CN102047814B - A method for cultivating Antrodia camphorata with micro-aeration section wood - Google Patents

Abstract

The invention relates to a method for cultivating antrodia camphorata by micro-ventilation cut log, which belongs to the field of fungus cultivation and is characterized in that liquid strains are inoculated on an antrodia camphorata section with the water content of 50-70 percent, and the fruiting bodies are cultivated by micro-ventilation cultivation at the temperature of 25-28 ℃ and the humidity of 85-95 percent. According to the method, the camphorwood is treated, liquid strains are obtained by liquid fermentation and propagation and inoculated on the surface of the camphorwood, a micro-ventilation mode is adopted, the cultivation is carried out by controlling the conditions of cultivation temperature, humidity, illumination and the like, the growth cycle is 11 months, the biotransformation rate reaches 15%, the polysaccharide content of the camphorwood is equivalent to that of wild antrodia camphorata, and the antrodia camphorata cultivated by cut logs is consistent with that of the wild antrodia camphorata in appearance, fragrance, bitterness, efficacy and the like.

Description

A method for cultivating Antrodia camphorata with micro-aeration section wood

technical field

The invention relates to a method for cultivating Antrodia camphorata, in particular to a method for cultivating antrodia camphorata with slightly air-permeable section wood, so as to produce fruiting bodies of Antrodia camphorata in large quantities.

Background technique

Antrodia cinnamomea is referred to as "Antrodia camphorata" for short, and its scientific name is Antrodia Camphorata. The fungus grows only on the unique Taiwan camphor tree (Cinnamomum kanehira), and is the only wood-rot fungus found on the camphor tree. Antrodia camphorata generally grows on the hollow inner wall of old trunks with rotten heartwood, and a small part grows on the dark and damp surface of camphor wood with fallen trunks. Will not grow on common camphor trees, white camphor and other similar tree species (Chen Tiqiang, Li Kaiben, Lin Zhangyu. Development status of edible (medicinal) fungi in Taiwan [J]. Fujian Agricultural Science and Technology, 2000, (1): 20-21.) . Cinnamomum camphora is a tall evergreen broad-leaved tree that grows in mountainous areas with an altitude of 450-2000m. It is mainly distributed in Fuxing Township and Jiaoban Mountain in Taoyuan; Nanzhuang Township and Sanwan Township in Miaoli; Turtle is a unique conservation tree species in Taiwan Province of my country. It is kept by the Forestry Department or Forest Management Bureau according to law. Those who steal camphor trees will be brought to justice for suspected violation of the Forest Conservation Law.

Antrodia camphorata is known as "yin and yang counterpart mushroom" because of its special and miraculous curative effect, and Taiwan folks call it "ruby in the forest". Studies in recent years have not only confirmed that Antrodia camphorata has no side effects at all, but also can activate cells, improve physical fitness, protect the liver, enhance immunity, and get rid of the diseases of civilization that modern people often suffer from. Antrodia camphorata contains a variety of physiologically active ingredients, such as polysaccharides, triterpenoids, superoxide dismutase (SOD) adenosine, proteins (including immune proteins), multivitamins, trace elements (calcium, phosphorus, 楮), nucleic acid , lectins, amino acids, cholesterol, lignin, blood pressure stabilizing substances, etc. According to the "Latest Research Results of Artificially Cultivated Antrodia Antrodiae Fruiting Bodies" by Taipei Medical University, when the concentration of artificially cultivated Antrodia camphorata fruiting bodies reaches 1500ug/mL, the reversal effect of cancer cells can almost reach 100% effective rate[ 4]. Antrodia camphorata is regarded as a unique and precious medicinal fungus, so it has extremely high research and commercial value, and is currently the most expensive wild fungus in Taiwan, and is called "Shenzhi" in Hong Kong and Macau.

In recent years, the miraculous curative effect of Antrodia camphorata fruiting bodies has gradually become known, and the demand is strong. Although the restoration work has been carried out in different regions, it is still too late. The price of natural Antrodia camphorata is as high as hundreds of thousands of NT dollars per 600 grams, and it is known as the most expensive in the world. Fungal herbs. The artificial cultivation of Antrodia camphorata has also become a research hotspot. Due to factors such as the lack of resources of the parasitic tree species of Antrodia camphorata, camphor tree, and the special growth environment, it is difficult to carry out artificial cultivation of Antrodia camphorata. At present, most of the researches on Antrodia camphorata focus on its liquid fermentation, but there are few reports on the artificial cultivation techniques of Antrodia camphorata. At present in Taiwan, the artificial cultivation of Antrodia camphorata can be roughly divided into three types: liquid fermentation method, solid cultivation method, and section wood cultivation method.

The liquid fermentation method is to obtain the mycelia through the liquid fermentation of Antrodia camphorata, and the cultivation can be completed in about 15 days. The speed is fast and the cost is low, but the medicinal effects of the cultivated mycelium and the fruiting bodies of Antrodia camphorata are very different.

The solid cultivation method is the space bag cultivation method, and the cultivation is completed in about three months. The Antrodia camphorata bacteria block is implanted into the space bag cultivation method, which is faster and less polluting.

The title of the invention "cultivation method of Antrodia camphorata" (ZL200610162766.5) involves inoculating Antrodia camphorata in a section of wood and cultivating fruiting bodies. It is mentioned in the invention that the effective substances in Antrodia camphorata are mainly triterpenoids and polysaccharides, but the invention The method is to use a solid medium to expand the bacterial strains, and then cultivate the fruiting body on the section wood, and only compare the content of triterpenoids in the artificially cultivated Antrodia camphorata and the wild Antrodia camphorata, and the same content in Antrodia camphorata. Polysaccharides with medicinal efficacy and high content are not involved.

Because of the special effects of the fruiting bodies of Antrodia camphorata and the difficulty in cultivating them, the strange objects of Antrodia camphorata can be inhabited. Therefore, it is necessary to enrich the methods for cultivating the fruiting bodies of Antrodia camphorata and provide cheap and good-quality Antrodia camphorata for the market.

Contents of the invention

The micro-permeable segment wood cultivation method adopted in the present invention strictly controls the cultivation conditions, such as: light, temperature, humidity, ventilation, etc., and successfully realizes the artificial cultivation of Antrodia camphorata, with a growth period of 11 months and a biotransformation rate of 15%. Its polysaccharide content is equivalent to that of wild Antrodia camphorata, and the cultivated Antrodia camphorata is consistent with wild Antrodia camphorata in terms of appearance, aroma, bitterness, and medicinal effect.

A method for cultivating Antrodia camphorata with micro-ventilation section wood, comprising the steps of:

(1) Antrodia camphorata is obtained by liquid fermentation, and (2) camphor wood section treatment: soak the camphor wood section in water to make the water content 50%-70%, and then put it into a sponge silica gel In a container with a plug seal, sterilize, (3) cover with a sponge silica gel plug after inoculation, (4) cultivate: A) at a temperature of 25°C-28°C and a humidity of 85%-95%, cultivate it to the surface of camphor wood in a dark place It is covered with mycelium; B) then place it at a daytime temperature of 25°C-28°C, a night temperature of 18°C-20°C, a temperature difference between day and night of about 8°C, a humidity of 85%-95%, and a CO2 concentration of 1.0%- 1.5%, fruiting bodies are cultivated under low light, and the light intensity during the daytime is 50-150Lux, and the light intensity at night is 0.001-0.02Lux.

The culture time of step A is 5-6 months, and the culture time of step B) is 3-4 months.

The processing of the camphor wood section also includes punching holes in the cut surface and/or side of the camphor wood section along the vertical direction.

The distribution of the pores is plum-shaped, and the depth of the pores is 5-10 cm.

The sterilization is at 121° C. for 90 minutes, and then naturally cooled to room temperature.

The container is a polypropylene bacteria bag.

The inoculation in the step (3) is to place the camphor wood in the liquid spawn after the liquid spawn is flowed down along each face of the camphor wood section.

The humidity is controlled by continuously adding water.

The making of described liquid strain comprises the steps:

a) Activation of strains: insert refrigerated Antrodia camphorata blocks into the parent species for cultivation based on a 28°C constant temperature and light-proof slant for 7-10 days;

b) primary seed liquid: insert the Antrodia camphorata strain obtained in a) into a container with a liquid fermentation medium, culture on a shaking table at 180 rpm/min, and cultivate at 28° C. for 7-12 days;

c) Secondary seed liquid: the pollution-free primary seed liquid is inoculated in a container with a liquid fermentation medium, the shaking table is 180rpm/min, and cultured at 28°C for 7-12 days to obtain a liquid strain.

The mother seed medium is glucose 2%, banana powder 0.5%, yeast extract 0.5%, peptone 0.5%, MgSO4 0.3%, KH2PO4 0.3%, citric acid 0.05%, (NH4)2SO4 0.05%, agar 2%, camphor Wood extract, pH4.5; The liquid fermentation medium is: glucose 2%, banana powder 0.5%, yeast extract 0.5%, peptone 0.5%, MgSO4 0.3%, KH2PO40.3%, citric acid 0.05%, (NH4 )2SO40.05%, camphor wood extract, pH4.5.

The inoculum amount during cultivation of the secondary seed liquid is 10%.

The present invention adopts liquid fermentation and multiplication to inoculate the surface of camphor wood through the treatment of camphor wood, adopts micro-ventilation mode, and cultivates by controlling cultivation temperature, humidity, light and other conditions, and adopts micro-ventilation mode , which not only ensures air permeability but also effectively prevents the contamination of bacteria, and the fruiting body of Antrodia camphorata is cultivated.

The liquid fermentation method of Antrodia camphorata is a conventional technology, and has been studied the most. In the present invention, the conventional method can be used, and the liquid fermentation scheme of the present invention can also be used. The camphor wood extract is added to the fermentation liquid of the present invention, so that the environment of liquid fermentation is somewhat similar to the environment of natural growth, and thus the polysaccharide content of the strain mycelium obtained by fermentation of the present invention is relatively high.

Cinnamomum camphora scientific name: Cinnamomum camphora (L) Presl. Classification: Lauraceae, alias: wood camphor, black camphor, camphor, camphor, fragrant stamen, camphor tree, what the present invention adopts is that camphor wood comes from the camphor tree of Xiaogan, Hubei Cinnamomum camphora, it is a type of Cinnamomum camphora (Linn.) Presl var.

Punching holes on the camphor wood section (plum blossom punching method) is conducive to the growth of mycelium in the camphor wood, shortens the time for germination, and improves air permeability.

According to the living environment of wild Antrodia camphorata in Taiwan, the present invention selects the artificial cultivation temperature, cultivation method and treatment method of cultivation raw materials of Antrodia camphorata, utilizes the Antrodia camphorata bacterial strain collected in this laboratory, adopts plum blossom punching method and soaking method to treat main The cultivation material camphor wood is treated and controlled and managed by imitating the living environment of wild Antrodia camphorata during the growth period of Antrodia camphorata mycelium and fruiting body, such as: low light, high temperature and temperature difference, high humidity (multiple water replenishment method), micro-ventilation etc. successfully carried out the artificial cultivation of Antrodia camphorata, and the growth period is about 11 months, which is significantly shorter than the 2-3 years of the artificially cultivated fruiting bodies of Antrodia camphorata, which laid the foundation for the pharmacological research of Antrodia camphorata fruiting bodies. .

Description of drawings

Figure 1 The initial stage of fruiting body growth

Figure 2 Fruiting body after ripening

Figure 3 Mycelium growth curve

Figure 4 The content of extracellular and extracellular crude polysaccharides

Figure 5 Total sugar determination standard curve

Figure 6 Reducing sugar determination standard curve

Figure 7 Effect of Antrodia camphorata cultivating fruiting body polysaccharides on NO production in peritoneal macrophages stimulated by LPS in S180 mice

Figure 8 Effect of Antrodia camphorata cultivated fruiting body polysaccharides on the phagocytosis of neutral red by S180 mouse macrophages

Fig. 9 Effect of Antrodia camphorata cultivated fruiting body polysaccharides on NK lethality in S180 mice

Figure 10 Effect of Antrodia camphorata cultivating fruiting body polysaccharides on the proliferation of spleen lymphocytes stimulated by ConA in S180 mice

Figure 11 Effect of Antrodia camphorata cultivating fruiting body polysaccharides on NO production in peritoneal macrophages stimulated by LPS in H22 mice

Figure 12 Effect of Antrodia camphorata cultivating fruiting body polysaccharides on the phagocytosis of neutral red by H22 mouse macrophages

Fig. 13 Effect of Antrodia camphorata cultivated fruiting body polysaccharides on NK lethality in H22 mice

Figure 14 Effect of Antrodia camphorata cultivating fruiting body polysaccharides on the proliferation of spleen lymphocytes stimulated by ConA in H22 mice

Marked on the figure: ^* P<0.05, ^** P<0.01

Detailed ways

Example

1 Materials and methods

1.1 Materials

1.1.1 Tested strains: Antrodia Camphorata: It is collected by the Provincial Key Laboratory of Applied Fungi of Qingdao Agricultural University and can be released to the public. This experiment is not limited to this strain, but also applicable to other strains.

1.1.2 Mother medium: 2% glucose, 0.5% banana powder, 0.5% yeast extract, 0.5% peptone, 0.3% MgSO4, 0.3% KH2PO4, 0.05% citric acid, (NH4)2SO4 0.05%, agar 2%, camphor wood extract, pH4.5

1.1.3 Liquid fermentation medium: 2% glucose, 0.5% banana powder, 0.5% yeast extract, 0.5% peptone, 0.3% MgSO4, 0.3% KH2PO4, 0.05% citric acid, (NH4)2SO4 0.05%, camphor Wood extract, pH4.5 Preparation of camphor extract: Take 500g of camphor wood debris, add 5000mL of water, cook until concentrated to 1/10 of the original volume, the resulting liquid is camphor extract, and the dosage is 100mL of liquid Add 50mL camphor wood extract to the fermentation medium.

1.2 Strain activation

Pick refrigerated Antrodia fungus blocks and insert them into the slant culture medium of the mother species, and seal them with cotton plugs to ensure good air permeability. Placed at 28°C for about 7-10 days at a constant temperature and protected from light for subsequent experiments.

1.3 Preparation of liquid strains

First-level seed liquid: pick Antrodia camphorata cultured with the mother seed medium and put it into a conical flask, shake it at 180rpm/min, and cultivate it at 28°C for 7 days.

Secondary seed solution: After microscopic examination and plate scribing to confirm that the first-level seed solution is free of pollution, it is used to make the second-level seed solution. The liquid volume of each 250mL Erlenmeyer flask is 100mL, and the inoculum volume is 10mL. Shaking table 180rpm/min, 9 days of liquid fermentation at 28°C, used for follow-up experiments.

1.4 Pretreatment of Antrodia camphorata cultivation materials

Wild Antrodia camphorata mostly grows in the rotten hollow interior of the camphor tree trunk or the damp surface of the fallen trunk. According to the growth environment of wild Antrodia camphorata, low light, high temperature and temperature difference, high humidity (multiple water replenishment method), micro-ventilation, and camphor wood segments were used to cultivate Antrodia camphorata in this experiment.

Cultivation raw material camphor wood comes from Cinnamomum camphora in Xiaogan, Hubei, which is a type of Cinnamomum camphora (Linn.) Presl var. linaloolif ear Fujita in Lauracae.

The raw material for Antrodia camphorata cultivation - camphor wood is pretreated. Saw the camphor wood into a stake with a length of about 20 cm, and make plum blossom-shaped holes in the vertical direction on the upper and lower cut surfaces and sides of the stake, with a depth of about 5-10 cm. Plum-blossom punching method is beneficial to the growth of mycelium in camphor wood, shortens the time for germination, and improves air permeability. The camphor wood that has been punched is fully submerged in water and soaked for 2-3 days, and the excess water is dried (the water content of camphor wood is controlled between 50%-70%), and packed into a polypropylene fungus bag, according to camphor wood The thickness of the wood section Each fungus bag contains about 1-2 wood sections. Seal it with a 18-23mm sponge silicone plug and tie it tightly with a rope, which not only ensures air permeability but also effectively prevents bacterial contamination. 121°C, sterilize for 90 minutes, and require the sterilizer to cool down to room temperature before opening, which has achieved the effect of full sterilization.

1.5 Inoculation and bagging

Fully mix the liquid fermented seed liquid in the ultra-clean workbench to ensure the consistency of the quality of the seed liquid. After the fungus bag is cooled, open the fungus bag, add 200mL Antrodia camphorata liquid fermented seed liquid into each bag, ensure that the seed liquid flows down along each surface of the wood section, seal it with a 18-23mm sponge silica gel plug, and tie it tightly with a rope. It ensures air permeability and effectively prevents bacterial contamination.

1.6 Management of mycelium growth period and fruiting period.

Place the inoculated fungus bag in a dark place with a temperature of 25°C-28°C for cultivation. The growth period of mycelium is about 6 months. Replenish water 5-7 times, and replenish 200-300mL of sterile water each time to ensure that the humidity during the growth of Antrodia camphorata is between 85% and 95%. After about 6 months, after the mycelium is covered with wood, place it in an environment where the daytime temperature is 25°C-28°C, the night temperature is 18°C-20°C, and the temperature difference between day and night is about 8°C. Waiting for fruiting, the growth of fruiting bodies is relatively slow, and this time is about 3-4 months. During the fruiting period, the humidity and ventilation of the growth environment of Antrodia camphorata should still be ensured. The CO2 concentration is 1.0%-1.5%.

2. Results and Analysis

The fruiting body of Antrodia camphorata cultivated by the method is closely attached to the surface of the wood, corky to woody, extremely hard in texture, flat and straight at the edge, rich in camphor fragrance, sessile, and plate-shaped in appearance. Bacterial pores on the surface of the fruiting bodies are large, numerous and dense. The fruiting body grows very slowly, and the surface of the fruiting body is bright red in the early stage of growth (Figure 1), and the color is yellowish brown after maturity (Figure 2). The biotransformation rate is about 15%.

Detection of polysaccharide content in experimental example

1. Determination of the growth of mycelia and the content of extracellular and extracellular crude polysaccharides during the liquid fermentation of Antrodia camphorata

1.1 Mycelium growth curve during liquid fermentation of Antrodia camphorata

After inoculation of the secondary seed liquid, the polysaccharide content and the dry weight of the bacteria balls in the liquid fermentation liquid of Antrodia camphorata were measured every 24 hours and the data were recorded. In this way, the relationship between mycelium dry weight and polysaccharide content is sought, and the optimal fermentation time is determined simultaneously (as shown in Figure 3).

1.2 Extraction of extracellular (fermentation broth) crude polysaccharides during the fermentation of Antrodia camphorata

Filter the fermented liquid with eight layers of gauze to collect the supernatant, heat and concentrate at 90°C to 1/5 of the original liquid, then centrifuge at 5000rpm for 5 minutes, collect the supernatant, add 3 times the volume of 95% alcohol, and precipitate at 4°C for 12 hours. The flocculent precipitate was precipitated, and the precipitate was washed several times with anhydrous acetone, acetone, and ether, and then vacuum-dried, and then placed in a phosphorus pentoxide dryer for further drying to obtain a dry product of extracellular crude polysaccharide, which is the Exopolysaccharides.

1.3 Extraction of intracellular (mycobacterium) crude polysaccharides in Antrodia camphorata liquid fermentation broth

Filter the fermentation broth with eight layers of gauze to collect mycelium, dry it at 60°C, take the dried mycelium and grind it with a mortar, put the mycelium in 90°C hot water according to the ratio of 1g mycelium to 50mL water Extract for 2 hours, filter out the extract, then add new water to the precipitate and extract for another 2 hours, then combine the extracts, heat and concentrate at 90°C to 1/5 of the original solution, and then follow the steps of exopolysaccharides to obtain cell Inner crude polysaccharides. The content of extracellular and extracellular crude polysaccharides is shown in Figure 4.

As shown in Figure 1, with the prolongation of fermentation time, the change trend of the mycelial biomass of Antrodia camphorata grows faster from the 4th day to the 8th day, and the biomass reaches the maximum value. The biomass decreased obviously at the beginning.

Combining Figure 1 and Figure 2, it can be found that the amount of intracellular crude polysaccharide is directly related to the yield of mycelium, and it changes with the change of mycelium weight. After 8 days, the amount of mycelium decreases accordingly. , but the reduction was faster, mainly because of the autolysis of mycelia, some polysaccharides were dissolved into the fermentation broth; and the amount of exopolysaccharides was also closely related to the yield of mycelia. When microorganisms grow, they can secrete an enzyme outside the cells, which can link monosaccharides and polysaccharides to produce glucans and form their own cell walls. This may be the reason for the rapid increase of exopolysaccharides during the vigorous growth of mycelium. After 8 days, although the weight of the mycelium decreased, the exopolysaccharide still increased, mainly because of the dissolution of the polysaccharide in the mycelium, but the increase was limited after that, and it began to decline soon, so it can be concluded that: The size of the production of Antrodia camphorata mycelium can reflect the height of polysaccharide production, and the following culture experiments can evaluate the production of polysaccharides with the number of mycelia production; in order to obtain higher production of mycelia and polysaccharides, the liquid fermentation of Antrodia camphorata (secondary stage) Seeds) are suitable for 8-9d cultivation.

2. Determination of polysaccharide content in fruiting body, mycelia and fermentation broth of Antrodia camphorata cultivation

2.1 Extraction of Polysaccharides from Fruiting Bodies, Mycelia and Fermentation Broth of Antrodia Camphorata Cultivation

Extraction methods of crude polysaccharides in Antrodia camphorata fermentation broth and crude polysaccharides in mycelia are as in 1.2 and 1.3, drying the extracted crude polysaccharides, dissolving them in water, centrifuging to get the supernatant for the determination of polysaccharide content; Antrodia camphorata cultivars The entity is extracted in 90°C hot water for 2 hours at the ratio of 1g fruiting body to 50mL water, and the supernatant obtained by centrifugation is directly used for the determination of polysaccharide content.

2.2 Determination of polysaccharide content in fruiting body, mycelium and fermentation broth of Antrodia camphorata cultivation

2.2.1 Anthrone-sulfuric acid colorimetric method for determination of total sugar content in fruiting body, mycelia and fermentation broth of Antrodia camphorata cultivation

(1) Principle: Strong acid can dehydrate sugars to produce furfural or hydroxymethylfurfural, and the product is dehydrated and condensed with anthrone to form furfural derivatives, which are blue-green in color and have maximum absorption at 620nm.

(2) Reagents:

① Glucose standard solution: 100μg/mL

Accurately weigh 100 mg of analytically pure glucose that was baked at 80°C to constant weight, put it in a small beaker, add a small amount of distilled water to dissolve it, and set the volume to 1 L for later use.

② Anthrone: Dissolve 0.2g of anthrone in 100mL of concentrated sulfuric acid and prepare it for immediate use

(3) Making of glucose standard curve:

Take 6 test tubes and add reagents according to Table 1:

Table 1

After adding the reagents, shake immediately, cool with ice water, and cover the mouth of the tube with a silica gel stopper to prevent evaporation. Accurately boil it for 10 minutes, take it out, cool it with running water, and let it stand at room temperature for 10 minutes. Use the No. 0 tube as a control, and measure the color at the maximum absorption wavelength of 620nm. Take the standard glucose content (μg) as the abscissa and the absorbance value as the ordinate to make a standard curve (see Figure 3).

(4) Determination of samples: Dissolve the extracted intracellular polysaccharides with distilled water and set the volume to 100 mL, dilute the exopolysaccharides to 10,000 times the original, draw 1 mL of the sample solution into a large test tube, and the subsequent operations are the same as the preparation of the standard curve.

2.2.23, 5-Dinitrosalicylic acid colorimetric method to determine the content of reducing sugar in fruiting body, mycelia and fermentation broth of Antrodia camphorata cultivation

(1) Principle: Under alkaline conditions, reducing sugar and 3,5-dinitrosalicylic acid are heated together, and 3,5-dinitrosalicylic acid is reduced to 3-amino-5-nitrosalicylic acid Acid (brown red substance), reducing sugar is oxidized to sugar acid and other products. Measure the absorbance value of the brown-red substance at the maximum absorption wavelength, check the standard curve and calculate, then the content of reducing sugar can be obtained.

(2) Reagents

①1mg/mL glucose standard solution

Accurately weigh 100 mg of analytically pure glucose that was baked at 80°C to constant weight, put it in a small beaker, add a small amount of distilled water to dissolve it, transfer it to a 100mL volumetric flask, dilute to 100mL with distilled water, mix well, and store in a refrigerator at 4°C for later use .

②3,5-Dinitrosalicylic acid (DNS) reagent

Add 6.3g of DNS and 262mL of 2M NaOH solution to 500mL of hot aqueous solution containing 185g of potassium sodium tartrate, add 5g of crystalline phenol and 5g of anhydrous sodium sulfite, stir to dissolve, add distilled water to 1000mL after cooling, and store in a brown bottle spare.

(3) Make a glucose standard curve:

Take 7 test tubes and add reagents according to Table 2:

Table 2

After adding the reagents, shake well and heat in a boiling water bath for 5 minutes, take it out and immediately cool to room temperature, dilute to 25mL with distilled water, and mix well. Colorimetric at the maximum absorption wavelength of 540nm. With the glucose content (mg) as the abscissa and the absorbance value as the ordinate, a standard curve was made. (See Figure 4).

(4) Determination of samples: Dissolve the extracted extracellular polysaccharides with distilled water and set the volume to 100mL, pipette 2mL of sample solution into a large test tube, and the subsequent operations are the same as the preparation of the standard curve.

2.2.3 Polysaccharide calculation formula: polysaccharide content = total sugar content - reducing sugar content

table 3

As shown in the above table 3, the measured polysaccharide content of the fruiting body of Antrodia camphorata cultivated in the micro-air-permeable segment is 20.4%, which is not much different from the reported polysaccharide content of 23.8% in the fruiting body of Antrodia camphorata.

3. Research on the anti-tumor and immunoregulatory effects of polysaccharides from fruiting bodies of Antrodia camphorata cultivation

3.1 Experimental materials

3.1.1 Experimental equipment

Inverted microscope, microscope, 96-well culture plate, 1ml gavage device, 5ml syringe, 1ml syringe, 10ml centrifuge tube, 5ml centrifuge tube, long-necked pipette, scissors, petri dish, counter, counting plate, carbon dioxide incubator (manufactured by MMM Group Galaxy B Scotland), microplate reader (model ELx800): autoclave, ultra-clean bench

3.1.2 Reagents

FBS (CLARK), streptomycin and penicillin double antibody solution (Hyclone), RPMI-1640 (GIBCO), thioglycolate medium, heparin, Griess reagent, LPS, ConA, DMSO, MTT staining solution, D-hanks solution , neutral red, sodium bicarbonate, trypan blue

3.1.3 Drugs

1. Cyclophosphamide (Clophosphamide), Jiangsu Hengrui Medicine Co., Ltd., specification 200mg/bottle (batch number 07032721).

2. Antrodia camphorata fruiting body polysaccharides: The fruiting bodies of Antrodia camphorata successfully cultivated by the micro-ventilation section wood cultivation method in our laboratory were dried and crushed into powder, extracted three times in hot water, and precipitated with three times the volume of 95% alcohol After obtaining Antrodia camphorata polysaccharide, the extraction rate of crude polysaccharide is 2.43% (polysaccharide weight/fruiting body dry weight)

3.1.4 Cells

YAC-1 (mouse lymphoma cells) cell line was purchased from Shanghai Chinese Academy of Sciences Cell Bank, with RPMI-1640 containing 10% FBS (CLARK), final concentration of 100ug/ml streptomycin and penicillin double antibody solution (Hyclone) (GIBCO) culture medium was continuously passaged.

3.1.5 Animals and tumor strains

KM mice, SPF grade, weighing 22±2g, male and female, were purchased from Qingdao Drug Quarantine Institute, Shandong Province; the tumor strains were mouse ascites-type sarcoma cell line S180 and ascites-type liver cancer cell line H22, purchased from Shandong Province Medicine Institute of Materia Medica, Academy of Sciences.

3.2 Experimental method

3.2.1 Establishment of transplanted tumor models in mice with sarcoma S180 and liver cancer H22

According to the research method of transplanted tumors, 0.2 mL of S180 sarcoma cell and H22 liver cancer cell suspension were inoculated in groups in the right forelimb armpit of each mouse, and the inoculation concentration was 2×106 cells/mL.

3.2.2 Test conditions

After inoculation, the mice were placed in a room temperature of 20-23°C, in a well-ventilated and well-lit environment. The mice were free to eat and drink. The drinking bottle was changed once a day, and the litter was changed once every three days. The pellet feed was provided by the New Drug Evaluation Center of Shandong University. .

3.2.3 Grouping and administration

3.2.3.1 Grouping:

The mice were divided into S180 group and H22 group after weighing, and each group was further divided into 5 groups, namely normal saline group (NS), cyclophosphamide group (CTX), low-dose group, middle-dose group, high-dose group Group, 10 in each group.

3.2.3.2 Administration method:

After 24 hours of inoculation, continuous oral gavage was carried out according to the above groups for 10 days, once a day at 9:00am, the volume of gavage was 20mL/kg, and the dosage (mg/kg·d) was as follows in Table 4:

Table 4: Dosage of 180 group and H22 group (mg/kg·d)

3.2.4 Determination of immune indicators

3.2.4.1 Determination of NO generation

(1) Preparation of macrophages: In the last 4 days of administration, each mouse was injected intraperitoneally with 2 mL of macrophage inducer (3% thioglycolate medium), and the mice were sacrificed, and 5 mL of heparin-containing cold D-hanks The peritoneal cavity was lavaged with fluid, the peritoneal fluid containing macrophages was aspirated, and the macrophages were obtained by centrifugation.

(2) Add 2x106/mL 100uL of the obtained macrophages to a 96-well culture plate, and use 1 μg/mL LPS as a positive control. After 20 hours of culture, take 50uL of the culture solution and mix it with the same volume of Griess reagent, and store it at room temperature in the dark. , cultivated for 5min. Then ELISA was used to measure the absorbance at 540nm.

3.2.4.2 Phagocytosis of macrophages

Adjust the obtained macrophages to 5×105/mL, take 100ul in a 96-well culture plate, culture in a 5% CO2 incubator at 37°C for 24 hours, then add 0.1% neutral red 100μL to each well of the culture plate , and then placed in the incubator for 30 minutes, washed the plate with warm D-hanks solution to wash away the extracellular neutral red, then added 200 μL of cell lysate to each well of the culture plate, placed it at 4°C overnight, and microplate reader 490nm Measure the OD value.

3.2.4.3 Spleen NK cell activity detection

(1) Preparation of effector cells: kill the mice by dislocation of the cervical spine, aseptically remove the spleen, wash it with D-Hanks solution, grind the spleen to make a single-cell suspension, filter through a 200-mesh steel wire mesh, and wash 3 times with D-Hanks solution , using RPMI-1640 culture medium to make cell suspension, trypan blue staining and counting the number of viable cells above 95%, and using RPMI-1640 complete culture medium containing 10% FBS to adjust the cell concentration to 2×107/mL.

(2) Preparation of target cells: The YAC-1 lymphoma cell line continuously subcultured was used as the target cells, the number of viable cells was above 95%, and the cell concentration was adjusted to 2×10 5 cells/ml.

(3) Determination of NK cell activity: Take 96-well culture plate and add 100 μL of effector cells and target cells, the effect-to-target ratio is 100:1. Separately set effector cells and target cells as a control. After culturing for 4 hours at 37°C and 5% CO2, add 20 μL of 5g/LMTT staining solution, cultured for another 2 hours, added DMSO digestion solution, measured the 570nm OD value of each well the next day, and calculated the NK cytotoxic activity according to the formula.

NK cell activity %={[OD mean value of target cell control group - (OD mean value of experimental group - effector cell mean value)]/OD mean value of target cell control group} × 100%

3.2.4.4 Determination of lymphocyte proliferation rate

Take a 96-well culture plate, add 100 μl of splenocytes with a concentration of 5×106 cells/ml to each well, add 10 μl of ConA(), add RPMI-1640 culture solution to the control well, and culture at 37°C and 5% CO2 for 72 hours. Add MTT staining solution, add DMSO digestion solution after culturing for another 4 hours, and measure the 570nm absorbance value OD with a microplate reader.

Lymphocyte proliferation rate (%)=(OD value of experimental well-OD value of control well)/OD value of control well×100%

3.2.5 Tissue processing:

The tumor tissue, spleen, thymus, and liver were aseptically collected, weighed after decontamination, and the tumor inhibition rate and the index of each organ were calculated according to the following formula to compare the effects of polysaccharides on immune organs. (Tumor inhibition rate ≥ 30% is judged to have a certain anticancer effect)

Tumor inhibition rate (%)=[average tumor weight of negative control group-average tumor weight of experimental group]/average tumor weight of control group×100%

Spleen index = spleen weight/body weight (mg/g)

Thymus index = thymus weight/body weight (mg/g)

Liver index = liver weight (g) × 100/body weight (g)

Note: The weighing method of immune organs is one of the methods for studying the body's immune status at present. It is generally believed that the decreased weight of immune organs is caused by immunosuppression, while the increased weight of immune organs is the performance of immune enhancement.

3.3 Results and analysis

3.3.1 Effects of Antrodia camphorata fruiting body polysaccharides on the daily activities of sarcoma S180 and liver cancer H22 tumor-bearing mice Mice respond quickly, and the back hair is bright and not easy to fall off. It can be seen that cyclophosphamide has adverse effects on the life of the mice, while the mice in the polysaccharide group live normally.

3.3.2 Inhibitory effect of polysaccharides from fruiting bodies of Antrodia camphorata cultivation on S180 transplanted solid tumors

Table 5: Inhibitory effect of polysaccharides from fruiting bodies of Antrodia camphorata cultivation on S180 transplanted solid tumors

Compared with normal saline negative control: ^** p<0.01

See Table 5 for comparisons of body weight changes and tumor inhibition rates before and after administration of mice in the normal saline group, cyclophosphamide group and polysaccharide group. Compared with the negative control of normal saline, cyclophosphamide, as a chemotherapeutic drug, has the most obvious tumor inhibitory effect on S180, reaching 70.54%, but it inhibits the increase of mouse body weight (p<0.01); Polysaccharides from fruiting body of Antrodia camphorata have good tumor inhibitory effect on S180 mice, and can promote the growth of mice. The tumor inhibition rate of low dose is 45.73%, and that of high dose is 50.38%. Especially, the tumor inhibition rate of medium dose reaches The result was 63.56%, far greater than the reported inhibition rate (58.8%) of the same dose of Antrodia camphorata mycelium polysaccharide on S180 sarcoma. It can be seen that polysaccharides from the cultivated fruiting body of Antrodia camphorata have a good inhibitory effect on S180, and it is not dose-dependent.

3.3.3 Effects of polysaccharides from fruiting body of Antrodia camphorata cultivation on liver index, spleen index and thymus index of S180 tumor-bearing mice

Table 6: Effects of Antrodia camphorata cultivated fruiting body polysaccharides on liver index, spleen index and thymus index in S180 tumor-bearing mice

Compared with normal saline negative control: ^** P<0.01, ^* P<0.5

See Table 6 for the comparison of liver index, spleen index and thymus index of S180 mice in normal saline group, cyclophosphamide group and polysaccharide group. Compared with the normal saline negative control, cyclophosphamide has a very obvious inhibitory effect on the immune organs of the spleen in mice, indicating that cyclophosphamide can reduce the immune ability of the body while inhibiting tumor growth, and the liver index has decreased (P<0.5 ), indicating that cyclophosphamide can have an adverse effect on the normal operation of the liver; while three doses of Antrodia camphorata polysaccharides exerted anti-tumor effects, the spleen index and thymus index of mice were increased to varying degrees, The improvement was most pronounced at high doses. It can be seen that the fruiting body polysaccharide of Antrodia camphorata can improve the immunity of the body. As the liver is a detoxification organ, the three doses of polysaccharides can promote the growth of the liver to a certain extent. The medium dose can significantly improve the liver index, and the high dose can significantly improve the liver index.

3.4 Effects of Antrodia camphorata cultivating fruiting body polysaccharides on the immune function of S180 tumor-bearing mice

3.4.1 Effects of polysaccharides from the fruiting body of Antrodia camphorata cultivation on the production of NO in peritoneal macrophages stimulated by LPS

Taking 5 treatments (mg/kg) as the abscissa and NO production as the ordinate, it can be seen from Figure 7 that after adding LPS (final concentration of 1 μg/ml) to stimulate peritoneal macrophages for 20 hours, the difference between different treatments and the control There was a very significant difference in NO release in normal saline group (NS). Among them, the release amount of NO in the medium-dose (PST 200mg/kg) group of Antrodia camphorata cultivated fruiting body polysaccharides was the highest at about 29uM, followed by the high-dose group (PST 400mg/kg) and the low-dose group (PST 100mg/kg). The release of NO in the cyclophosphamide group was the lowest, and it was significantly lower than that in the normal saline group. This shows that polysaccharides from the fruiting bodies of Antrodia camphorata can significantly improve the ability of mouse macrophages to release NO, while cyclophosphamide can significantly inhibit the ability of mouse macrophages to release NO. LPS is a kind of endotoxin and specific antigen, which can stimulate macrophages to produce inflammation, and NO can eliminate inflammation. The fruiting body polysaccharide of Antrodia camphorata can improve the ability of macrophages to release NO, which shows that the fruiting body polysaccharide of Antrodia camphorata cultivation can Improving the immune ability of mice against inflammation.

3.4.2 Phagocytosis of macrophages

Taking 5 treatments (mg/kg) as the abscissa and OD490 as the ordinate, it can be seen from Figure 8 that after the middle dose and high dose of polysaccharides act on mouse peritoneal macrophages, their phagocytosis of neutral red is comparable to that of the normal saline group. Compared with it, it has been significantly improved; low-dose polysaccharides also significantly improved the phagocytosis of peritoneal macrophages on neutral red; peritoneal macrophages of mice treated with cyclophosphamide significantly inhibited their phagocytosis. It can be seen that polysaccharides from the fruiting bodies of Antrodia camphorata can effectively activate the phagocytosis of phagocytes, and the phagocytosis of the high-dose group is the strongest (0.38).

3.4.3 Determination of NK cell lethality

Taking 5 treatments (mg/kg) as the abscissa and NK cell viability (%) as the ordinate, the effects of NK lethality isolated from spleens of different treatments are shown in FIG. 9 . NK is a natural killer cell, which can directly kill tumor cells and has direct significance in inhibiting tumor growth and metastasis. Compared with the normal saline group, the lethality of mice spleen NK treated by the three polysaccharide groups of low, medium and high polysaccharide groups all had extremely significant differences, among which the lethality of the medium dose group (PST 200) reached 69.78%, followed by high dose group, low dose group. The cyclophosphamide group significantly inhibited the lethality of NK, which was significantly different from the normal saline group, from 46.59% to 30.29%.

3.4.4 Proliferation of splenic lymphocytes

Taking 5 treatments (mg/kg) as the abscissa, and taking the lymphocyte proliferation rate (%) as the ordinate, ConA can promote cell mitosis. Effect, as shown in Figure 10: both the medium-dose and high-dose polysaccharide groups can significantly activate the proliferation of splenic lymphocytes, and the proliferation index of the middle-dose group reached 52.46%, and the high-dose group reached 45.09%. Cyclophosphamide group significantly inhibited the proliferation of spleen lymphocytes. What ConA stimulates is the proliferation of T lymphocytes, and the immunity mediated by T lymphocytes is cellular immunity in vivo. It can be seen that polysaccharides from fruiting bodies of Antrodia camphorata can improve the cellular immunity of S180 mice.

3.5 Inhibitory effect of polysaccharides from fruiting bodies of Antrodia camphorata cultivation on liver cancer H22

Table 7: Inhibitory effect of polysaccharides from fruiting bodies of Antrodia camphorata cultivation on liver cancer H22

Compared with normal saline negative control: ^** p<0.01

See Table 7 for comparisons of body weight changes and tumor inhibition rates of H22 mice in the normal saline group, cyclophosphamide group and polysaccharide group before and after administration. Compared with the normal saline negative control, cyclophosphamide, as a chemotherapeutic drug, has the most obvious tumor inhibitory effect on liver cancer H22, reaching 74.13%. However, it inhibits the increase of mouse body weight (p<0.01); each dose The fruiting body polysaccharides cultivated by Antrodia camphorata have good tumor-inhibitory effects on H22 mice with liver cancer, and can promote the growth of mice. Tumor rate reached 68.10%, showing no dose dependence.

Compared with relevant literature, the tumor inhibition rates of Antrodia camphorata liquid fermentation powder polysaccharide (100mg/kg/day and 200mg/kg/day) to liver cancer H22 were 34% and 52% respectively; The fruiting body powder of Antrodia camphorata has a tumor-inhibiting rate of 74.24% in mice bearing liver cancer H22 [2]; the fruiting body polysaccharides cultivated by Antrodia camphorata in this experiment have obvious advantages in inhibiting liver cancer H22.

3.6 Effects of Antrodia camphorata cultivating fruiting body polysaccharides on liver index, spleen index and thymus index in H22 mice

Table 8: Effects of polysaccharides from fruiting body of Antrodia camphorata cultivation on liver index, spleen index and thymus index in H22 mice

Compared with normal saline negative control: ^** P<0.01, ^* P<0.5

See Table 8 for the comparison of liver index, spleen index and thymus index in polysaccharide group, cyclophosphamide group, and normal saline group. Compared with the normal saline negative control, cyclophosphamide has a very obvious inhibitory effect on the immune organs of the spleen in mice, indicating that cyclophosphamide can reduce the immune ability of the body while inhibiting tumor growth, and the liver index has decreased (P<0.5 ), indicating that cyclophosphamide can adversely affect the normal functioning of the liver; while three doses of Antrodia camphorata cultivated fruiting body polysaccharides in this experiment exerted anti-tumor effects, they also had different effects on the spleen index and thymus index of mice. The degree of increase is related to the dose, and the increase of the spleen index is the most obvious in high doses. It can be seen that the cultivation of polysaccharides from the fruiting body of Antrodia camphorata can improve the immunity of the body. As the liver is a detoxification organ, the three doses of polysaccharides can promote the growth of the liver to a certain extent. The medium dose can significantly improve the liver index, and the high dose can significantly improve the liver index.

3.7 Effects of Antrodia camphorata cultivating fruiting body polysaccharides on the immune function of liver cancer H22 tumor-bearing mice

3.7.1 Effects of Polysaccharides from Fruiting Bodies of Antrodia Camphorata Cultivation on NO Production in Peritoneal Macrophages Stimulated by LPS

Taking 5 treatments (mg/kg) as the abscissa and NO production as the ordinate, it can be seen from Figure 11 that after adding LPS (final concentration of 1 μg/mL) to stimulate peritoneal macrophages for 20 hours, the difference between different treatments and the control There was a very significant difference in NO release in normal saline group (NS). The release amount of NO in the medium-dose (PST 200mg/kg) group of Antrodia camphorata cultivated fruiting body polysaccharide was about 31uM. The release of NO in the cyclophosphamide group was the lowest, and it was significantly lower than that in the normal saline group. This shows that polysaccharides from the fruiting bodies of Antrodia camphorata can significantly improve the ability of mouse macrophages to release NO, while cyclophosphamide can significantly inhibit the ability of mouse macrophages to release NO. LPS is a kind of endotoxin and specific antigen, which can stimulate macrophages to produce inflammation, and NO can eliminate inflammation. The fruiting body polysaccharide of Antrodia camphorata can improve the ability of macrophages to release NO, which shows that the fruiting body polysaccharide of Antrodia camphorata cultivation can Improving the immune ability of mice against inflammation.

3.7.2 Phagocytosis of macrophages

Taking 5 treatments (mg/kg) as the abscissa and OD490 as the ordinate, it can be seen from Figure 12 that after the middle dose and high dose of polysaccharides act on mouse peritoneal macrophages, their phagocytosis of neutral red is comparable to that of the normal saline group. Compared with it, it has been significantly improved; low-dose polysaccharides also significantly improved the phagocytosis of peritoneal macrophages on neutral red; peritoneal macrophages of mice treated with cyclophosphamide significantly inhibited their phagocytosis. It can be seen that polysaccharides from the fruiting body of Antrodia camphorata can effectively activate the phagocytosis of phagocytes, and the phagocytosis of the high-dose group is the strongest (0.36).

3.7.3 Determination of NK cell lethality

As shown in Fig. 13, 5 treatments (mg/kg) are taken as the abscissa, and NK cell viability (%) is taken as the ordinate. Compared with the normal saline group, the lethality of mice spleen NK treated by the three polysaccharide groups of low, medium and high polysaccharide groups all had extremely significant differences, among which the lethality of the medium dose group (PST 200) reached 69.78%, followed by high dose group, low dose group. Cyclophosphamide group significantly inhibited the lethality of NK, which was significantly different from that of normal saline group.

3.7.4 Proliferation of splenic lymphocytes

Taking the 5 treatments (mg/kg) as the abscissa and the lymphocyte proliferation rate (%) as the ordinate, the effect of Antrodia camphorata cultivation polysaccharides on the proliferation of spleen lymphocytes stimulated by ConA is shown in Figure 14: High-dose and high-dose polysaccharide groups can significantly activate the proliferation of splenic lymphocytes, and the proliferation index of the middle-dose group reaches 36.41%. Cyclophosphamide group significantly inhibited the proliferation of spleen lymphocytes. What ConA stimulates is the proliferation of T lymphocytes, and the immunity mediated by T lymphocytes is cellular immunity in vivo. It can be seen that the polysaccharides of fruiting bodies cultivated by Antrodia camphorata can improve the cellular immunity of H22 mice with liver cancer.

4 Conclusion

The anti-tumor ability of Antrodia polysaccharides does not directly affect tumor cells, but stimulates macrophages, T lymphocytes, B lymphocytes, and natural killer cells to enhance immune function and achieve anti-tumor functions.

The polysaccharide in the present invention can significantly improve the phagocytosis of macrophages, the release of NO, the proliferation of activated T lymphocytes, and the cytotoxicity of NK cells. The improvement of these immune indicators shows that improving the immune function of immune cells is the key to the cultivation of Antrodia camphorata. An important mechanism of action of fruiting body polysaccharides on tumors. Kanazawa M, Zhang H, etc. also showed that the anticancer effect of polysaccharides is achieved by improving the immune function of the body.

The present invention shows that the polysaccharide cultivated fruiting bodies of Antrodia camphorata can enhance the cellular immune function of mice regardless of the dose. Based on the above immune indexes, the middle dose group, namely 200mg/kg·d, had the most obvious effect, showing a non-dose dependence. This study is of great significance and lays a theoretical foundation for the further research and application of polysaccharides from the cultivated fruiting bodies of Antrodia camphorata. The successful cultivation of the fruiting bodies of Antrodia camphorata has enriched the resources of Antrodia camphorata.

Claims (6)

1. the method for the segment wood cultivated camphor tree sesame of little ventilation comprises the steps:

(1) the camphor tree sesame makes liquid spawn through liquid fermentation, and (2) camphorwood section is handled: the camphorwood section is soaked in the water, and making its water content is 50%-70%; Put it into again in the container that seals with the sponge silica gel plug; Sterilization, sponge silica gel plug on (3) inoculation bonnet, cultivate (4): A) in 25 ℃-28 ℃ of temperature; Humidity 85%-95%, the lucifuge place is cultured to the camphorwood surface and covers with mycelium; B) being placed on daytime temperature again is 25 ℃-28 ℃, 18 ℃-20 ℃ of nocturnal temperatures, and day and night the temperature difference is about 8 ℃, humidity 85%-95%, CO ₂Low-light is turned out fruit body in the environment of concentration 1.0%-1.5%, and intensity of illumination was 50-150Lux between said low-light was cultivated in a few days, and night, intensity of illumination was 0.001-0.02Lux,

The preparation of said liquid spawn comprises the steps:

A) actication of culture: cryopreserved camphor tree sesame piece is inserted mother culture media in 28 ℃ of constant temperature lucifuge slant culture 7-10 days;

B) primary seed solution: have access in the container of liquid fermentation medium a) cultivating the camphor tree sesame bacterial classification that obtains, shaking table 180rpm/min cultivated 7-12 days for 28 ℃;

C) secondary seed solution: free of contamination primary seed solution is inoculated in the container of liquid fermentation medium, and shaking table 180rpm/min cultivates for 28 ℃ and promptly obtained liquid spawn in 7-12 days,

Said mother culture media is glucose 2%, banaina 0.5%, yeast extract 0.5%, peptone 0.5%, MgSO ₄0.3%, KH ₂PO ₄0.3%, citric acid 0.05%, (NH ₄) ₂SO ₄0.05%, agar 2%, camphorwood immersion liquid, pH4.5;

Said liquid fermentation medium is: glucose 2%, banaina 0.5%, yeast extract 0.5%, peptone 0.5%, MgSO ₄0.3%, KH ₂PO ₄0.3%, citric acid 0.05%, (NH ₄) ₂SO ₄0.05%, camphorwood immersion liquid, pH4.5,

Said camphorwood immersion liquid refers to get camphorwood chip 500g, adds entry 5000mL, and boiling is until 1/10 of the simmer down to original volume, and gained liquid is the camphorwood immersion liquid, and consumption is that every 100mL liquid fermentation medium adds the immersion liquid of 50mL camphorwood.

Said camphorwood section is handled the tangent plane and/or the side that also are included in the camphorwood section and is vertically punched,

Said inoculation refers to liquid spawn is placed liquid spawn with camphorwood again after under each surface current of camphorwood section,

Said steps A) incubation time is 5-6 month, step B) incubation time is 3-4 month.

2. method according to claim 1, said pore size distribution are the plum blossom shape, hole depth 5-10cm.

3. method according to claim 1, said sterilization are 121 ℃, 90 minutes, naturally cool to room temperature again.

4. method according to claim 1, said container are polypropylene bacterium bag.

5. method according to claim 1, said humidity are to control through constantly adding water.

6. inoculum concentration is 10% when method according to claim 1, the cultivation of said secondary seed solution.

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