CN113796260B - Poria (Wolfiporia cocos) YX1, and culture medium and cultivation method thereof - Google Patents

Abstract

The invention discloses a Poria cocos (Wolfiporacocos) YX1, which is preserved in China center for type culture Collection with the preservation number of CCTCCNO: m2021434. The invention also discloses a stock culture medium and a mother culture medium of the poria cocos (wolfiporicoocs) YX 1. The invention also discloses a cultivation method of the poria cocos (wolfiporicoocs) YX 1. The poria cocos (wolfiporiaccos) YX1 screened by the method has stable economic characters, high yield, high quality and stable genetic genes; and a high-yield and stable-production culture medium of Poria cocos (Wolfiporacocos) YX1 is screened out; and provides a scientific and practical cultivation method.

Description

A kind of Poria cocos (Wolfiporia cocos) YX1 and its culture medium and cultivation method

technical field

The present invention relates to the technical field of Poria cocos, in particular to a kind of Poria cocos (Wolfiporia cocos) YX1 and its culture medium and cultivation method.

Background technique

The protection of wild pine wood decay fungus germplasm resources and the stability of genetic information transmission are the raw materials to be used in breeding work, including wild and cultivated species or strains. At present, the research on pine wood decay fungus is mostly focused on pharmacological function, processing technology, polysaccharide, metabolism, etc., while the improved seeds, medium, raw materials, cultivation methods and cultivation time used in the cultivation of pine wood decay fungus, especially for pine wood decay fungus. The research on germplasm resources and raw material substitution of pine wood decay fungi is lagging behind. This has also become the main factor restricting the sustainable, rapid and healthy development of my country's pine wood decay fungus industry. Poria cocos in pine wood decaying fungi has nutritional and nourishing value. Poria cocos is often eaten and can soothe the nerves and live a long life. It is one of the traditional Chinese medicinal materials in my country. At the same time, Poria is also an important pine wood decaying fungus. The production and sales volume of Poria cocos in China is more than 17,000 to 30,000 tons every year. The sales of Poria cocos in Yuexi, Anhui ranks first in the country. The products are sold at home and abroad. With the rapid rise and development of the Poria cocos industry, the planting area continues to expand.

At present, on the whole, due to the weak collection and protection of wild resources, some precious mycoplasma resources cannot be saved, and wild Poria cocos is almost extinct. The traditional Poria cocos cultivation method uses masson pine wood segments as the main raw material. With the rapid rise and development of the Poria cocos industry, the amount of pine wood segments has increased sharply, a large number of pine trees have been felled, and the price of pine wood segments is high, and the production cost of Poria cocos is relatively high. In addition, the traditional Poria cocos cultivation method using masson pine wood segments as the main raw material will limit the cultivation method and cultivation time of Poria cocos. Miscellaneous bacteria. There is an urgent need for a high-yield and stable-yielding cultivated variety, a culture medium, and a cultivation method to solve the problems such as long-term unstable quality of Poria cocos and insufficient supply of raw materials.

SUMMARY OF THE INVENTION

Based on the technical problems existing in the background, the present invention proposes a Poria cocos (Wolfiporia cocos) YX1 and its culture medium and cultivation method. The present invention selects a Poria cocos (Wolfiporia cocos) YX1, and develops Poria cocos germplasm resources for it. The identification and analysis of its kinship and genetic diversity showed that Wolfiporia cocos YX1 has good economic characters, high yield and high quality, and stable genetic genes; Finally, a high-yield and stable-yielding seed medium was selected; it also provided a scientific and practical cultivation method, changed the traditional planting mode, and solved many problems such as traditional cultivation with masson pine wood segments as the main raw material, limited cultivation methods and cultivation time, and solved the problem of Poria cocos. The long-term quality of fungi is unstable and the supply of raw materials is insufficient.

The present invention proposes a kind of Poria cocos (Wolfiporia cocos) YX1, which is deposited in the China Type Culture Collection Center, and its deposit number is CCTCC NO: M 2021434, and the address of the China Type Culture Collection Center is Wuhan University in Wuhan, China, where the deposit is Date is April 22, 2021.

The present invention also proposes the original seed medium of the above-mentioned Wolfiporia cocos YX1, the raw materials by weight percentage include: main material ≥ 60%, auxiliary material 0-38%, sucrose 0.5-2%, gypsum 0.5-1%, 0-1% of natural resin, 0-0.1% of potassium dihydrogen phosphate, 0-0.0002% of vitamin B, 0-0.1% of grammylon, wherein the total weight percentage of each raw material is 100%, and the main materials are sawdust, pine branches , a mixture of at least one of pine needles and pine cones with pine chips or pine chips.

Preferably, the auxiliary material is at least one of rice bran, wheat bran, corncob, and cottonseed husk.

Preferably, the natural resin is: at least one of mastic gum resin, shellac, rosin resin, and amber.

Preferably, the weight percentage of the main material is greater than or equal to 70%.

Preferably, the mixing humidity of the stock medium is 50-60%.

The above-mentioned mixing humidity refers to the water content of the medium after adding water to the medium and mixing.

The pine trees of the above-mentioned "pine branches, pine needles, pine cones, and pine sawdust" can be masson pine, Simao pine, red pine, Huangshan pine, Huashan pine, and five-needle pine.

The original seed culture medium of the above-mentioned Wolfiporia cocos YX1 can also be added with vitamin C, anti-mildew and phytophthora additives (such as gram mould king, etc.), food mother raw tablets, etc.; gram mould king, food mother raw tablets can be purchased from the market have to.

For example, the stock medium of the above-mentioned Wolfiporia cocos YX1 can be:

①The raw materials by weight percentage include: 60-80% of pine sawdust, 18-37% of wheat bran, 1-2% of sucrose, and 0.5-1% of gypsum, wherein the total weight of each raw material is 100%;

②The raw materials include by weight percentage: 60-80% of pine sawdust, 18-37% of miscellaneous sawdust, 1-2% of sucrose, and 0.5-1% of gypsum, wherein the total weight of each raw material is 100%;

③The raw materials by weight percentage include: 50-60% of pine branches, 10-33% of pine sawdust, 5-15% of rice bran, 5-15% of wheat bran, 3-8% of corncob, 0.5-1% of sucrose, 0.5-1% of gypsum 1%, wherein the total weight percentage of each raw material is 100%;

④ Its raw materials include by weight percentage: 10-20% of pine sawdust, 46-58% of pine needles, 5-15% of rice bran, 5-15% of wheat bran, 5-10% of corncob, 0.5-1% of sucrose, 0.5-1% of gypsum 1%, wherein the total weight percentage of each raw material is 100%;

⑤The raw materials include by weight percentage: 50-78% of pine sawdust, 5-10% of pine needles, 5-10% of pine cones, 5-20% of wheat bran, 5-10% of corncob, 0.5-1% of sucrose, 0.5% of gypsum -1%, wherein the total weight percentage of each raw material is 100%;

⑥The raw materials include: 65-78% of pine sawdust, 20-31% of cottonseed husk, 1-2% of sucrose, 0.5-1% of gypsum, 0.1-1% of rosin resin, 0-0.1% of potassium dihydrogen phosphate, Vitamin B10-0.0002%, grammylon 0.05-0.1%, wherein the total weight percentage of each raw material is 100%.

The present invention also proposes the above-mentioned female culture medium of Wolfiporia cocos YX1, the raw material of which is: 1000 mL of pH=5-6 aqueous solution contains: 150-200 g of potato, 20-30 g of glucose, 15-20 g of agar powder, Potassium dihydrogen phosphate 0-1g, magnesium sulfate 0-0.5g;

Alternatively, 1000 mL of pH=5-6 aqueous solution contains: 20-30 g of glucose, 10-15 g of peptone, 15-20 g of agar powder, 0-1 g of potassium dihydrogen phosphate, and 0-0.5 g of magnesium sulfate.

The water in the above-mentioned parent seed medium can be sterile water or distilled water.

The parent seed refers to the pure mycelium obtained by artificial culture from spores, fruit bodies, sclerotia or mycelium within the substrate, and the culture medium used for the culture is the parent seed medium.

The stock seed refers to the mycelium grown on the stock medium by inoculating the parent seed.

The present invention also proposes the cultivation method of above-mentioned Poria cocos (Wolfiporia cocos) YX1, comprises the steps:

S1, by Poria cocos (Wolfiporia cocos) YX1 successively through mother seed medium, original seed medium culture to obtain Poria cocos (Wolfiporia cocos) YX1 original seed;

S2. Put the fungus bag containing the original species of Wolfiporia cocos YX1 on one end of the pine segment, then seal the fungus bag and fix it on the pine segment, and put the fungus bag on the other end of the pine segment and keep it open. , adjust the moisture content of the pine wood segment to 40-50%, cultivate at 28-32°C for 40-50 days, and then bury the pine wood segment in the cellar to grow ling;

Wherein, the parent seed medium is the parent seed medium of the above-mentioned Poria cocos (Wolfiporia cocos) YX1, and the original seed medium is cultured as the original seed medium of the above-mentioned Wolfiporia cocos (Wolfiporia cocos) YX1.

Preferably, in S2, the empty bacteria bag is a bacteria bag with both ends open.

Preferably, in S2, at the end of the pine wood segment of the empty mushroom bag, the empty mushroom bag extends 7-8 cm from the end of the pine wood segment.

Preferably, in S2, the middle position of the pine wood segment is not covered with a fungus bag.

In the above-mentioned S2, the size of the bacterial bag can be selected according to the volume of the pine wood section, so that one end of the pine wood section is sleeved with the bacterial bag containing the original seed and the original seed medium; the other end of the pine wood section is covered with an empty bacterial bag with both ends open , and fasten one end of the empty fungus bag to the pine wood section, the other end of the empty fungus bag is kept open, and the middle position of the pine wood segment is not covered with the fungus bag.

In above-mentioned S1, S2, carry out original seed culture in the bacterium bag that original seed culture medium is housed, to Poria cocos (Wolfiporia cocos) YX1 original seed mycelium overgrown bacterial bag is to obtain Poria cocos (Wolfiporia cocos) YX1 original seed; Then the fungus bag of the above-mentioned original hyphae of Wolfiporia cocos (Wolfiporia cocos) YX1 is covered on one end of the pine segment, and the step in S2 is continued; generally, 350-450g of Wolfiporia cocos YX1 is used for every 6kg of pine segment. The bag of the original mycelium.

In the above S2, the weight of the pine wood segment is preferably more than 6kg, and the diameter of the pine wood segment can be 8cm, 9cm, 10cm, 20cm and the like.

The material of the bacteria bag can be polypropylene, polyethylene and the like.

The above-mentioned mother seed medium and original seed medium are all sterilized; the above-mentioned bacterial bags are all sterilized.

Beneficial effects:

1. The present invention provides a kind of Poria cocos (Wolfiporia cocos) YX1 with high yield and stable yield, which enriches the germplasm resources of Poria cocos, its hereditary traits are stable, has the advantage of stable yield, and the quality of Poria cocos is good, the structure is regular, and High yield and high yield; and by screening out suitable parent seed medium and original seed medium, the yield of Poria cocos can be improved and maintained in good quality, and the output can be kept stable; the main materials of the original seed medium are weeds, pine branches and pine needles. , pine cone, pine sawdust, the raw materials are cheap, which can reduce the cost of Poria cultivation;

2. The cultivation method of inoculating the original seed at one end of the pine segment and covering the open empty fungus bag at the other end (referred to as the cultivation method of inoculating one end of the pine segment for short) solves the problem that the traditional cultivation mode is easy to contaminate the miscellaneous bacteria, and the structure is regular and Poria cocos Good quality, high yield, avoid bacterial contamination, and easy management, can realize factory cultivation;

3. In addition, traditional cultivation mode uses sclerotium inoculation, inoculates and cultivates in the lower pit when the weather is mild in April-May every year, and the inoculation amount is larger and the inoculation time is limited; and the present invention uses the bacterium bag that the original seed is housed to inoculate , after inoculation, cultivate it for a period of time at a suitable temperature to promote the growth of Poria cocos mycelium, and then cultivate it in the cellar. The inoculation period is not limited by the climate and can be carried out indoors, which is suitable for unified management of the factory, and can be inoculated in advance; Promoting the growth of Poria cocos mycelium can reduce the amount of inoculum; the invention solves many problems such as long-term unstable quality of the traditional Chinese medicinal material Poria fungus and insufficient supply of raw materials.

Description of drawings

Fig. 1 is the culturing characteristic picture of Poria cocos (Wolfiporia cocos) YX1 mycelium, wherein, a is the front of the petri dish, b is the back of the petri dish, c is the picture of 10 times of objective lens, and d is the picture of 20 times of objective lens.

Fig. 2 is a genetic phylogenetic analysis diagram of different strains of Wolfiporia cocos YX1, wherein, * represents Wolfiporia cocos YX1.

Figure 3 is a picture during the cultivation process of Example 5.

4 is a picture of the sclerotia of Poria cocos in Example 5.

FIG. 5 is a picture of the sclerotia of Poria cocos in Example 6. FIG.

Fig. 6 is the picture in the cultivation process of embodiment 7, wherein, a is the pine wood section that one end is inoculated with original seed and seals the fungus bag, and b is the pine wood segment that both ends are covered with fungus bag.

FIG. 7 is a picture of Example 5 when the ling is formed.

FIG. 8 is a picture of Example 7 when the ling is formed.

9 is a picture of the sclerotia of Poria cocos in Example 7.

Detailed ways

Hereinafter, the technical solutions of the present invention will be described in detail through specific embodiments.

Example 1

Screening of Wolfiporia cocos YX1

From the pine forest area of Chingzhai Village, Zhongguan Town, Yuexi County, Anqing City, Anhui Province (30°85'N, 116°35'E), under the decayed pine stump, the sclerotium dormant of the brown rot fungus Poria cocos was collected, and the The mycelium was then inoculated into the mother seed medium of different formulas in the ultra-clean workbench, and cultivated to observe the growth of Poria mycelium; then the well-selected mother seed was inoculated into the original seed medium of different formulations respectively. , carry out culture, observe the growth of Poria cocos mycelium, and screen good original species; then continue to transfer for 2 generations to obtain pure Poria cocos mycelium, and preserve the above-obtained Wolfiporia cocos YX1 in the Chinese typical culture collection in Wuhan Center (CCTCC), the deposit number is CCTCC NO: M 2021434.

Example 2

Poria cocos YX1 obtained in Example 1 was subjected to morphological identification of Poria mycelium.

1. Concrete method: inoculate the Poria cocos (Wolfiporia cocos) YX1 obtained in Example 1 in a petri dish containing a culture medium, cultivate for 4-5 days, and then insert the glass slide at a 45° tilt into the culture medium, and cultivate after 12h. Take out the glass slide, put a cover glass on the optical microscope and electron microscope, and observe the macroscopic and microscopic growth characteristics such as the formation, color, structure, and separation of Poria hyphae under the objective lens of 10 times or 20 times the field of view. As shown in Figure 1, Figure 1 is a picture of the culture characteristics of the mycelium of Wolfiporia cocos YX1, wherein a is the front of the petri dish, b is the back of the petri dish, c is a picture of 10 times the objective lens, and d is the objective lens 20 times the picture.

2. Results: The mycelium of Wolfiporia cocos YX1 includes monokaryotic mycelium and dikaryotic mycelium. The mycelium of Poria cocos is composed of many branched hyphae, and the hyphae are crisscrossed. Densely runs through the substrate or spreads and grows on the surface of the substrate, the mycelium is white villi and a lock-like joint phenomenon can be seen. There is a diaphragm in the hyphae that divides the hyphae into multiple linear cells, and the width of the hyphae is 2-5 μm. When cultured in a petri dish, colonies in the form of concentric rings are often seen, and the hyphae have strong vitality, covering the dish in 4-5 days.

3. Morphological and structural characteristics of Poria cocos in different developmental stages, showing three different morphological characteristics, namely mycelium, sclerotia and fruiting body. The above-mentioned Poria mycelium was further cultivated into sclerotia and fruiting bodies, and its macroscopic and microscopic growth characteristics were observed.

4. Results: The sclerotia of Wolfiporia cocos YX1 were spherical and ellipsoid; the size was uniform; it was a dormant body with hyphae aggregated. When fresh, it is soft and easy to tear, and the moisture content does not exceed 30%. After drying, it is hard and not easy to tear. Generally, the moisture content is less than 18%; There is a layer of shell-like skin, which is similar to the soil color when fresh, light brown, dark brown after drying, and the flesh of Poria is white.

The fruiting body of Wolfiporia cocos YX1 is produced on the surface of the sclerotium, and can also be seen on the older mycelium. Pale yellow. The pore tubes of the fruiting body are densely honeycomb-shaped, with a diameter of 0.5-2 μm and a depth of 2-3 μm. The orifices are polygonal to irregular, and become dentate when mature. The fruit layer is born on the surface of the inner wall of the pore tube, and is composed of a basidium without cysts. The basidium is cudgel-shaped, with a size of 19-22 μm×5-7 μm. Each mature basidium has 4 stalks, and each stalk produces 1 basidiospore. The spores are oblong or nearly cylindrical, sometimes slightly curved, with a crooked tip, the size is 6-11μm × 2.5-4μm, gray-white.

Example 3

Molecular biological identification was performed on the Wolfiporia cocos YX1 obtained in Example 1.

1. Extract the total genome DNA of Wolfiporia cocos YX1: put 10-30 mg of fresh hyphae or sclerotia of Wolfiporia cocos YX1 into a centrifuge tube, and fully grind it on a cell disrupter of the sample preparation system. The powder was crushed at 5000 rpm for 30 seconds, and repeated 2-3 times to obtain a broken wall sample; then, according to the CTAB method or the UNIQ-10 column fungal genome extraction kit (manufacturer: Sangon Bioengineering (Shanghai) Co., Ltd., No.: B511375), the total genomic DNA sample was extracted and stored in a -20°C refrigerator for future use;

2. Obtain the rDNA sequence, 28S sequence, and EF sequence of Wolfiporia cocos YX1: Take the total genome DNA obtained in step 1 as a template, and then carry out polymerase chain PCR amplification. The total system is 25 μl, using three pairs of The primers ITS1, ITS4; NL1, NL4; EF1F, EF1R were used for sequence analysis of Poria cocos. The PCR reaction program was pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 55 °C for 45 s, and extension at 72 °C for 2 min, a total of 30 cycles, and finally at 72 °C Prolonged for 10 min and terminated at 4°C; the target PCR product was detected by agarose gel electrophoresis, and the product was sequenced to obtain the rDNA sequence of Wolfiporia cocos YX1 of 1652bp, 28S sequence of 583bp, and EF sequence of 634bp.

The nucleotide sequence of the rDNA of the above-mentioned Wolfiporia cocos YX1 is shown in SEQ ID No.1, the nucleotide sequence of 28S is shown in SEQ ID No.2, and the nucleotide sequence of the transcription elongation factor EF is shown in SEQ ID No.2 ID No.3.

The components of the PCR reaction amplification system (25 μl) are shown in Table 1, and the sequences of primers ITS1, ITS4; NL1, NL4; EF1F, EF1R are shown in Table 2.

Table 1 PCR reaction amplification system (25μl)

reagent Dosage (μl) 10×PCR Buffer 2.5 Bsa 0.5 dNTP (2.5mM) 2.0 Primer 1 (10μmol/L) 0.5 Primer 2 (10μmol/L) 0.5 rTaq DNA polymerase (5U·μL) 0.2 template DNA 1.0

Table 2 Sequences of primers ITS1, ITS4; NL1, NL4; EF1F, EF1R

3. Submit the rDNA sequence, 28S sequence, and EF sequence of Wolfiporia cocos YX1 in GenBank of NCBI, BLAST the sequences of similar strains, and use ClustalW 2.0 software for comprehensive comparison, and finally use the software MEGA The phylogenetic tree was constructed by the NJ method in 6.1. The results are shown in Figure 2. Fig. 2 is a diagram showing the genetic phylogenetic analysis of different strains of Wolfiporia cocos YX1, wherein * represents Wolfiporia cocos YX1.

It can be seen from Figure 2: using MEGA6.1 software to construct a phylogenetic tree NJ clustering analysis, Poria cocos (Wolfiporia cocos) YX1 and 14 other strains of Poria cocos are the most closely, clustered together, Pachymahoelen, Wolfiporia cocos, Poria cocos and Macrohyporia cocos are synonymous strains of Poria cocos. Their genetic distances were also relatively close. Four ITS sequences of Poria cocos with synonyms clustered together, and the sequences of 3 Trichoderma, 1 Fungal sp.P6540 and 1 WolfiPoria cocos YX1 28S were clustered together and were the closest. There is also a cluster of 3 strains of Trichoderma koningii and WolfiPoria cocos YX1 EF transcription elongation factors. The ITS sequence, 28S sequence and EF sequence of Poria cocos are distinguished on the phylogenetic tree, but there are genetic connections among them, and the homology is more than 90%. It was confirmed that Wolfiporia cocos YX1 was a new strain.

Example 4 Screening medium

When cultivating and screening Wolfiporia cocos YX1, the mother seed medium and original seed medium of Wolfiporia cocos YX1 were screened. Cultivated yield of YX1.

1. Screening was carried out on the basis of the basic mother seed medium, and multiple formulas were obtained as shown in Table 3. With the same inoculum amount, all were incubated at 28°C for 5-7 days, and the results were shown in Table 3.

Table 3 The formula and culture results of the parent seed medium

As can be seen from Table 3: when the incubation time is the same, the mycelium growth speed is all faster than the basal medium in the enrichment mother seed medium of No. 1-3 and the peptone glucose medium; especially the enrichment mother seed of No. 1 Culture medium and peptone glucose medium, when the culture time is 120h, the growth rate of mycelium reaches more than 76.49±0.43mm. Compared with the basic medium, the culture time of these two mediums in the petri dish is shortened by 24h, and the growth rate is more than 2.1mm faster. .

2. take the pine tree appendages such as pine sawdust as ingredients, screen the original seed medium of Poria cocos (Wolfiporia cocos) YX1, obtain 6 groups of formulas as shown in table 4 (all prepare 100 catties), prepare the original seed by each group of formulas culture medium and inoculated.

The specific steps are: take each component and mix well, then add water and mix well, then press the original medium separately, pile it up for 10-12 hours, and then spray water until the material is squeezed by the fingers but no water is produced (control each group of original medium) The mixing humidity of the culture medium is 50-60%), then bagged and weighed, sterilized at 121 °C for more than 6 hours at high temperature and high pressure, and then cooled overnight, and transferred to Wolfiporia cocos YX1 early the next morning. Then control the temperature to 28-32 ℃, and cultivate until the mycelium is covered with the bacterial bag, and observe the culture results of each group as shown in Table 4.

Table 4 Formulation of stock medium and culture results

Remarks: The above natural resins are: at least one of mastic gum resin, shellac, rosin resin and amber.

It can be seen from Table 4: the shortest culture time of the first group is 24d, and the auxiliary material is wheat bran, which makes it easier to observe the growth of mycelium in appearance. Considering the price and cost, the formula of the first group is cheaper; the second group Only the main ingredient is pine sawdust, and no auxiliary materials are added. The growth rate of mycelium is slower than that of other groups, but the formula is simple and the cost is low. The main ingredients of group 3 are pine sawdust and pine branches. The culture time is longer, the longest culturing time is 38d, but in fact the mycelium has grown inside the pine branches, but the appearance is not easy to observe; in groups 4-5, the main materials are pine sawdust, pine needles, and pine cones, which are convenient and cheap to obtain, and the auxiliary materials are rice bran, Wheat bran and corncob are easy to grow mycelia of Poria cocos, and the growth rate is relatively fast; group 6 has the most comprehensive and rich nutrition, the time to fill the fungus bag is 26.6d, and the average growth rate is 77.26±0.28;

It can be seen that the 6 groups of original culture medium can grow mycelium, the average culture time of the mycelium is 30.33d, and the average growth rate is 71.99±0.30mm; stock medium;

When the original seed medium of the above-mentioned Poria cocos (Wolfiporia cocos) YX1 is prepared with pine tree appendages such as pine sawdust, the content of the main material cannot be less than 60%; The mixture of at least one of miscellaneous sawdust, pine branches, pine needles and pine cones and pine sawdust is used as the main material; the content of the auxiliary material is preferably 0-38%, and the auxiliary material can be at least one of rice bran, wheat bran, corncob, cottonseed husk . The original seed medium can also be supplemented with vitamin B2, vitamin C, anti-fungal Phytophthora additives (such as Kemengwang, etc.), mother-eating raw tablets, etc.

Embodiment 5 traditional pine wood section cultivation method

The traditional pine wood section cultivation method of Poria cocos (Wolfiporia cocos) YX1, comprises the following steps:

From November to December, select pine wood sections with a diameter of ≥ 9cm and a length of about 60cm, with some covers removed, and dried for later use;

From April to May of the second year, choose continuous sunny weather. On the prepared ling field, dig cellars with a length of about 1m, a depth and a width of about 50cm, and the interval between each cellar is about 20cm. (depending on the slope) and excavate a drainage ditch about 40cm wide; then put about 25kg barrels in each cellar, commonly known as "lower cellar" (1 barrel refers to 1 pine wood section, depending on the size of the pine wood section to determine the barrel The amount of addition, generally put 3-5 cylinders per cellar);

According to the method of S1 in Example 7, the culture medium of the original hyphae of Wolfiporia cocos (Wolfiporia cocos) YX1 was obtained; The 6kg pine wood section uses 400g of culture medium covered with tuckahoe (Wolfiporia cocos) YX1 original species mycelium, as shown in Figure 3), and then covers the soil with a thickness of 4-6cm, and the sclerotia begins to form in the cellar for a period of time by keeping warm and moisturizing. At this time, the soil surface cracks into tortoise cracks, and the cracks should be covered with soil in time to prevent the Poria cocos from emerging from the soil surface.

The above-mentioned heat preservation, moisturizing, and soil covering can make the mycelium grow and develop normally under dark conditions, which is conducive to the formation of sclerotia; the thickness of the covering soil is controlled at 4-6cm, which is neither tight nor loose. Poria traditional pine cultivation takes pine trees as the main source of nutrients, and mainly uses the trunks and stumps of pine trees for cultivation.

Figure 3 is a picture during the cultivation process of Example 5.

In the above-mentioned traditional pine wood section cultivation method, after the mycelium grows on the pine wood section, the treatment of inducing the tuckahoe can be carried out, thereby improving the output of Poria; The soil was dug up, and it was found that the lower part of the pine wood segment (that is, the end that was not inoculated with the original seed) had white mycelium growing, and the smell of Poria cocos could be confirmed. The first or second generation sclerotia cultivated by the strain (the size of the sclerotium is 4-8cm, fat and mature, the fresh skin is thin and pale red, the flesh is white, the pulp is juicy, and it is not easy to rot and deteriorate. After digging out, put it in a refrigerator of about 4 °C Internal preservation and standby), peel off the outer skin of the sclerotia, insert the peeled sclerotia into the crack of the pine wood with a wooden toothpick at the end of the pine wood section that has not been inoculated with the original species, and then bury it in the soil.

Example 6

Poria cocos farmers all over the country generally use the CGMCC Poria cocos (Wolfiporia cocos) 5.78 strain of the Chinese Academy of Sciences Microbial Culture Collection and Management Center for extensive cultivation.

The original species of Wolfiporia cocos 5.78 was obtained by culturing the strain of Wolfiporia cocos 5.78, and then inoculated with the original seed of Wolfiporia cocos 5.78 according to the procedure of Example 5 and cultivated. And compare the situation of the cultivation of Geling in Examples 5-6, the results are shown in Table 5 and Figures 4-5. FIG. 4 is a picture of the sclerotia of Poria cocos in Example 5; FIG. 5 is a picture of the sclerotia of Poria cocos in Example 6.

Table 5 The results of the traditional pine wood segment cultivation of Example 5-6

Remarks: 1. Yield refers to the ratio of dry weight to fresh weight of sclerotia of Poria sclerotiorum; 2. Tuckauri time refers to the time from the next cellar to the first picking of Poria, and each pine segment is picked twice.

It can be seen from Table 5 and Figures 4-5 that the formation time of Wolfiporia cocos YX1 is 7 days shorter than that of 5.78 strains, the formation rate of Wolfiporia cocos YX1 is 99.76%, and the formation rate of Wolfiporia cocos YX1 is 99.76%. ) The average sclerotia of YX1 is 0.152kg heavier than that of 5.78 strains, the yield of Wolfiporia cocos YX1 is 12.4% higher than that of 5.78 strains, and the flesh of Wolfiporia cocos YX1 is better than 5.78 strains , Poria cocos (Wolfiporia cocos) YX1 obtained sclerotia skin less fleshy;

The Poria cocos obtained by the Poria cocos YX1 of the present invention has the advantages of short formation time, high formation rate, heavy average sclerotia yield, large individual size of the Poria cocos, less water content, smooth, clean, fine lines, fine skin and white flesh. Strong sclerotia, no sediment, high yield, less skin and more meat.

Embodiment 7 Cultivation method for inoculation of one end of pine wood section

A method for inoculating and cultivating one end of tuckahoe (Wolfiporia cocos) YX1, comprising the following steps:

S1. Prepare the parent culture medium (1000mL of pH=6 aqueous solution contains: glucose 30g, peptone 15g, agar powder 20g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.5g), sterilize at 121°C, 0.105Mpa for more than 6h; Then, 2g of Wolfiporia cocos YX1 was inoculated into the cooled 10 mL parent seed medium, and cultured at 28°C for 5 days to obtain the parent seed of Wolfiporia cocos YX1;

Prepare the original seed medium (raw materials by weight percentage include: 80% of pine sawdust, 18.5% of wheat bran, 1% of sucrose, 0.5% of gypsum; the humidity of the mixture is 50-60%), placed in a bacterial bag, and stored at a high temperature of 121 ° C Autoclave sterilization for more than 6h, and then naturally cool overnight; then take the tuckahoe (Wolfiporia cocos) YX1 parent seed and inoculate it in 400g of the original seed medium, and cultivate at 28-32 ° C until the mycelium is covered with the bacteria bag (about 24-38 days). ) to obtain the original species of Wolfiporia cocos YX1;

S2, take and select the pine wood segment with a diameter of ≥9cm and a length of about 60cm that has been partially covered, and cover the fungus bag covered with the original hyphae of Wolfiporia cocos YX1 in S1 on one end of the pine wood segment (per 6kg pine wood segment). Use 400g fungus bag covered with tuckahoe (Wolfiporia cocos) YX1 original species mycelium), then use a rubber band to tie the fungus bag on the pine wood section (as shown in Figure 6a); then cover both ends on the other end of the pine wood section. Open the empty fungus bag, fix one end of the empty fungus bag on the pine wood section with tape, keep the other end of the empty fungus bag open, and extend the empty fungus bag from one end of the pine wood section by 7-8cm, and the pine wood section. The middle position of the pine wood is not covered with a fungus bag (as shown in Figure 6b), transfer to a culture room with a temperature of 28-32 °C and a relative humidity of 70-80% RH, and cover the surface of the pine section with newspaper to keep the moisture content of the pine section at 40-50 %, cultured for 40 days to grow mycelium; then buried the pine wood segment in the pit to grow the ling.

Fig. 6 is the picture in the cultivation process of embodiment 7, wherein, a is the pine wood section that one end is inoculated with original seed and seals the fungus bag, and b is the pine wood segment that both ends are covered with fungus bag.

The operation and management of digging the cellar in the above-mentioned embodiment 7, burying the pine wood section in the cellar, and burying the back in the cellar are all identical with those of the traditional pine wood section cultivation method in Example 5.

Buried in the cellar in above-mentioned embodiment 7, Poria sclerotia grows finished product in about 3-4 months.

In the above S1, during the sterilization of the culture medium, the flame should not be turned off to cool down in the middle to ensure complete sterilization.

In above-mentioned S1, the inoculation amount of Poria cocos (Wolfiporia cocos) YX1 female species in the original seed medium is not specified; generally every 400g of original seed medium is inoculated with Poria cocos (Wolfiporia cocos) YX1 female seed 50-100g.

In above-mentioned S2, at one end of the pine wood section of the inoculated original species, the bacterial bag is tightly sealed on the pine wood section with a rubber band, which can prevent the infection of other miscellaneous bacteria in the soil; Due to problems such as excessive humidity, the bag is contaminated with bacteria or detached from the wood, resulting in the need to replace the new bacteria bag in the later stage.

In above-mentioned S2, the one end of the pine wood section of the inoculated original species should have cracks, gaps or new wounds, which can make the pine wood easier to grow bacteria.

In the above-mentioned S2, the middle position of the pine wood section is not covered by the fungus bag, so that the pine wood section can be fully absorbed into the nutrient and water in the soil, and the surrounding of the pine wood section maintains the transportation and exchange of nutrients with the outside world.

In the above-mentioned S2, the other end of the pine wood section is covered with an empty bacteria bag with both ends open, and one end of the empty bacteria bag is fixed on the pine wood section, and the other end of the empty bacteria bag is kept in an open state, and the empty bacteria bag is elongated. One end of the pine wood section is 7-8 cm, which can make the pine wood section fully absorb the nutrients and water in the soil, and maintain the transportation and exchange of nutrients with the outside world around the pine wood section. , so that the mycelium grows white and dense.

In the above S2, before the pine wood section goes down to the cellar, one end of the empty fungus bag is covered, and the empty fungus bag extending 7-8 cm from one end of the pine wood segment can be fastened with a rubber band. When the cross section of the pine wood segment is covered with mycelium, the The rubber band is loosened, and the scorpion is placed in the lower cellar to grow, which can further isolate the pollution, make the tuckahoe not easy to contaminate the bacteria, and the quality of the tuckahoe is good.

Poria cocos can also be cultivated by punching holes on the pine sections, so as to avoid bacterial contamination and reduce the amount of inoculation; the specific steps of punching cultivation can be as follows: take 60-70cm long Punch holes on the pine section (the punching position can be: a the center of the cross section of the pine section, b the side of the pine section at a distance of 2-4cm from the cross section, c the center of the cross section of the pine section and the pine section at a distance of 2-4cm from the cross section Section side), the hole diameter is about 4cm, the hole depth is 4-6cm, and then immediately plug the original species of Poria cocos mycelia, then paste the hole sealing sticker or sealing wax, and cultivate for about 60-70 days until the pine wood segment is covered with mycelium, Adjust the moisture content of the pine wood segment to be about 40-50%, then bury it in the cellar and grow the ling.

The sclerotia of Example 5 and Example 7 were counted, and the results were shown in Table 6 and Figures 7-9.

Fig. 7 is the picture when the embodiment 5 is established; Fig. 8 is the picture when the embodiment 7 is established; Fig. 9 is the Poria sclerotia picture of the embodiment 7.

The sclerotia result of table 6 embodiment 5, embodiment 7

As can be seen from Table 6, Figure 7-9, the sclerotia obtained in Example 5 and Example 7 are not much different, and the method of Example 7 is not easy to contaminate miscellaneous bacteria, the original seed inoculum can be reduced, and it is convenient for indoor cultivation, concise and convenient. , to avoid being limited by the planting time; and in Example 7, when the tuckahoe was formed, the Poria cocos was white and free of contaminating bacteria, while in Example 5, when the pine tree was formed, the cross section of the pine wood section was not protected, and the pine wood section was directly buried in the soil, Poria is susceptible to miscellaneous bacteria.

The above description is only a preferred embodiment of the present invention, but the protection scope of the present invention is not limited to this. The equivalent replacement or change of the inventive concept thereof shall be included within the protection scope of the present invention.

sequence listing

<110> Anqing Normal University

Anhui Yuelan Pharmaceutical Co., Ltd.

<120> A kind of Poria cocos (Wolfiporia cocos) YX1 and its culture medium and cultivation method

<130> 2021

<141> 2021-09-14

<160> 3

<170> SIPOSequenceListing 1.0

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<211> 1652

<212> DNA

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ttgggttatc gtgtccggtc ggagtgggtt catggcacac gttgtaacga cacgcgtgtg 180

atgccctcac gtaacccagt cgagccctga gtggatcgcg cggaagatgc tgtccggtga 240

gcgaagccaa tcgcgacccg ggccgtcctg ttcggaggca agcagatatc cgttagtgtg 300

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gacccgcttg gattgacctg ttgcaccgtc tacagtcatc ttccgagtgt gtgcaatggg 540

agagaacgaa gcccgcgatt ggggaattcg agatgccctc ccatttattg gggcgtgggg 600

aggtttgtgt actcccagac cagctccgag tcgtgccgcc gtctactact acagaccttt 660

tgtctgagat atctggtcga tgccgtcgag cacgtcacaa gtcattctcg aattcatatc 720

cgtccgtcta tgacaggcgc ggcccactac ggacgtgaga gttcgttaac acgggtcgcg 780

agcgtccgtt aacacgggcg tgagcgtccg ttacggtacc gtctttctcg atagacaaaa 840

gaccctttgg cacccctttc acataccaca cccgtgcacc tatttgccgc ggtgcaagga 900

cccacgttcg gtccttccgc gcgtgtgaag accctctgcc cgcggcgccc ttacaaaccc 960

cataatgtca gaacgttgtc ccgatataac aatgaaagag tttaataaca actctcagcg 1020

acggatctct tggctctcgc atcgatgaag aacgcagcga aatgcgataa gtaatgtgaa 1080

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gttccctgga acgggacgcc acagagggtg agagccccgt ctggctggcc accgagcctc 180

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tgaaaagcac cttgaaaaga gggttaaaca gtacgtgaaa ttgttgaaag ggaagcgctt 360

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gcgtcacacc ccgcttggcc tgtctacccc tcctttggca gcaaattttt ctgctgcctc 180

gtttgacttt agtggggtgt caatgttttt ttggcaaccc cgctattgcc actgtccctc 240

atccatcgtc ccaacaaaat gcactcattc aatcgcatcg tcttttgact cgatttctct 300

atggttcgtt gtgctaatca tgcttcaatc aataggaagc cgccgaactc ggcaagggtt 360

ctttcaagta tgcgtgggtt cttgacaagc tcaaggccga gcgtgagcgt ggtatcacca 420

tcgacattgc cctctggaag ttcgagactc ccaagtacta tgtcaccgtc attggtatgt 480

tattcctggc tcttgacacg tcgaaatcat cattctaacg tgccaataca gacgctcccg 540

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tgattatcgc tgccggtact ggtgaagttc gagg 634

Claims (10)

1. Poria cocos (Poria cocos) fungusWolfiporia cocos) YX1, which is characterized in that the preservation number is CCTCC NO: m2021434.

2. The Poria cocos bacterium (F) (Poria cocos bacterium) of claim 1Wolfiporia cocos) The stock culture medium of YX1 is characterized by comprising the following raw materials in percentage by weight: the main material is more than or equal to 60 percent, the auxiliary material is 0-38 percent, the cane sugar is 0.5-2 percent, the gypsum is 0.5-1 percent, the natural resin is 0-1 percent, the monopotassium phosphate is 0-0.1 percent, the vitamin B is 0-0.0002 percent, and the gram mould king is 0-0.1 percent, wherein the total weight percentage of all the raw materials is 100 percent, and the main material is a mixture of pine sawdust and at least one of miscellaneous wood chips, pine branches, pine needles and turpentine or pine sawdust.

3. Poria cocos bacterium (hoelen) according to claim 2Wolfiporia cocos) The stock culture medium of YX1 is characterized in that the auxiliary material is at least one of rice bran, wheat bran, corncob and cottonseed hull.

4. Poria cocos bacterium (hoelen) according to claim 2 or 3Wolfiporia cocos) The stock culture medium of YX1 is characterized in that the natural resin is: at least one of mastic gum resin, shellac, rosin resin, and amber.

5. Poria cocos bacterium (hoelen) according to claim 2 or 3Wolfiporia cocos) The stock culture medium of YX1 is characterized in that the weight percentage of the main material is more than or equal to 70 percent.

6. Poria cocos bacterium (hoelen) according to claim 2 or 3Wolfiporia cocos) The stock culture medium of YX1 is characterized in that the mixing humidity of the stock culture medium is 50-60%.

7. The Poria cocos bacterium (F) (Poria cocos bacterium) of claim 1Wolfiporia cocos) The mother culture medium of YX1 is characterized in that the raw materials are as follows: 1000mL of an aqueous solution having pH =5-6 contains: 150 g of potato, 20-30g of glucose and 15-2 g of agar powder0g, 0-1g of monopotassium phosphate and 0-0.5g of magnesium sulfate;

alternatively, a 1000mL aqueous solution having pH =5-6 contains: 20-30g of glucose, 10-15g of peptone, 15-20g of agar powder, 0-1g of monopotassium phosphate and 0-0.5g of magnesium sulfate.

8. The Poria cocos bacterium (F) (Poria cocos bacterium) of claim 1Wolfiporia cocos) The cultivation method of YX1 is characterized by comprising the following steps:

s1, adding PoriaWolfiporia cocos) YX1 sequentially culturing in mother culture medium and stock culture medium to obtain PoriaWolfiporia cocos) YX1 stock;

s2, adding PoriaWolfiporia cocos) Sleeving a fungus bag of a stock strain YX1 at one end of a pine section, sealing the fungus bag, fixing the fungus bag on the pine section, sleeving a hollow fungus bag at the other end of the pine section, keeping the opening state, adjusting the water content of the pine section to 40-50%, culturing at 28-32 ℃ for 40-50 days, burying the pine section in a cellar, and growing polyporus;

wherein the mother culture medium is the Poria cocos (Schw.) wolf fungus (Poria cocos (Schw.) wolf) of claim 7Wolfiporia cocos) YX1, wherein the stock culture medium is the Poria cocos (Poria cocos) strain of claim 2Wolfiporia cocos) Stock culture medium of YX 1.

9. Poria cocos bacterium (hoelen) according to claim 8Wolfiporia cocos) The cultivation method of YX1 is characterized in that in S2, the empty fungus bag is a fungus bag with two open ends; in S2, the empty fungus bag extends 7-8cm beyond the end of the pine section, where the empty fungus bag is sleeved.

10. Poria cocos bacterium (hoelen) according to claim 8 or 9Wolfiporia cocos) The cultivation method of YX1, wherein the middle position of pine wood segment is not covered with fungus sack in S2.

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