Culture method of tricholoma matsutake strain

Abstract

The invention relates to a tricholoma matsutake strain culture method, which comprises: (1) inoculating a tricholoma matsutake stipe tissue to a mother strain culture medium, and culturing to obtain a mother strain; (2) inoculating the mother strain obtained in the step (1) into a liquid shake-flask culture medium for culturing to obtain a liquid shake-flask strain; and (3) inoculating the liquid shake-flask strain obtained in the step (2) to a solid culture medium, and culturing to obtain a solid strain. According to the culture method, the tricholoma matsutake mother strain and the liquid strain are firstly cultured, then the solid culture medium is inoculated with the liquid strain to be cultured into the solid strain, and the culture method solves the problems that the tricholoma matsutake strain is long in culture period, low in success rate and low in activity and purity.

Description

Technical Field

[0001] The invention relates to the technical field of strain culture, and in particular to a method for culturing a matsutake strain. Background Art

[0002] Matsutake (Tricholoma matsutake), scientifically known as Tricholoma matsutake, also known as pine mushroom, combined mushroom, and Taiwan mushroom, belongs to the Basidiomycota, Trichodermaceae. It is an ectomycorrhizal fungus on trees such as pine and oak. It has a unique and rich aroma. It is a rare and precious natural medicinal fungus in the world. It is known as the "king of fungi" and is a second-level endangered protected species in my country.

[0003] Matsutake contains 18 kinds of amino acids, 14 essential trace elements for the human body, 49 active nutrients, 5 unsaturated fatty acids, 8 vitamins, 2 glycoproteins, rich dietary fiber and a variety of active enzymes. It also contains 3 precious active substances, namely double-chain pine mushroom polysaccharide, pine mushroom polypeptide and the world's unique anti-cancer substance - pine mushroom alcohol. It is the most precious natural medicinal fungus in the world.

[0004] The nutritional and medicinal value of Matsutake is extremely high. Modern medicine shows that Matsutake has many functions such as improving immunity, fighting cancer and tumors, treating diabetes and cardiovascular diseases, anti-aging and beauty, promoting gastrointestinal function and protecting the liver. Therefore, it is widely used in the research and development of medicines, health products and cosmetics around the world.

[0005] Matsutake is rare, precious, and low in yield. It is mainly produced in Yunnan, Sichuan, Guizhou, and Northeast China. Matsutake variants have also been found in the United States and Europe, but they are of poor quality and have lower yields. At present, wild matsutake cannot be fully cultivated artificially worldwide. According to statistics, the global annual production of matsutake is only 2,000 tons, and due to climate change and people's uncontrolled picking, the production is still showing a decreasing trend year by year.

[0006] However, the demand for matsutake mushrooms at home and abroad is still increasing, resulting in a situation where the matsutake industry is in short supply. Based on this, researchers selected wild matsutake mushrooms in Yunnan and hybridized them with Pleurotus eryngii (old man's head) to obtain artificial hybrid matsutake mushrooms. Through HPLC chromatogram analysis, the RT value of wild matsutake mushrooms has a certain similarity with artificial hybrid matsutake mushroom products, which is close to 35%. Wild matsutake mushrooms have limited production due to climate and seasonal conditions. Artificial cultivation of matsutake mushrooms increases production to meet people's needs. Therefore, it is urgent to provide a method for cultivating matsutake mushrooms that can effectively shorten the culture cycle, improve activity and success rate, and increase production. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a method for cultivating Tricholoma matsutake strains. The cultivation method provided by the present invention first cultivates Tricholoma matsutake mother strains and liquid strains, and then inoculates the liquid strains on a solid culture medium to cultivate solid strains. The cultivation method solves the problems of long cultivation cycle, low success rate, low activity and purity of Tricholoma matsutake strains.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] The present invention provides a method for culturing Tricholoma matsutake strains, the method comprising the following steps:

[0010] (1) inoculating the stipe tissue of Tricholoma matsutake onto a mother culture medium for culturing to obtain a mother culture;

[0011] (2) inoculating the mother strain obtained in step (1) into a liquid shake flask culture medium for culturing to obtain a liquid shake flask strain;

[0012] (3) inoculating the liquid shake flask strain obtained in step (2) onto a solid culture medium for cultivation to obtain a solid strain.

[0013] The present invention provides a cultivation method from mother culture, liquid culture to solid culture. The method comprises the following steps: firstly cultivating the mother culture and liquid culture of Tricholoma matsutake, and then inoculating the liquid culture onto a solid culture medium to cultivate the solid culture. The above three cultivation links are combined to obtain Tricholoma matsutake strains with the advantages of short cultivation cycle, high cultivation success rate, high purity and strong activity.

[0014] Preferably, in step (1), the raw materials for preparing the mother culture medium include: potatoes, glucose, corn flour, bran, peptone, yeast extract, magnesium sulfate, potassium dihydrogen phosphate, vitamin B1 and agar powder.

[0015] In the present invention, the mother culture medium comprises a combination of potato, corn flour and bran, and the three components work synergistically to enhance the activity of the strain and shorten the culture period.

[0016] Preferably, based on 1L of the culture medium system, the raw materials for preparing the mother culture medium include 150-200g potatoes, 15-20g glucose, 20-30g corn flour, 30-50g bran, 2-5g peptone, 2-3g yeast extract, 0.5-1g magnesium sulfate, 1-2g potassium dihydrogen phosphate, 30-50mg vitamin B1 and 16-20g agar powder.

[0017] In the above values, potatoes can be 160g, 170g, 180g, 190g, etc.; glucose can be 16g, 17g, 18g, 19g, etc.; corn flour can be 22g, 24g, 26g, 28g, etc.; bran can be 32g, 35g, 38g, 40g, 42g, 45g, 48g, etc.; peptone can be 2.5g, 3g, 3.5g, 4g, 4.5g, etc.; yeast extract can be 2.2g, 2.4 g, 2.6g, 2.8g, etc.; magnesium sulfate can be 0.6g, 0.7g, 0.8g, 0.9g, etc.; potassium dihydrogen phosphate can be 1.2g, 1.4g, 1.6g, 1.8g, etc.; vitamin B1 can be 32mg, 35mg, 38mg, 40mg, 42mg, 45mg, 48mg, etc.; agar powder can be 16.5g, 17g, 17.5g, 18g, 18.5g, 19g, 19.5g, etc.

[0018] Preferably, in step (2), the raw materials for preparing the liquid shake flask culture medium include: potatoes, glucose, corn flour, bran, peptone, yeast extract, magnesium sulfate, potassium dihydrogen phosphate, vitamin B1 and agar powder.

[0019] In the present invention, the liquid shake flask culture medium comprises a combination of potato, corn flour and bran, and the three components work synergistically to enhance the activity of the strain and shorten the culture cycle; in addition, the addition of agar powder to the liquid shake flask culture medium can increase the density of the culture medium and reduce the shear force of stirring, thereby increasing the density and activity of the liquid strain mycelium.

[0020] Preferably, based on 1L of the culture medium system, the raw materials for preparing the liquid shake flask culture medium include 150-200g of potatoes, 15-20g of glucose, 20-30g of corn flour, 30-50g of bran, 3-5g of peptone, 2-3g of yeast extract, 0.5-1g of magnesium sulfate, 1-2g of potassium dihydrogen phosphate, 30-50mg of vitamin B1 and 1-2g of agar powder.

[0021] In the above values, potatoes can be 160g, 170g, 180g, 190g, etc.; glucose can be 16g, 17g, 18g, 19g, etc.; corn flour can be 22g, 24g, 26g, 28g, etc.; bran can be 32g, 35g, 38g, 40g, 42g, 45g, 48g, etc.; peptone can be 2.5g, 3g, 3.5g, 4g, 4.5g, etc.; yeast extract can be 2.2g, 2.4g, 2.6g, 2.8g, etc.; magnesium sulfate can be 0.6g, 0.7g, 0.8g, 0.9g, etc.; potassium dihydrogen phosphate can be 1.2g, 1.4g, 1.6g, 1.8g, etc.; vitamin B1 can be 32mg, 35mg, 38mg, 40mg, 42mg, 45mg, 48mg, etc.; agar powder can be 1.2g, 1.4g, 1.6g, 1.8g, etc.

[0022] Preferably, the method for preparing the mother culture medium or liquid shake flask medium comprises the following steps: boiling potatoes, corn flour and bran respectively, combining the filtrates, adding glucose, peptone, yeast extract, magnesium sulfate, potassium dihydrogen phosphate, vitamin B1 and agar powder, and boiling until the agar is completely melted to obtain the mother culture medium or liquid shake flask medium.

[0023] Preferably, in step (1), the stipe tissue is the mushroom flesh tissue of 2mm×2mm×2mm-3mm×3mm×3mm (for example, it can be 2.2mm×2.2mm×2.2mm, 2.4mm×2.4mm×2.4mm, 2.6mm×2.6mm×2.6mm, 2.8mm×2.8mm×2.8mm, etc.) in the middle part of the stipe of the Matsutake fruiting body.

[0024] Preferably, in step (1), the culture temperature is 22-26°C (for example, it can be 22.5°C, 23°C, 23.5°C, 24°C, 24.5°C, 25°C, 25.5°C, etc.), and the culture time is 20-25 days (for example, it can be 20 days, 21 days, 22 days, 23 days, 24 days, etc.).

[0025] Preferably, in step (2), the culturing comprises static culturing and agitated culturing performed sequentially.

[0026] Preferably, the static culture temperature is 22-26°C (for example, 22.5°C, 23°C, 23.5°C, 24°C, 24.5°C, 25°C, 25.5°C, etc.), and the time is 2-3 days (for example, 2 days, 2.5 days, 3 days, etc.).

[0027] Preferably, the temperature of the agitated culture is 22-26°C (for example, 22.5°C, 23°C, 23.5°C, 24°C, 24.5°C, 25°C, 25.5°C, etc.), and the time is 5-7 days (for example, 5 days, 6 days, 7 days, etc.).

[0028] Preferably, the stirring rate of the stirring culture is 500-1500 rpm (for example, it can be 500 rpm, 600 rpm, 800 rpm, 1000 rpm, 1200 rpm, 1400 rpm, 1500 rpm, etc.).

[0029] Preferably, in step (3), the components of the solid culture medium include cooked wheat, sawdust, bran, soybean meal, gypsum, lime and water.

[0030] Preferably, the wood chips comprise oak wood chips.

[0031] Preferably, the components of the solid culture medium include, by weight, 53.5-81.2 parts of cooked wheat, 10-30 parts of sawdust, 5-10 parts of bran, 3-5 parts of soybean meal, 0.5-1 part of gypsum, 0.3-0.5 part of lime and 40-60 parts of water.

[0032] Among the above values, wheat can be 55 parts, 60 parts, 65 parts, 70 parts, 75 parts, 80 parts, etc.; wood chips can be 12 parts, 15 parts, 18 parts, 20 parts, 22 parts, 25 parts, 28 parts, etc.; bran can be 6 parts, 7 parts, 8 parts, 9 parts, etc.; soybean meal can be 3.2 parts, 3.5 parts, 3.8 parts, 4 parts, 4.2 parts, 4.5 parts, 4.8 parts, etc.; gypsum can be 0.6 parts, 0.7 parts, 0.8 parts, 0.9 parts, etc.; lime can be 0.32 parts, 0.35 parts, 0.38 parts, 0.4 parts, 0.42 parts, 0.45 parts, 0.48 parts, etc.; water can be 42 parts, 45 parts, 48 ​​parts, 50 parts, 52 parts, 55 parts, 58 parts, etc.;

[0033] Preferably, the method for preparing the solid culture medium comprises the following steps: cooking the wheat with water, draining the water, pre-wetting the sawdust with water, and mixing the processed wheat and sawdust with bran, soybean meal, gypsum and lime to obtain the solid culture medium.

[0034] Preferably, in step (3), the culture temperature is 22-26°C (for example, it can be 22.5°C, 23°C, 23.5°C, 24°C, 24.5°C, 25°C, 25.5°C, etc.), and the culture time is 20-30 days (for example, it can be 22 days, 24 days, 26 days, 28 days, etc.).

[0035] Preferably, the culturing method comprises the following steps:

[0036] (1) inoculating a 2 mm × 2 mm × 2 mm-3 mm × 3 mm × 3 mm mushroom tissue in the middle of the stipe of a Tricholoma matsutake fruiting body on a mother culture medium, and culturing at 22-26° C. for 28-32 days to obtain a mother culture medium; based on 1 L of the culture medium system, the raw materials for preparing the mother culture medium include 150-200 g of potato, 15-20 g of glucose, 20-30 g of corn flour, 30-50 g of bran, 2-5 g of peptone, 2-3 g of yeast extract, 0.5-1 g of magnesium sulfate, 1-2 g of potassium dihydrogen phosphate, 30-50 mg of vitamin B1, and 16-20 g of agar powder;

[0037] (2) inoculating the mother culture obtained in step (1) into a liquid shake flask culture medium, culturing at 22-26° C. for 2-3 days, and then stirring and culturing at 22-26° C. and 500-1500 rpm for 5-7 days to obtain a liquid shake flask culture; based on 1 L of the culture medium, the raw materials for preparing the liquid shake flask culture medium include 150-200 g of potato, 15-20 g of glucose, 20-30 g of corn flour, 30-50 g of bran, 3-5 g of peptone, 2-3 g of yeast extract, 0.5-1 g of magnesium sulfate, 1-2 g of potassium dihydrogen phosphate, 30-50 mg of vitamin B1 and 1-2 g of agar powder;

[0038] (3) inoculating the liquid shake flask strain obtained in step (2) onto a solid culture medium, and culturing at 22-26° C. for 20-30 days to obtain a solid culture medium; the solid culture medium comprises, by weight, 53.5-81.2 parts of wheat, 10-30 parts of sawdust, 5-10 parts of bran, 3-5 parts of soybean meal, 0.5-1 part of gypsum, 0.3-0.5 parts of lime, and 40-60 parts of water.

[0039] Compared with the prior art, the present invention has at least the following beneficial effects:

[0040] The present invention provides a cultivation method from mother culture, liquid culture to solid culture, which comprises cultivating the mother culture and liquid culture first, and then inoculating the liquid culture onto a solid culture medium to cultivate the solid culture. The above three cultivation links are combined to obtain a matsutake strain having the advantages of a short cultivation cycle, a high cultivation success rate, a high purity and a strong activity. DETAILED DESCRIPTION

[0041] For the convenience of understanding the present invention, the present invention lists the following embodiments. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0042] The artificial hybrid matsutake used in the following examples is hybridized by wild matsutake from Yunnan and Pleurotus eryngii (Old Man's Head). Through HPLC analysis, the RT value of wild matsutake has a certain similarity with the artificial hybrid matsutake product, which is close to 35%.

[0043] Example 1: Preparation of mother culture medium

[0044] This preparation example provides a mother culture medium. Taking the system of the culture medium as 1L, the raw materials for preparing the mother culture medium include 150g potato, 15g glucose, 20g corn flour, 30g bran, 3g peptone, 2g yeast extract, 0.5g magnesium sulfate, 1g potassium dihydrogen phosphate, 30mg vitamin B1 and 16g agar powder;

[0045] The preparation method of the mother culture medium comprises the following steps: washing, peeling, weighing, and cutting fresh potatoes into thin slices and putting them into a large beaker, adding a certain amount of water for boiling, boiling the potatoes until they are cooked but not mushy, filtering and obtaining the filtrate, and simultaneously boiling corn flour and bran and then filtering and obtaining the filtrate, combining the filtrates, adding glucose, agar powder, peptone, yeast extract, magnesium sulfate, potassium dihydrogen phosphate and vitamin B1, and boiling until the agar is completely dissolved and the pH value is 5.0±1.0, so as to obtain the mother culture medium; and packaging the mother culture medium into triangular bottles, sterilizing at 121° C. for 30 minutes, and pouring the mother culture medium into a culture dish for standby use after cooling.

[0046] Example 2: Preparation of mother culture medium

[0047] This preparation example provides a mother culture medium. Taking the system of the culture medium as 1L, the raw materials for preparing the mother culture medium include 200g potatoes, 20g glucose, 30g corn flour, 50g bran, 5g peptone, 3g yeast extract, 1g magnesium sulfate, 2g potassium dihydrogen phosphate, 50mg vitamin B1 and 20g agar powder; the preparation method refers to the mother culture medium preparation example 1.

[0048] Example 3: Preparation of mother culture medium

[0049] This preparation example provides a mother culture medium. Taking the system of the culture medium as 1L, the raw materials for preparing the mother culture medium include 175g potatoes, 17.5g glucose, 25g corn flour, 40g bran, 4g peptone, 2.5g yeast extract, 0.75g magnesium sulfate, 1.5g potassium dihydrogen phosphate, 40mg vitamin B1 and 18g agar powder; the preparation method refers to the mother culture medium preparation example 1.

[0050] Example 4: Preparation of mother culture medium

[0051] This preparation example provides a mother culture medium, which differs from the mother culture medium preparation example 1 only in that potatoes are not included in the preparation raw materials, and their amount is proportionally distributed to corn flour and bran; the preparation method refers to the mother culture medium preparation example 1.

[0052] Example 5: Preparation of mother culture medium

[0053] This preparation example provides a mother culture medium, which differs from the mother culture medium preparation example 1 only in that corn flour is not contained in the preparation raw materials, and its amount is proportionally distributed to potatoes and bran; the preparation method refers to the mother culture medium preparation example 1.

[0054] Example 6: Preparation of mother culture medium

[0055] This preparation example provides a mother culture medium, which differs from the mother culture medium preparation example 1 only in that the preparation raw materials do not contain bran, and its amount is proportionally distributed to potatoes and corn flour; the preparation method refers to the mother culture medium preparation example 1.

[0056] Liquid shake flask culture medium preparation example 1

[0057] This preparation example provides a liquid shake flask culture medium. Taking the system of the culture medium as 1L, the raw materials for preparing the liquid shake flask culture medium include 150g of potato, 15g of glucose, 20g of corn flour, 30g of bran, 3g of peptone, 2g of yeast extract, 0.5g of magnesium sulfate, 1g of potassium dihydrogen phosphate, 30mg of vitamin B1 and 1g of agar powder; the preparation method refers to the mother culture medium preparation example 1.

[0058] Liquid shake flask culture medium preparation example 2

[0059] This preparation example provides a liquid shake flask culture medium. Taking the system of the culture medium as 1L, the raw materials for preparing the liquid shake flask culture medium include 200g of potatoes, 20g of glucose, 30g of corn flour, 50g of bran, 3g of peptone, 3g of yeast extract, 1g of magnesium sulfate, 2g of potassium dihydrogen phosphate, 50mg of vitamin B1 and 2g of agar powder; the preparation method refers to the mother culture medium preparation example 1.

[0060] Liquid shake flask culture medium preparation example 3

[0061] This preparation example provides a liquid shake flask culture medium. Taking the system of the culture medium as 1L, the raw materials for preparing the liquid shake flask culture medium include 175g of potato, 17.5g of glucose, 25g of corn flour, 40g of bran, 4g of peptone, 2.5g of yeast extract, 0.75g of magnesium sulfate, 1.5g of potassium dihydrogen phosphate, 40mg of vitamin B1 and 1.5g of agar powder; the preparation method refers to the mother culture medium preparation example 1.

[0062] Liquid shake flask culture medium preparation example 4

[0063] This preparation example provides a liquid shake flask culture medium, which differs from the liquid shake flask culture medium preparation example 1 only in that the preparation raw materials do not contain potatoes, and their amount is proportionally distributed to corn flour and bran; the preparation method refers to the mother culture medium preparation example 1.

[0064] Liquid shake flask culture medium preparation example 5

[0065] This preparation example provides a liquid shake flask culture medium, which differs from the liquid shake flask culture medium preparation example 1 only in that corn flour is not contained in the preparation raw materials, and its amount is proportionally distributed to potatoes and bran; the preparation method refers to the mother culture medium preparation example 1.

[0066] Liquid shake flask culture medium preparation example 6

[0067] This preparation example provides a liquid shake flask culture medium, which differs from the liquid shake flask culture medium preparation example 1 only in that the preparation raw materials do not contain bran, and its amount is proportionally distributed to potatoes and corn flour; the preparation method refers to the mother culture medium preparation example 1.

[0068] Liquid shake flask culture medium preparation example 7

[0069] This preparation example provides a liquid shake flask culture medium, which differs from the liquid shake flask culture medium preparation example 1 only in that the preparation raw materials do not contain agar powder, and the preparation method refers to the mother culture medium preparation example 1.

[0070] Solid culture medium preparation example 1

[0071] This preparation example provides a solid culture medium, which includes 53.5 parts of wheat, 30 parts of oak wood chips, 10 parts of bran, 5 parts of soybean meal, 1 part of gypsum, 0.5 parts of lime and 50 parts of water;

[0072] The preparation method of the culture medium comprises the following steps: soaking and washing full dry wheat, cooking it until it is cooked but not mushy, drying it in the sun and draining it for later use, and pre-wetting sawdust with water in advance; adding the pre-wetted sawdust, bran, soybean meal, gypsum and lime into the wheat and mixing them to obtain the solid culture medium; sterilizing the solid culture medium with high-pressure steam, the sterilization pressure is 0.1Mpa, the sterilization temperature is 121°C, the sterilization time is 1.5h, and then setting it aside.

[0073] Solid culture medium preparation example 2

[0074] This preparation example provides a solid culture medium, which includes 81.2 parts of wheat, 10 parts of oak wood chips, 5 parts of bran, 3 parts of soybean meal, 0.5 parts of gypsum, 0.3 parts of lime and 55 parts of water; the preparation method refers to Solid Culture Medium Preparation Example 1.

[0075] Solid culture medium preparation example 3

[0076] This preparation example provides a solid culture medium, which includes 60 parts of wheat, 20 parts of oak wood chips, 7.5 parts of bran, 4 parts of soybean meal, 0.75 parts of gypsum, 0.4 parts of lime and 45 parts of water; the preparation method refers to Solid Culture Medium Preparation Example 1.

[0077] Example 1

[0078] This embodiment provides a method for culturing Tricholoma matsutake strains, the culturing method comprising the following steps:

[0079] (1) Selecting high-quality artificial hybrid matsutake fruiting bodies, wiping the matsutake stems with a cotton ball containing 75% alcohol, taking a 2 mm × 2 mm × 2 mm mushroom tissue block from the middle of the stem with a scalpel, placing it in a culture dish filled with the mother culture medium provided in the mother culture medium preparation example 1, and culturing it in an incubator at 25° C. for 25 days until the mycelium fills the culture dish, thereby obtaining the mother culture;

[0080] (2) inoculating the mother culture obtained in step (1) into the liquid shake flask culture medium provided in Example 1 for preparing the liquid shake flask culture medium, and culturing the culture at 25° C. for 2 days. After the mycelium germinates and is free of contamination, placing the shake flask on a magnetic stirrer for stirring and culturing. Stirring and culturing at 25° C. and 1000 rpm for 7 days, a liquid shake flask culture is obtained.

[0081] (3) The liquid shake flask strain obtained in step (2) was inoculated into the solid culture medium provided in Solid Culture Medium Preparation Example 1 at an inoculum rate of 5%, and cultured in a dark environment at 25° C. for 30 days, with ventilation twice a day for 15 minutes each time, to obtain a solid strain.

[0082] Example 2

[0083] This embodiment provides a method for culturing Tricholoma matsutake strains, the culturing method comprising the following steps:

[0084] (1) Selecting high-quality artificial hybrid matsutake fruiting bodies, wiping the matsutake stems with a cotton ball containing 75% alcohol, taking a 2.5 mm × 2.5 mm × 2.5 mm mushroom tissue block from the middle of the stem with a scalpel, placing it in a culture dish filled with the mother culture medium provided in Example 2 for preparing the mother culture medium, and culturing it in an incubator at 25° C. for 20 days until the mycelium fills the culture dish, thereby obtaining the mother culture;

[0085] (2) inoculating the mother culture obtained in step (1) into the liquid shake flask culture medium provided in Example 2 for preparing the liquid shake flask culture medium, and culturing the culture at 26° C. for 2 days. After the mycelium germinates and is free of contamination, the shake flask is placed on a magnetic stirrer for stirring and culturing. The culture is stirred at 25° C. and 1500 rpm for 5 days to obtain a liquid shake flask culture.

[0086] (3) The liquid shake flask strain obtained in step (2) was inoculated onto the solid culture medium provided in Solid Culture Medium Preparation Example 2 at an inoculum rate of 10%, and cultured in a dark environment at 25° C. for 20 days with ventilation twice a day for 15 minutes each time to obtain a solid strain.

[0087] Example 3

[0088] This embodiment provides a method for culturing Tricholoma matsutake strains, the culturing method comprising the following steps:

[0089] (1) Selecting high-quality artificial hybrid matsutake fruiting bodies, wiping the matsutake stems with a cotton ball containing 75% alcohol, taking a 3 mm × 3 mm × 3 mm mushroom tissue block from the middle of the stem with a scalpel, placing it in a culture dish filled with the mother culture medium provided in the mother culture medium preparation example 3, and culturing it in an incubator at 25° C. for 23 days until the mycelium fills the culture dish, thereby obtaining the mother culture;

[0090] (2) inoculating the mother culture obtained in step (1) into the liquid shake flask culture medium provided in Example 3 for preparing the liquid shake flask culture medium, and culturing the culture at 25° C. for 3 days. After the mycelium germinates and is free of contamination, placing the shake flask on a magnetic stirrer for stirring and culturing. Stirring and culturing at 25° C. and 500 rpm for 6 days to obtain a liquid shake flask culture;

[0091] (3) The liquid shake flask strain obtained in step (2) was inoculated onto the solid culture medium provided in Solid Culture Medium Preparation Example 3 at an inoculum rate of 7.5%, and cultured in a dark environment at 25° C. for 25 days with ventilation twice a day for 15 minutes each time to obtain a solid strain.

[0092] Example 4

[0093] This embodiment provides a method for culturing Tricholoma matsutake strains, which differs from Embodiment 1 only in that, in step (1), the mother strain culture medium is replaced with the culture medium provided in Mother Strain Culture Medium Preparation Example 4, and the rest is as in Embodiment 1.

[0094] Example 5

[0095] This embodiment provides a method for culturing Tricholoma matsutake strains, which differs from Embodiment 1 only in that, in step (1), the mother strain culture medium is replaced with the culture medium provided in Mother Strain Culture Medium Preparation Example 5, and the rest is as in Embodiment 1.

[0096] Example 6

[0097] This embodiment provides a method for culturing Tricholoma matsutake strains, which differs from Embodiment 1 only in that, in step (1), the mother strain culture medium is replaced with the culture medium provided in Mother Strain Culture Medium Preparation Example 6, and the rest is as in Embodiment 1.

[0098] Example 7

[0099] This embodiment provides a method for culturing Tricholoma matsutake strains, which differs from Embodiment 1 only in that, in step (2), the liquid shake flask culture medium is replaced with the culture medium provided in Liquid Shake Flask Culture Medium Preparation Example 4, and the rest is as in Embodiment 1.

[0100] Example 8

[0101] This embodiment provides a method for culturing Tricholoma matsutake strains, which differs from Embodiment 1 only in that, in step (2), the liquid shake flask culture medium is replaced with the culture medium provided in Liquid Shake Flask Culture Medium Preparation Example 5, and the rest is as in Embodiment 1.

[0102] Example 9

[0103] This embodiment provides a method for culturing Tricholoma matsutake strains, which differs from Embodiment 1 only in that, in step (2), the liquid shake flask culture medium is replaced with the culture medium provided in Liquid Shake Flask Culture Medium Preparation Example 6, and the rest is as in Embodiment 1.

[0104] Example 10

[0105] This embodiment provides a method for culturing Tricholoma matsutake strains, which differs from Embodiment 1 only in that, in step (2), the liquid shake flask culture medium is replaced with the culture medium provided in Liquid Shake Flask Culture Medium Preparation Example 7, and the rest is referred to Embodiment 1.

[0106] Comparative Example 1

[0107] This comparative example provides a method for culturing Tricholoma matsutake strains, which differs from Example 1 only in that step (2) is not performed, and the mother strain obtained in step (1) is directly inoculated onto a solid culture medium for culturing.

[0108] Test Example 1

[0109] Activity test

[0110] Test method: Observe the time required for artificial hybrid matsutake mycelium to grow fully on three culture media, in days.

[0111] The activity test results of the mother culture, liquid shake flask culture and solid culture provided in Examples 1-10 and Comparative Example 1 are shown in Table 1.

[0112] Table 1

[0113]

[0114]

[0115] (1) It can be seen from Examples 1 to 10 that the culture method from mother culture, liquid culture to solid culture provided by the present invention can obtain Tricholoma matsutake strains with higher activity within a shorter culture period, and is particularly suitable for the culture of artificial hybrid Tricholoma matsutake strains.

[0116] (2) By comparing Example 1 with Examples 4-9, it can be seen that the present invention can achieve a better technical effect of improving the activity of the strain by further rationally compounding potatoes, corn flour and bran in the mother culture medium and the liquid shake flask culture medium, and the three components synergistically enhance the effect, and the growth cycle of the matsutake mycelium is further shortened;

[0117] By comparing Example 1 with Example 10, it can be seen that adding a small amount of agar powder to the liquid shake flask culture medium in the present invention can increase the density of the culture medium, reduce the shear force of stirring, increase the density and vitality of the liquid bacterial strain mycelium, and shorten the culture period.

[0118] (3) By comparing Example 1 with Comparative Example 1, it can be seen that the present invention combines mother culture with liquid culture, so that the solid strain obtained has higher activity and the growth cycle is further shortened.

[0119] Test Example 2

[0120] Survival test

[0121] Test method: Observe the survival rate of artificial hybrid matsutake in three culture media, with percentage (%) as the unit.

[0122] The test results of the survival rates of the strains provided in Examples 1-10 and Comparative Example 1 are shown in Table 2.

[0123] Table 2

[0124]

[0125] It can be seen from Examples 1 to 10 that the culture method from mother culture, liquid culture to solid culture provided by the present invention can obtain Tricholoma matsutake strains with a higher survival rate within a shorter culture period, and is particularly suitable for the culture of artificial hybrid Tricholoma matsutake strains.

[0126] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for cultivating Tricholoma matsutake strains, characterized in that: The culture method comprises the following steps: (1) inoculating the stipe tissue of Tricholoma matsutake onto a mother culture medium for culturing to obtain a mother culture; (2) inoculating the mother strain obtained in step (1) into a liquid shake flask culture medium for culturing to obtain a liquid shake flask strain; (3) inoculating the liquid shake flask strain obtained in step (2) onto a solid culture medium for cultivation to obtain a solid strain.

2. The culture method according to claim 1, characterized in that In step (1), the raw materials for preparing the mother culture medium include: potatoes, glucose, corn flour, bran, peptone, yeast extract, magnesium sulfate, potassium dihydrogen phosphate, vitamin B1 and agar powder; Preferably, based on 1L of the culture medium system, the raw materials for preparing the mother culture medium include 150-200g potatoes, 15-20g glucose, 20-30g corn flour, 30-50g bran, 2-5g peptone, 2-3g yeast extract, 0.5-1g magnesium sulfate, 1-2g potassium dihydrogen phosphate, 30-50mg vitamin B1 and 16-20g agar powder.

3. The culture method according to claim 1 or 2, characterized in that In step (2), the raw materials for preparing the liquid shake flask culture medium include: potatoes, glucose, corn flour, bran, peptone, yeast extract, magnesium sulfate, potassium dihydrogen phosphate, vitamin B1 and agar powder; Preferably, based on 1L of the culture medium system, the raw materials for preparing the liquid shake flask culture medium include 150-200g of potatoes, 15-20g of glucose, 20-30g of corn flour, 30-50g of bran, 3-5g of peptone, 2-3g of yeast extract, 0.5-1g of magnesium sulfate, 1-2g of potassium dihydrogen phosphate, 30-50mg of vitamin B1 and 1-2g of agar powder.

4. The culture method according to claim 2 or 3, characterized in that: The preparation method of the mother culture medium or liquid shake flask culture medium comprises the following steps: boiling potatoes, corn flour and bran respectively, combining the filtrates, adding glucose, peptone, yeast extract, magnesium sulfate, potassium dihydrogen phosphate, vitamin B1 and agar powder, and boiling until the agar is completely melted to obtain the mother culture medium or liquid shake flask culture medium.

5. The culture method according to any one of claims 1 to 4, characterized in that: In step (1), the stipe tissue is the 2mm×2mm×2mm-3mm×3mm×3mm flesh tissue in the middle part of the stipe of the Tricholoma matsutake fruiting body; Preferably, in step (1), the culture temperature is 22-26° C. and the culture time is 20-25 days.

6. The culture method according to any one of claims 1 to 5, characterized in that: In step (2), the culturing includes static culturing and stirring culturing performed sequentially; Preferably, the temperature of the static culture is 22-26°C and the time is 2-3 days; Preferably, the temperature of the agitated culture is 22-26°C and the time is 5-7 days; Preferably, the stirring rate of the stirring culture is 500-1500 rpm.

7. The culture method according to any one of claims 1 to 6, characterized in that: In step (3), the components of the solid culture medium include cooked wheat, sawdust, bran, soybean meal, gypsum, lime and water; Preferably, the components of the solid culture medium include, by weight, 53.5-81.2 parts of cooked wheat, 10-30 parts of sawdust, 5-10 parts of bran, 3-5 parts of soybean meal, 0.5-1 part of gypsum, 0.3-0.5 part of lime and 40-60 parts of water.

8. The culture method according to claim 7, characterized in that: The preparation method of the solid culture medium comprises the following steps: boiling wheat with water, draining the water, pre-wetting sawdust with water, and mixing the processed wheat and sawdust with bran, soybean meal, gypsum and lime to obtain the solid culture medium.

9. The culture method according to any one of claims 1 to 8, characterized in that: In step (3), the culture temperature is 22-26° C. and the culture time is 20-30 days.

10. The culture method according to any one of claims 1 to 9, characterized in that: The culture method comprises the following steps: (1) inoculating a 2 mm × 2 mm × 2 mm-3 mm × 3 mm × 3 mm mushroom tissue in the middle of the stipe of a Tricholoma matsutake fruiting body on a mother culture medium, and culturing at 22-26° C. for 28-32 days to obtain a mother culture medium; based on 1 L of the culture medium system, the raw materials for preparing the mother culture medium include 150-200 g of potato, 15-20 g of glucose, 20-30 g of corn flour, 30-50 g of bran, 2-5 g of peptone, 2-3 g of yeast extract, 0.5-1 g of magnesium sulfate, 1-2 g of potassium dihydrogen phosphate, 30-50 mg of vitamin B1, and 16-20 g of agar powder; (2) inoculating the mother culture obtained in step (1) into a liquid shake flask culture medium, culturing at 22-26° C. for 2-3 days, and then stirring and culturing at 22-26° C. and 500-1500 rpm for 5-7 days to obtain a liquid shake flask culture; based on 1 L of the culture medium, the raw materials for preparing the liquid shake flask culture medium include 150-200 g of potato, 15-20 g of glucose, 20-30 g of corn flour, 30-50 g of bran, 3-5 g of peptone, 2-3 g of yeast extract, 0.5-1 g of magnesium sulfate, 1-2 g of potassium dihydrogen phosphate, 30-50 mg of vitamin B1 and 1-2 g of agar powder; (3) inoculating the liquid shake flask strain obtained in step (2) onto a solid culture medium, and culturing at 22-26° C. for 20-30 days to obtain a solid culture medium; the solid culture medium comprises, by weight, 53.5-81.2 parts of wheat, 10-30 parts of sawdust, 5-10 parts of bran, 3-5 parts of soybean meal, 0.5-1 part of gypsum, 0.3-0.5 parts of lime, and 40-60 parts of water.

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