Rose-red vein boletus strain preservation culture medium and preparation method thereof

Abstract

The invention discloses a culture medium for preserving boletus edulis and a preparation method thereof, and belongs to the technical field of boletus cultivation. The culture medium for preserving boletus edulis comprises the following components by mass percentage: 8-12% fresh oats, 15-25% grains, 15-25% soil, 15-25% sawdust, 8-12% rice husks, 8-12% silkworm excrement, 3-8% vermiculite, 2-4% limestone powder, 1-3% peat soil, 0.2-0.6% betaine, 0.05-0.15% KCl, 0.1-0.15% MgSO4, 0.05-0.12% MnSO4, 0.01-0.03% licorice antioxidant, and 0.2-0.4% EM microbial agent. The technical solution of the invention can effectively solve the problems of short preservation time, poor strain stability, and easy mutation and degradation of boletus edulis in existing preservation methods.

Description

Technical Field

[0001] The invention belongs to the technical field of boletus cultivation, and particularly relates to a culture medium for preserving boletus edulis strains and a preparation method thereof. Background Art

[0002] Boletus mushrooms are numerous and widely distributed, with over 800 species reported worldwide. my country boasts a rich boletus resource, with over 390 species of boletus with tubes or gills, 199 of which are edible. Boletus mushrooms are beloved by consumers worldwide for their nutritious and delicious taste. However, since most boletes are symbiotic, living in a symbiotic relationship with their host plant, artificial cultivation is challenging. Currently, there are no reports of successful artificial cultivation of other boletes, with the exception of the dark brown clematis and the Chinese saprophytic boletus. The dark brown clematis variety can be cultivated saprophytically under mushroom house conditions, allowing for year-round large-scale production. The rose-veined boletus is rich in protein, crude fat, total sugars, crude fiber, various minerals, and amino acids, making it a highly nutritious edible fungus. Its delicious flavor and vibrant color make it a popular choice among consumers. The distribution range of Boletus rosaceus is narrow. It grows together with Boletus rufipogonus in an abandoned loquat orchard. Its yield is scarce and it is a rare and endangered species. No other distribution areas or research reports on this species have been found.

[0003] Rose-veined Boletus is a new strain recently discovered with cultivation potential. The mycelium of Rose-veined Boletus grows slowly and has harsh growth conditions. It becomes inactivated after being stored at a low temperature of 4°C for 2 months. Commonly used preservation methods include low-temperature slant preservation, liquid paraffin preservation, and aqueous solution preservation. However, the existing preservation methods all have problems such as short preservation time, easy contamination, easy aging, poor strain stability, and easy mutation and degeneration.

[0004] Rose-veined boletus is a medium-to-high temperature edible fungus. Its mycelium grows slowly and is easily oxidized. Its low-temperature storage time is short. In addition, its distribution range is narrow and its yield is small. Therefore, in order to preserve the germplasm resources of rose-veined boletus and continuously study it, the preservation of the strain is particularly important. Summary of the Invention

[0005] The technical problem to be solved by the present invention is that the existing method for preserving Boletus edulis has the problems of short preservation time, poor strain stability, and easy mutation and degeneration.

[0006] The technical solution adopted by the present invention to solve the technical problem is as follows: a culture medium for preserving boletus edulis strains comprises the following components by mass percentage: 8-12% of fresh oats, 15-25% of grains, 15-25% of soil, 15-25% of sawdust, 8-12% of rice husks, 8-12% of silkworm excrement, 3-8% of vermiculite, 2-4% of limestone powder, 1-3% of peat soil, 0.2-0.6% of betaine, 0.05-0.15% of KCl, 0.1-0.15% of MgSO4, 0.05-0.12% of MnSO4, 0.01-0.03% of liquorice antioxidants, and 0.2-0.4% of EM bacterial agent.

[0007] Furthermore, fresh oats 10%, grains 20%, soil 20%, sawdust 20%, husks 10%, silkworm litter 10%, vermiculite 4%, limestone powder 3%, peat soil 2%, betaine 0.4%, KCl 0.1%, MgSO4 0.13%, MnSO4 0.1%, licorice antioxidant 0.02%, and EM bacterial agent 0.25%.

[0008] The particle diameter of the soil is 0.1-1 cm, the particle diameter of the sawdust is 0.5-2 cm, the particle diameter of the limestone powder is ≤0.5 mm, and the particle diameter of the peat soil is ≤0.5 mm.

[0009] The preparation method of the above-mentioned culture medium for preserving the boletus edulis comprises the following steps:

[0010] a. Prepare raw materials by Rose Red Vein Boletus culture medium;

[0011] b. Fresh sawdust and husks were fully pre-wetted with water at room temperature and covered with film for 24h, peat soil and silkworm litter were added and mixed to control the moisture content to 55-60%, and then EM bacteria were added and fermented for two weeks covered with plastic film to obtain a fermentation product;

[0012] c. The fresh grains were soaked at room temperature for 24h and then removed and drained, and then mixed with soil, vermiculite, limestone powder, the fermented product obtained in step b, fresh oats, and the mixture was piled for 48 hours to obtain;

[0013] d. The mixture obtained in step c was placed in polypropylene bags and autoclaved at 120-125 ° C for 85-95min before removal. The licorice antioxidants were completely dissolved in ethanol and then completely dissolved in deionized water. KCl, MgSO4, MnSO4 and betaine were mixed and then sprayed on the sterilized mixture and stirred evenly to control the moisture content to 60-70%;

[0014] e. The mixture after step d was placed in a test tube, sealed with a silicone stopper, sterilized at 120-125 ° C for 28-32 min, taken out, and placed in a sterile inoculation chamber to cool to room temperature to obtain a culture medium for preserving boletus edulis.

[0015] Furthermore, in the above step d, the polypropylene bag has a size of 25 cm×45 cm×0.05 mm and is sterilized under high pressure at 121° C. for 90 min.

[0016] Furthermore, in the above step d, the ethanol concentration is 75%.

[0017] Furthermore, in the above step d, KCl, MgSO4, MnSO4 and betaine are completely dissolved and mixed in a mass ratio of the total mass of KCl, MgSO4, MnSO4 and betaine to water = 1:10.

[0018] Furthermore, in the above step e, the test tube has a specification of 30 mm×150 mm and is sterilized under high pressure at 121° C. for 30 min.

[0019] Preservation method of rose-veined boletus strain: select the area of ​​the culture dish where the mycelium grows more neatly at the edge of the plate, punch a hole with a 2 cm diameter hole, and then inoculate it into the culture medium prepared according to the above-mentioned method for preparing the rose-veined boletus strain preservation medium. Cultivate at 25°C. When the mycelium grows to 3 / 5 of the bag, seal it with paraffin and store it in a 5°C refrigerator.

[0020] The beneficial effects of the present invention are as follows: the culture medium for preserving boletus edulis strains and the preparation method thereof provided by the present invention; fresh oats, grains, soil, sawdust, and husks are macromolecular nutrient protectants; small molecule protectants such as betaine; osmotic regulator KCl; antioxidants such as licorice antioxidants; silkworm excrement can provide good organic matter and other nutrients; alkaloids, flavonoids, sterols, and vitamin E in the silkworm excrement are good antioxidants; limestone powder can regulate the stable pH value of the culture medium and change the air permeability of the matrix; vermiculite can provide good air permeability and water retention; peat soil contains a large amount of humic acid, which is conducive to the growth of mycelium; and the EM bacterial agent can promote the fermentation of sawdust, husks, and silkworm excrement, soften the cellulose in the sawdust and husks, reduce the content of tannin in the sawdust, and reduce the pH value in the silkworm excrement.

[0021] The rose-veined boletus strain preservation culture medium and the preparation method thereof of the present invention select fresh oats, grains, soil, sawdust, husks, KCl, MgSO4, MnSO4, betaine, licorice antioxidants, silkworm excrement, peat soil, limestone powder, vermiculite, and EM fermentation bacteria agent for combined use. The various components complement each other's advantages. The rose-veined boletus strain preservation culture medium and the preparation method thereof of the present invention can greatly extend the preservation time of the rose-veined boletus strain, and the strain has good stability, strong vitality, and is not easy to degenerate. DETAILED DESCRIPTION

[0022] The technical solution of the present invention can be implemented specifically in the following manner.

[0023] The culture medium for preserving boletus edulis has the following components in percentage by mass: 8-12% fresh oats, 15-25% grains, 15-25% soil, 15-25% sawdust, 8-12% rice husks, 8-12% silkworm litter, 3-8% vermiculite, 2-4% limestone powder, 1-3% peat soil, 0.2-0.6% betaine, 0.05-0.15% KCl, 0.1-0.15% MgSO4, 0.05-0.12% MnSO4, 0.01-0.03% licorice antioxidant, and 0.2-0.4% EM bacterial agent.

[0024] Preferably, the composition comprises 10% fresh oats, 20% grains, 20% soil, 20% sawdust, 10% husks, 10% silkworm litter, 4% vermiculite, 3% limestone powder, 2% peat soil, 0.4% betaine, 0.1% KCl, 0.13% MgSO4, 0.1% MnSO4, 0.02% licorice antioxidant, and 0.25% EM bacterial agent.

[0025] The particle diameter of the soil is 0.1-1 cm, the particle diameter of the sawdust is 0.5-2 cm, the particle diameter of the limestone powder is ≤0.5 mm, and the particle diameter of the peat soil is ≤0.5 mm.

[0026] The present invention also provides a method for preparing the above-mentioned Boletus edulis strain preservation culture medium, comprising the following steps:

[0027] a. Prepare raw materials by Rose Red Vein Boletus culture medium;

[0028] b. Fresh sawdust and husks were fully pre-wetted with water at room temperature and covered with film for 24h, peat soil and silkworm litter were added and mixed to control the moisture content to 55-60%, and then EM bacteria were added and fermented for two weeks covered with plastic film to obtain a fermentation product;

[0029] c. The fresh grains were soaked at room temperature for 24h and then removed and drained, and then mixed with soil, vermiculite, limestone powder, the fermented product obtained in step b, fresh oats, and the mixture was piled for 48 hours to obtain;

[0030] d. The mixture obtained in step c was placed in polypropylene bags (size 25cm × 45cm × 0.05mm), autoclaved at 120-125 ℃ for 85-95min, and then removed. The licorice antioxidants were completely dissolved in ethanol and then completely dissolved in deionized water, KCl, MgSO4, MnSO4 and betaine were mixed, and then sprayed on the sterilized mixture and stirred evenly to control the moisture content to 60-70%;

[0031] e. The mixture after step d was placed in a test tube (size 30mm × 150mm), sealed with a silicone stopper and sterilized at 120-125 ° C for 28-32min, then taken out and placed in a sterile inoculation chamber to cool to room temperature to obtain a culture medium for preserving the boletus edulis.

[0032] Preferably, in the above step d, autoclaving is performed at 121° C. for 90 min; the ethanol concentration is 75%; and KCl, MgSO 4 , MnSO 4 and betaine are completely dissolved and mixed in a mass ratio of the total mass of KCl, MgSO 4 , MnSO 4 and betaine to water of 1:10.

[0033] Preferably, in the above step e, high pressure sterilization is performed at 121° C. for 30 min.

[0034] Preservation method of rose-veined boletus strain: select the area of ​​the culture dish where the mycelium grows more neatly at the edge of the plate, punch a hole with a 2 cm diameter hole, and then inoculate it into the culture medium prepared according to the above-mentioned method for preparing the rose-veined boletus strain preservation medium. Cultivate at 25°C. When the mycelium grows to 3 / 5 of the bag, seal it with paraffin and store it in a 5°C refrigerator.

[0035] The technical solutions and effects of the present invention are further illustrated below through practical examples.

[0036] Example

[0037] The components of the culture medium for preserving Boletus rosidii in the embodiments of the present invention are, by mass percentage, 10% fresh oats, 20% grains, 20% soil, 20% sawdust, 10% rice husks, 10% silkworm excrement, 4% vermiculite, 3% limestone powder, 2% peat, 0.4% betaine, 0.1% KCl, 0.13% MgSO₄, 0.1% MnSO₄, 0.02% licorice antioxidant, and 0.25% EM microbial agent. The licorice antioxidant was purchased from Chongqing Tianrun Biological Products Co., Ltd.; the EM microbial agent was purchased from Shanxi Changtai Animal Pharmaceutical Co., Ltd. in accordance with standard Q / SCY010-213; and the vermiculite was purchased from Lingshou County Xuhai Mineral Products Trading Co., Ltd. in accordance with standards JC / T810-2008 and JC / 441-1996.

[0038] (1) Effects of different preservation methods on the preservation effect and time of strains

[0039] Example: A portion of the culture dish where the mycelium grew more neatly at the edge of the dish was selected, and a hole was punched with a 2 cm diameter hole. The hole was then inoculated into a culture medium prepared according to the above-mentioned method for preparing a culture medium for the preservation of Boletus edulis. The culture was incubated at 25°C. When the mycelium grew to 3 / 5 of the bag, the bag was sealed with paraffin and stored in a 5°C refrigerator.

[0040] Comparative Example: The conventional aqueous solution preservation method and the low-temperature slant preservation method were used to preserve the strains.

[0041] Low-temperature slant preservation method: Use PDA enriched culture medium (formulation components: 200g potatoes, 2g beef extract, 1g MgSO4, 1.5g KH2PO4, 20g glucose, 20g agar powder, 1L tap water, natural pH; preparation method: place the prepared culture medium into a 500ml Erlenmeyer flask and autoclave at 121°C for 30 minutes. Pour 30ml of the sterilized culture medium into each culture dish using a sterilized volumetric flask and cool and solidify for use.) Select the area on the edge of the culture dish where the mycelium of the strain is growing more neatly, punch a hole with a 2cm diameter hole, and inoculate it onto the slant of the test tube. Incubate at 25°C, and store the grown slant test tube in a 4°C refrigerator.

[0042] Aqueous solution preservation method: Use distilled water to prepare NaCl solution, sterilize it by high pressure at 121℃ for 20 minutes, select the area on the edge of the culture dish where the mycelium grows more neatly, punch it with a 2 cm diameter puncher, and inoculate it into a sterilized 5 mL cryovial. Repeat the operation, put 5 culture blocks in each cryovial, use a pipette to transfer 3-4 mL of NaCl solution into each cryovial, so that the NaCl solution immerses the culture block, seal it, and store it in a 4℃ refrigerator.

[0043] The experimental results of the examples and comparative examples are shown in Table 1 (Note: "+" indicates mycelial growth).

[0044] Table 1 Preservation effect

[0045]

[0046] (2) Effect of limestone powder on strain preservation

[0047] Example: A 2 cm diameter hole was punched in a culture dish where mycelium grew neatly at the edge of the dish. The hole was then inoculated into a test tube culture prepared using the above-described Boletus rosaceae culture preservation medium and preparation method. The culture was incubated at 25°C. When the mycelium reached 3 / 5 of its volume, the tube was sealed with paraffin and stored in a 5°C refrigerator. Comparative Example: No limestone powder was added to the Boletus rosaceae culture preservation medium. The remaining steps were the same as in Example. The experimental results are shown in Table 2 (Note: "+" indicates mycelium growth).

[0048] Table 2 Preservation effect

[0049]

[0050]

[0051] (3) Effect of Licorice Antioxidants on the Preservation of Bacteria

[0052] Example: A 2 cm diameter hole was punched in a culture dish where mycelium grew neatly at the edge of the dish. The hole was then inoculated into a test tube culture prepared using the aforementioned Boletus rosaceae culture preservation medium and preparation method. The culture was incubated at 25°C. When the mycelium reached 3 / 5 of its volume, the tube was sealed with paraffin and stored in a 5°C refrigerator. Comparative Example: Licorice antioxidant was omitted from the Boletus rosaceae culture preservation medium. The remaining steps were the same as in Example. The experimental results are shown in Table 3 (Note: "+" indicates mycelium growth).

[0053] Table 3 Preservation effect

[0054]

[0055] (4) Effect of silkworm excrement on bacterial strain preservation

[0056] Example: A 2 cm diameter hole was punched in a culture dish where mycelial growth was relatively uniform at the edge of the dish. The hole was then inoculated into a test tube culture prepared using the aforementioned culture medium and preparation method for the Boletus rosaceae strain. The tube was incubated at 25°C. When the mycelium reached 3 / 5 of its volume, the tube was sealed with paraffin and stored in a 5°C refrigerator. Comparative Example: Silkworm excrement was omitted from the culture medium for the Boletus rosaceae strain. The remaining steps were the same as in Example. The experimental results are shown in Table 4 (Note: "+" indicates mycelial growth).

[0057] Table 4 Preservation effect

[0058]

[0059]

[0060] (5) Effect of fermentation treatment on strain preservation

[0061] Example: A 2 cm diameter hole was punched in a culture dish where mycelial growth was relatively uniform at the edge of the dish. The hole was then inoculated into a test tube culture prepared using the aforementioned Boletus ruberoidii culture preservation medium and preparation method. The culture was incubated at 25°C. When the mycelium reached 3 / 5 of its volume, the tube was sealed with paraffin and stored in a 5°C refrigerator. Comparative Example: The fermentation treatment in step b was omitted when preparing the Boletus ruberoidii culture preservation medium. The remaining steps were the same as in Example. The experimental results are shown in Table 5 (Note: "+" indicates mycelial growth).

[0062] Table 5 Preservation effect

[0063]

Claims

1. A method for preparing a culture medium for preserving the culture of Boletus edulis, characterized in that The steps include: a. Prepare raw materials according to the composition of the culture medium for preserving boletus cultures; The components of the culture medium for preserving the boletus rosaceae are as follows by mass percentage: 8-12% fresh oats, 15-25% grains, 15-25% soil, 15-25% sawdust, 8-12% rice husks, 8-12% silkworm litter, 3-8% vermiculite, 2-4% limestone powder, 1-3% peat soil, 0.2-0.6% betaine, 0.05-0.15% KCl, 0.1-0.15% MgSO4, 0.05-0.12% MnSO4, 0.01-0.03% licorice antioxidant, and 0.2-0.4% EM bacterial agent. b. Fresh sawdust and husks were fully pre-wetted with water at room temperature and covered with film for 24 hours. Peat soil and silkworm litter were added and mixed to control the moisture content to 55-60%. EM inoculants were then added and fermented for two weeks covered with plastic film to obtain a fermented product. c. The fresh grains were soaked at room temperature for 24h and then removed and drained, and then mixed with soil, vermiculite, limestone powder, the fermented product obtained in step b, fresh oats, and the mixture was piled for 48 hours to obtain; d. The mixture obtained in step c was placed in a polypropylene bag and sterilized at 120-125 ° C for 85-95 min. After removal, the licorice antioxidant was completely dissolved in ethanol and mixed with KCl, MgSO4, MnSO4 and betaine completely dissolved in deionized water. The mixture was then sprayed on the sterilized mixture and stirred evenly to control the moisture content to 60-70%. e. The mixture after step d was placed in a test tube, sealed with a silicone stopper, sterilized at 120-125 ° C for 28-32 min, taken out, and placed in a sterile inoculation chamber to cool to room temperature to obtain a culture medium for preserving boletus edulis.

2. The method for preparing the culture medium for preserving the boletus edulis according to claim 1, wherein: In step a, the components of the rose red vein boletus culture preservation medium are as follows by mass percentage: 10% fresh oats, 20% grains, 20% soil, 20% sawdust, 10% husks, 10% silkworm litter, 4% vermiculite, 3% limestone powder, 2% peat soil, 0.4% betaine, 0.1% KCl, 0.13% MgSO4, 0.1% MnSO4, 0.02% licorice antioxidant, and 0.25% EM bacterial agent.

3. The method for preparing the culture medium for preserving the boletus edulis according to claim 1 or 2, wherein: The particle diameter of the soil is 0.1-1 cm, the particle diameter of the sawdust is 0.5-2 cm, the particle diameter of the limestone powder is ≤0.5 mm, and the particle diameter of the peat soil is ≤0.5 mm.

4. The method for preparing the culture medium for preserving the boletus edulis according to claim 1, wherein: In step d, the polypropylene bag has a size of 25 cm×45 cm×0.05 mm and is sterilized under high pressure at 121° C. for 90 min.

5. The method for preparing the culture medium for preserving Boletus edulis according to claim 1, wherein: In step d, the ethanol concentration is 75%.

6. The method for preparing the culture medium for preserving Boletus edulis according to claim 1, wherein: In step d, KCl, MgSO 4 , MnSO 4 and betaine are completely dissolved and mixed in a mass ratio of the total mass of KCl, MgSO 4 , MnSO 4 and betaine to water = 1:

10.

7. The method for preparing the culture medium for preserving Boletus edulis according to claim 1, wherein: In step e, the test tube has a specification of 30 mm×150 mm and is sterilized under high pressure at 121° C. for 30 min.

8. A method for preserving Boletus edulis, characterized in that: Select the area where the mycelium of the strain grows more neatly at the edge of the petri dish, punch a hole with a 2 cm diameter hole, and then inoculate it into the rose-red vein boletus strain preservation medium prepared by the preparation method according to any one of claims 1 to 7, culture it at 25°C, and when the mycelium grows to 3 / 5 of the bag, seal it with paraffin and store it in a 5°C refrigerator.

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