In the nitrogen source basal medium, 10 g/L of yeast extract powder is added as a nitrogen source. All other operations are the same as in embodiment 3.

Example 5

The shake flask of embodiment 4 is at 30 ° C and 120 rpm in one

cultured until mycelium balls are formed in large numbers. The shaving head

High-speed scissors are sterilized in an autoclave in advance, then into the

Built-in high-speed scissors, sterilized with alcohol and placed in ultra-clean bench for UV sterilization. The liquid culture containing the above mycelia balls is sheared and crushed for 60 seconds at a high speed of the fifth stage to obtain a suspension containing a large number of minute mycelia dispersed.

The suspension is diluted ten times and the light absorbance value is 0.480 at 410 nm using a blank culture solution as a control. 0.5 mL of the suspension is aspirated with a sterile gun, into a shake flask (250 mL) with 150 mL liquid

culture medium and cultivated for 10 days at 30°C and 120 rpm in a shaking stove. The measurements after the end of the fermentation agreed with the

Embodiment 1 match.

Example 6

Same as embodiment 5, the difference is that the labor intensity of the

high-speed shearing is set to 6, and all other operations are the same as in embodiment 5.

Example 7

Same as embodiment 5, the difference is that the working time of the

high-speed shearing is extended to 120s, and all other operations are the same as in embodiment 5.

Example 8

Same as Working Example 5, the difference is that the light absorbance value of the diluted suspension is set to 0.341, and all other operations are the same as Working Example 5.

Embodiment 9

Same as Working Example 5, the difference is that 1.0 mL of the suspension is aspirated with a sterile tip, and all other operations are the same as in

Example 5.

Example 10 Fermentation culture with wheat bran (10 g/L, 30 mesh) as an adjuvant

Weigh and take an appropriate amount of wheat bran, soak for a

A period of time in distilled water, filtering under reduced pressure to obtain the filter residue, repeating for 3 times and drying the filter residue in an oven at 60°C, the completely dried wheat bran is crushed in a crusher, sieved to 30 mesh and collected. the excipient base medium is off

Maltosel0 g/L, yeast extract powder 10 g/L, potassium dihydrogen phosphate 1 g/L and

Magnesium sulfate 0.5 g/L and 10 g/L wheat bran (grain size: 30 mesh) are added as an adjuvant. All other operations are the same as in embodiment 1.

Example 11 Fermentation culture with wheat bran (∼8 g/L, 50 mesh) as an adjuvant

In the excipient base medium, 8 g/L wheat bran (grain size: 50 mesh) is added as an adjuvant. All other operations are the same as in embodiment 5.

Comparative example 1 Fermentation culture with dextrose as carbon source

Same as embodiment 1, the difference is that the maltose through the

Glucose is replaced, and all other operations are the same as in embodiment 1.

Comparative Example 2 Fermentation culture using cane sugar as a carbon source

Same as embodiment 1, the difference is that the maltose through the

Cane sugar is replaced, and all other operations are the same as in embodiment 1.

Comparative Example 3 Fermentation culture with lactose as carbon source

Same as embodiment 1, the difference is that the maltose through the

Lactose is replaced, and all other operations are the same as in embodiment 1.

Comparative Example 4 Fermentation Culture Using Soluble Starch as Carbon Source

Same as embodiment 1, the difference is that the maltose is replaced with the soluble starch, and all other operations are the same as embodiment 1.

Comparative example 5 Fermentation culture with peptone as nitrogen source

Same as embodiment 3, the difference is that the yeast extract powder is replaced with the peptone and all other operations are the same as in FIG

Example 3.

Comparative Example 6 Fermentation culture with beef paste as nitrogen source

Same as embodiment 3, the difference is that the yeast extract powder is replaced with the beef paste, and all other operations are the same as in FIG

Example 3.

Comparative Example 7 Fermentation culture with soy flour cake as nitrogen source

Same as Embodiment 3, the difference is that the yeast extract powder is replaced with the soybean flour cake, and all other operations are the same as in FIG

Example 3.

Comparative Example 8 Fermentation culture with urea as nitrogen source

Same as embodiment 3, the difference is that the yeast extract powder is replaced with the urea, and all other operations are the same as in FIG

Example 3.

Comparative Example 9 Fermentation culture using the optimal carbon and

Sources of nitrogen without adding wheat bran

Same as embodiment 4, the difference is that the time of

Fermentation culture is shortened to 10 d.

Table 1 Average diameter and mass of mycelium in fermentation broth and potency

average 0000 licher extracellular

Designation Dry weight Total area Total polyp ng Diameter of © vonoide henole

Sample" of mycelium (g/L) Polysaccharic (mg/mL) (mg/mL)

Mycelium balls de (mg/mL) en (cm) 'Execution 1 4990 680 g example 0.79 0.0406 0.1339 1

Design 4.55 5.9 example 0.68 0.0318 0.1383 2

Execution 5.13 6.26 example 0.79 0.0394 0.1385 3

Design 4.58 6.03 example 0.82 0.0468 0.1460 4

Design 5.59 5.72 example 0.33 0.0645 0.1643

Design 6.16 6.89 example 0.24 0.0711 0.1681 6

Design 6.34 6.99 example 0.29 0.0709 0.1614 7

Version 5.55 5.70 0.35 0.0678 0.1597 example

Design 6.22 6.68 example 0.29 0.073 0.1694 9

Design 6.03 6.35 example 0.31 0.0698 0.1660 10

Design 6.86 7.04 example 0.21 0.0783 0.1745 11

Comparison 4.7 1.96

I 0.91 0.0377 0.1250 sexample1

Comparison 4.83 0.99 oo 0.85 0.0418 0.1311 example2

Comparison 3.15 1.3

I 0.59 0.0306 0.1295 example3

Comparison 2.45 0.2 nn 0.61 0.0411 0.1239 sexample4

Comparison 4.26 4.67

I 0.92 0.0415 0.1315 example5

Comparison 3.97 5.02

I 0.84 0.0428 0.1244 example6

Comparison 1.44 3.18

I 0.34 0.0356 0.0598 sexample7

Comparison 0.28 0.4

I 0.13 0.0105 0.0445 sexample8

Comparison 3.69 3.54

I 0.76 0.0386 0.1477 example9

From Experimental Examples 1-4 and the first eight Comparative Examples, it can be seen that the optimum carbon and nitrogen sources for promoting Inonotus hispidus are for

Fermentation and production of extracellular polysaccharides are maltose and yeast powder. The reason lies in the different metabolic pathways of the different

Nutrients within the mycelium and the various extracellular active products that can be formed. In this strain, maltose and yeast powder promote intracellular synthesis and extracellular compared to other carbon and nitrogen sources

Secretion of active products such as biomolecules stronger. The one used in fermentation

Stem shape is also crucial for the growth of the mycelium and the secretion of the

Products. It can be seen from the table that in Working Examples 5-9, mycelium obtained by the high-speed shear fracture is used as the stem, which makes it easier to control the inoculation amount compared to agar blocks and the number of

Increased mycelial growth sites, thereby increasing mycelial growth and utilization of

Culture solution are accelerated and the fermentation time is greatly reduced. In addition, the addition of a certain amount of wheat bran can increase the yield of extracellular polysaccharides and the production of metabolites, which is mainly due to the coarse fibrous structure of wheat bran, which allows the

Allows mycelium to grow on it to reduce entanglements. through the

Reducing the diameter of the mycelial aggregates, wheat bran increases the contact area with the fermentation broth, allowing more active products to be produced. For

Inonotus hispidus can therefore optimize the carbon and nitrogen source of the liquid culture medium, improve the inoculation procedure and add

Wheat bran significantly increase the yield of active metabolites, which makes the cultivation of the

strain on a large scale and the extraction of the products, as well as enabling the further development of drugs based on Inonotus hispidus that have an antioxidant effect and inhibit the proliferation of tumor cells.

Obviously, the above embodiments only serve to clearly explain the

Examples of the present invention rather than limiting the embodiment of the present invention. Those of ordinary skill in the art can make changes or modifications to other forms based on the explanation above. It is both unnecessary and impossible to list all of the embodiments. All performed under the spirit and principles of the present invention

Modifications, equivalent substitutions and improvements should be considered within the scope of the

Claims of the present invention are considered to be covered.

Claims (10)

Expectations A liquid fermentation culture method of Inonotus hispidus, comprising the steps of: plate culture: inoculating the activated Inonotus hispidus strain in the agar plate culture medium for cultivation; Preparation of culture medium: Recipe of carbon source culture medium: carbon source 5-20g/L, yeast extract powder 5-20g/L, potassium dihydrogen phosphate 0.5-5g/L, magnesium sulfate 0.1-2g/L, recipe of nitrogen source culture medium: maltose 5-20 g/L, nitrogen source 5-20 g/L, potassium dihydrogen phosphate 0.5-5 g/L, magnesium sulfate 0.1-3 g/L; Mycelium dispersion: Agar blocks obtained from the plate culture are used as strains, inoculated into the above carbon source culture medium and/or nitrogen source culture medium, cultured until mycelium balls are formed, and the liquid culture containing the mycelium balls is made high Speed sheared and broken to finally obtain a mushroom suspension containing dispersed mycelium; Inoculation of the mycelium and liquid fermentation culture: the concentration of the crushed mushroom suspension is adjusted according to the light absorption value, then the mushroom suspension is inoculated into the above culture medium, in which the wheat bran is added to obtain the inoculated culture solution for liquid fermentation culture. 2. The method according to claim 1, characterized in that Inonotus hispidus selected from China General Microbiological Culture Collection Center with the number CGMCC3.15188; wherein in the step of inoculating the mycelium and the liquid fermentation culture, the amount of added wheat bran in the culture solution is 6-10 g/L. 3. The method according to claim 1, characterized in that the carbon source in the step of preparing the culture medium is at least one of dextrose, cane sugar, maltose, lactose and soluble starch, wherein the nitrogen source is at least one of yeast extract powder, peptone, beef paste, soybean cake and urea . 4. The method according to claim 1, characterized in that in the step of preparing the culture medium, the recipe of the carbon source culture medium is carbon source 10-15 g/L, yeast extract powder 10-15 g/L, potassium dihydrogen phosphate 0.5-3 g/L, Magnesium sulfate 0.1-1 g/L, the nitrogen source culture medium formulation containing maltose 5-15 g/L, nitrogen source 5-15 g/L, potassium dihydrogen phosphate 0.5-3 g/L and magnesium sulfate 0.1-1 g/L contains. 5. The method according to claim 1, characterized in that the culture temperature in the step of mycelium dispersion is 25-35°C; wherein in the mycelium dispersing step, the high-speed shears used in the high-speed shearing has a work intensity of 5 to 6; the high-speed shear has a working time of 60 to 120s. 6. The method according to claim 1, characterized in that in the step of inoculating the mycelium and the liquid fermentation culture, the adjusted light absorption value of the mushroom suspension is 0.3 to 0.5. 7. The method according to claim 1, characterized in that in the step of inoculating the mycelium and the liquid fermentation culture, the amount of the mushroom suspension inoculum is 0.5-1%. The method according to claim 1, characterized in that the liquid fermentation culture in the step of inoculating the mycelium and the liquid fermentation culture is that the obtained inoculated culture solution is further cultured for 8 to 15 days. 9. The method according to claim 1, characterized in that the activated Inonotus hispidus strain is obtained by mixing the malt juice culture medium with freeze-dried mushroom powder so that the freeze-dried mycelium is dissolved into a suspension state, and then the mycelium suspension into a solid culture medium, and wherein the colonies which grow strongly are selected and inoculated into a new solid culture medium for cultivation, thereby obtaining a well-grown strain. 10. The method according to claim 1, characterized in that in the step of plate culture, the culture conditions include culturing for 8 to 10 days at 22-28°C under shielding from light; wherein the culture temperature in the mycelium dispersing step is 25-35°C.