A method for preserving Boletus aereus strains and a preservation medium
A simple and cost-effective cultivation medium using small grains, wood chips, and bran maintains the viability and quality of black oyster mushroom strains for four and a half years, addressing the challenges of preservation and selection complexity.
Patent Information
- Application Number
- CN202210385341.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-04-13
- Publication Date
- 2025-07-15
- Estimated Expiration
- 2042-04-13
AI Technical Summary
Black boletus strains are prone to self-soluble under low temperature conditions. The existing technology is cumbersome and costly, making it difficult to store easily and for a long time. The breeding procedures for excellent strains are complex, the workload is large, and it is not easy to promote.
A preservation culture medium consisting of wheat grains, mixed wood chips, pine wood chips, bran and gypsum is used to prepare and preserve black boletus sclerosis to form a closely intertwined dormant body to resist the adverse environment.
The long-term preservation of the vitality and excellent performance of the black boletus sclerotia is achieved for up to four and a half years, which simplifies operation, reduces costs, and maintains the vitality and cultivation performance of the strain.
Smart Images
Figure BDA0003593418020000101
Abstract
Description
Technical Field
[0001] The present invention relates to the field of preservation of fungal strains, and particularly to a method for preserving Boletus aereus strains and a preservation medium. Background Art
[0002] The quality of fungal strains is directly related to the yield and quality of edible mushroom cultivation, and even affects the success or failure of production. The Boletus aereus strain is one of the key links in its artificial cultivation and bionic cultivation. Boletus aereus belongs to thermophilic basidiomycetes, and its mycelium is very sensitive to low temperature and will autolyze and die under low temperature conditions. The optimum temperature for mycelial growth is 28-30°C. When the temperature is lower than 15°C or higher than 30°C, the mycelial growth rate will drop sharply, which also indicates that it is difficult for Boletus aereus to be preserved under the condition of 0-6°C like other strains. Although there are studies on long-term low-temperature preservation of Boletus aereus strains in the prior art, they usually require pre-cooling and segmented treatment, or additional addition of various protective agent components, which have the disadvantages of cumbersome operation and increased cost. Currently, the commonly used test tube slant subculture preservation method in production preserves the strains at 15-20°C and transfers the tubes once every 3-6 months. This technology provides a simple and effective short-term preservation method, but also increases the risk of contamination of the strains and variation of their morphological and physiological characteristics.
[0003] In addition, most of the current excellent strains of Boletus aereus are obtained through natural selection, that is, by widely collecting fruiting bodies from different regions, different growth environments and different development periods to isolate strains, and systematically testing various aspects such as the mycelial growth rate, culture characteristics, adaptability to the cultivation environment, the early or late fruiting period, the shape, size, color, texture, etc. of the fruiting bodies of each strain, and finally screening out excellent strains. This breeding method has complex procedures, large workload and is not easy to promote and apply. Therefore, it is extremely important to design a method that can simply and effectively preserve Boletus aereus strains and maintain their excellent traits. Summary of the Invention
[0004] The purpose of the present invention is to provide a method for preserving Boletus aereus strains and a preservation medium to solve the problems existing in the above-mentioned prior art. This preservation method can preserve the viability and excellent properties of Boletus aereus sclerotia for up to four and a half years, solves the problem that Boletus aereus is not easy to be simply and long-term preserved, and provides technical support for the large-scale subculture and propagation of Boletus aereus.
[0005] To achieve the above purpose, the present invention provides the following solution:
[0006] The present invention provides a preservation medium for Boletus aereus sclerotia, comprising the following raw materials in parts by weight: 45-48 parts of wheat grains, 15-18 parts of miscellaneous wood chips, 15-17 parts of pine wood chips, 18-19 parts of wheat bran, 1-2 parts of gypsum and 1-2 parts of white sugar.
[0007] The present invention also provides a method for preparing the above-mentioned preservation medium for the sclerotium of Boletus aereus, comprising the following steps:
[0008] (1) Boil wheat grains until they are saturated with water absorption without breaking the skin, and then filter and dry them;
[0009] (2) Mix the remaining raw materials, add lemon water thereto, and finally add the filtered and dried wheat grains and stir evenly to obtain the preservation medium for the sclerotium of Boletus aereus.
[0010] Further, the concentration of the lemon water in step (2) is 20 mg / L, and the mass ratio of the lemon water to the remaining raw materials is 1:1.4.
[0011] The present invention also provides a method for preserving the strain of Boletus aereus, comprising the steps of inoculating the strain of Boletus aereus into the preservation medium for the sclerotium of Boletus aereus according to claim 1 to culture sclerotia, and then preserving them under the condition of 15-18 °C.
[0012] Further, the strain of Boletus aereus is the mother strain obtained by culturing Boletus aereus on a mother culture medium.
[0013] Further, the mother culture medium comprises the following raw materials: by weight, 30 parts of starch, 1 part of peptone, 20 parts of glucose, 1 part of potassium dihydrogen phosphate, and 16 parts of agar, with the balance being water.
[0014] Further, the mother culture medium is prepared according to the following steps: first dissolve the starch in water, then add peptone, potassium dihydrogen phosphate, and glucose and dissolve them, then add agar and stir evenly, and finally adjust the pH to 5.0 and make up the volume to 1 L with water.
[0015] Further, the culture conditions are as follows: first, culture at 28-30 °C with the humidity controlled at 60%-70% in the dark for 30 d, then adjust the temperature to 25-27 °C, and continue to culture at a humidity of 55%-65% and a light intensity of 50-100 lx for 20-30 d.
[0016] Further, the preservation conditions are to preserve in the dark with the humidity controlled at 50%-60%.
[0017] The present invention discloses the following technical effects:
[0018] (1) The sclerotium preservation medium provided by the present invention uses wheat grains as the main nutrient substrate to provide a carbon source. Through the operations of boiling and draining, the wheat grains can fully absorb water, which is beneficial for thorough autoclaving and can fully release the nutrients in the wheat grains. Miscellaneous wood chips provide lignocellulose, wheat bran provides a nitrogen source, and citric acid is used to adjust the pH value. The carbon-nitrogen ratio of the medium is 20-25:1, which is suitable for the growth of Boletus aereus mycelium. The pine wood chips contain aromatic substances that can induce the formation of sclerotia by the mycelium of Boletus aereus. The sclerotium of Boletus aereus is a tightly intertwined dormant body storing nutrients, which can resist adverse environments better than its mycelium and is easy to maintain the activity of the strain. Miscellaneous wood chips, pine wood chips and wheat bran are fillers for the pores between wheat grains, which can further absorb the surface moisture of wheat grains, adjust the water content of wheat grains, prevent wheat grains from sticking together, and provide space for the normal growth of mycelium.
[0019] (2) The preservation method of the present invention is simple in operation, the components of the preservation medium are easy to obtain, and the production cost is low. Moreover, there is no significant difference in the vitality and cultivation performance of the sclerotia of Boletus aereus preserved by the preservation method of the present invention and the original sclerotia. It can preserve the vitality and excellent properties of the sclerotia of Boletus aereus for up to four and a half years, solving the problem that Boletus aereus is not easy to be simply and long-term preserved, and providing technical guarantee for the large-scale subculture and propagation of Boletus aereus. Detailed implementation manners
[0020] The various exemplary implementation manners of the present invention will be described in detail below. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation manners of the present invention.
[0021] It should be understood that the terms described in the present invention are only for describing particular implementation manners and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0022] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes the preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.
[0023] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.
[0024] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0025] Unless otherwise specified, the test methods used in the examples are conventional methods; the reagents used are commercially available reagents unless otherwise specified.
[0026] The black boletus used in the following examples is a dark brown clematis boletus (Phlebopus portentosus) collected by the applicant, which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee on February 28, 2022, with a deposit number of CGMCC NO.40107, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. Other common black boletus on the market are also applicable to the preservation method of the present invention.
[0027] Example 1
[0028] 1. Mother stock culture
[0029] 1) Mother culture medium formula: 30 g starch, 1 g peptone, 20 g glucose, 1 g potassium dihydrogen phosphate, 16 g agar, and water to 1 L, pH 5.0.
[0030] 2) Preparation of mother culture medium: Weigh each component according to the formula in 1), first add starch into water to dissolve, then add peptone, potassium dihydrogen phosphate, and glucose to dissolve, finally add agar while heating and stirring, then add water to make up to 1L, and adjust the pH to 5.0 with 1N hydrochloric acid. Divide into 18mm×180mm test tubes while hot, each test tube containing 10-12ml, and plug with silicone stoppers.
[0031] 3) Sterilization: Place the test tube filled with culture medium vertically into the sterilizer and sterilize at 121°C for 20 to 30 minutes.
[0032] 4) Preparation of the inclined surface of the mother culture medium test tube: After sterilization, open the sterilizer and place the test tube at a certain slope (about 10°C) while it is still hot, with the front end of the culture medium inclined surface 40 to 50 mm away from the test tube mouth. The test tube will be formed into an inclined surface after cooling.
[0033] 5) Tissue isolation: Select Boletus aereus fruit bodies with normal morphological development, no pests or diseases, the edges of the caps not yet unfolded, and weighing 60 - 80 g as the isolation material. Disinfect the surface with 75% alcohol. Under aseptic conditions, cut the cap with a blade, and cut a 5 mm × 5 mm tissue block at the junction of the stipe and the cap and inoculate it into the center of the medium in 2). After inoculation, place it in an incubator at 28 - 30 °C in the dark for 15 - 20 d, check the mycelial growth every 2 - 3 d, and select those with good growth as the mother culture.
[0034] 2. Sclerotium culture
[0035] 1) Sclerotium medium formula: By weight, wheat grains 48%, miscellaneous wood chips 16%, pine wood chips 16%, wheat bran 18%, gypsum 1%, white sugar 1%.
[0036] 2) Preparation of sclerotium medium: Weigh each component according to the formula in 1). First, boil the wheat grains with water until they are thoroughly cooked and fully absorb water, with the skin intact and the inside transparent without white residue, and then filter them. Stir the miscellaneous wood chips, pine wood chips, wheat bran, gypsum, and white sugar evenly, add lemon water with a concentration of 20 mg / L according to the ratio of material to water quality of 1:1.4 to adjust the moisture content of the culture material, and finally add the wheat grains and stir evenly.
[0037] 3) Tube filling: Use a 32 mm × 200 mm test tube, fill the material to 1 / 2 of the test tube length, with the requirement of loose filling at the bottom and tight filling at the top, wipe the tube wall clean, and plug it tightly with a silica gel plug.
[0038] 4) Sterilization: Place the test tubes filled with the culture material vertically in the autoclave and sterilize at 126 °C for 2.5 h.
[0039] 5) Inoculation: When the temperature of the material drops below 30 °C, inoculate the mother culture cultured in 1 under aseptic conditions.
[0040] 6) Cultivation: At 28 - 30 °C, control the humidity at 60% - 70% and culture in the dark for 30 d, then adjust the temperature to 25 - 27 °C, control the humidity at 55% - 65%, and the light intensity at 50 - 100 lx and continue to culture for 20 - 30 d. A large number of sclerotia can be produced on the surface of the culture material and the test tube wall.
[0041] 3. Sclerotium preservation
[0042] Wrap the test tubes with sclerotia cultured in 2 tightly with kraft paper and store them in the dark at 15 °C and a humidity of 50% - 60% for 42 months.
[0043] 4. Sclerotium subculture
[0044] Under aseptic conditions, inoculate the sclerotia stored for a long time above into the test tubes of the mother culture medium, inoculate one sclerotium into each test tube, and culture at 28 °C.
[0045] Example 2
[0046] 1. The steps for mother spawn cultivation are the same as those in Example 1, with differences in 2. sclerotium cultivation, 3. sclerotium preservation, and 4. sclerotium subculture steps, which are as follows:
[0047] 2. Sclerotium cultivation
[0048] 1) Formula of sclerotium culture medium: By weight, wheat grains 45%, miscellaneous wood chips 18%, pine wood chips 15%, wheat bran 19%, gypsum 2%, white sugar 1%.
[0049] 2) Preparation of sclerotium culture medium: Weigh each component according to the formula in 1). First, boil the wheat grains thoroughly with water to let them fully absorb water until the skin is intact and the inside is transparent without white residue, then filter them dry, and stir evenly with miscellaneous wood chips, pine wood chips, wheat bran, gypsum, and white sugar. Add lemon water with a concentration of 20 mg / L according to the ratio of material to water quality of 1:1.4 to adjust the water content of the culture material, and finally add wheat grains and stir evenly again.
[0050] 3) Tube filling: Use a 32 mm×200 mm test tube, fill the material to 2 / 3 of the test tube length, with the requirement of loose at the bottom and tight at the top, wipe the tube wall clean, and plug it tightly with a silica gel plug.
[0051] 4) Sterilization: Place the test tubes filled with the culture material vertically in the autoclave and sterilize at 126 °C for 2.5 h.
[0052] 5) Inoculation: When the material temperature drops below 30 °C, inoculate the mother spawn cultured in 1 under sterile conditions.
[0053] 6) Cultivation: Cultivate at 28 - 30 °C with the humidity controlled at 60% - 70% in the dark for 30 d, then adjust the temperature to 25 - 27 °C, control the humidity at 55% - 65%, and continue to cultivate at a light intensity of 50 - 100 lx for 20 - 30 d. A large number of sclerotia can be produced on the surface of the culture material and the test tube wall.
[0054] 3. Sclerotium preservation
[0055] Wrap the test tubes of sclerotia cultured in 2 tightly with kraft paper and store them in the dark at 16 °C with a humidity of 50% - 60% for 45 months.
[0056] 4. Sclerotium subculture
[0057] Under sterile conditions, inoculate the sclerotia preserved for a long time above into the test tubes of the mother spawn culture medium, inoculate one sclerotium into each test tube, and cultivate at 30 °C.
[0058] Example 3
[0059] 1. The steps for mother spawn cultivation are the same as those in Example 1, with differences in 2. sclerotium cultivation, 3. sclerotium preservation, and 4. sclerotium subculture steps, which are as follows:
[0060] 2. Sclerotium culture
[0061] 1) Medium formula: By weight, 47% wheat grains, 15% miscellaneous wood chips, 17% pine wood chips, 18% wheat bran, 1% gypsum, 2% white sugar.
[0062] 2) Preparation of sclerotium medium: Weigh each component according to the formula in 1). First, cook the wheat grains thoroughly with water until they absorb enough water, with the skin intact and the inside transparent without white residue. Then filter them dry and mix evenly with miscellaneous wood chips, pine wood chips, wheat bran, gypsum, and white sugar. Add lemon water with a concentration of 20 mg / L according to the ratio of material to water quality of 1:1.4 to adjust the moisture content of the culture medium. Finally, add wheat grains and mix evenly.
[0063] 3) Tube filling: Use a 32 mm × 200 mm test tube, fill the material to 1 / 2 of the test tube length, with the requirement of loose at the bottom and tight at the top. Wipe the tube wall clean and plug it tightly with a silica gel plug.
[0064] 4) Sterilization: Place the test tubes filled with the culture material vertically into the autoclave and sterilize at 126 °C for 2.5 h.
[0065] 5) Inoculation: When the temperature of the material drops below 30 °C, inoculate the mother culture that has been cultured in 1 under sterile conditions.
[0066] 6) Cultivation: Cultivate at 28 - 30 °C with the humidity controlled at 60% - 70% in the dark for 30 d, then adjust the temperature to 25 - 27 °C, the humidity to 55% - 65%, and the light intensity to 50 - 100 lx and continue to cultivate for 20 - 30 d. A large number of sclerotia can be produced on the surface of the culture material and the test tube wall.
[0067] 3. Sclerotium preservation
[0068] Wrap the test tubes of sclerotia cultured in 2 tightly with kraft paper and store them in the dark at 18 °C with a humidity of 50% - 60% for 54 months.
[0069] 4. Subculture of sclerotium
[0070] Under sterile conditions, inoculate the sclerotia preserved for a long time above into the test tubes of the mother culture medium, inoculate one sclerotium into each test tube, and cultivate at 30 °C.
[0071] Comparative Example 1
[0072] The difference from Example 1 is that in 2) Preparation of medium in sclerotium culture, pine wood chips are adjusted to oak wood chips.
[0073] Comparative Example 2
[0074] The difference from Example 1 is that in the sclerotium culture, the medium formula is: 200 g of potato, 1 g of peptone, 20 g of glucose, 1 g of potassium dihydrogen phosphate, 16 g of agar, and 1 L of water.
[0075] Comparative Example 3
[0076] The difference from Example 1 is that in the sclerotium preservation, the preservation temperature is 10°C.
[0077] Effect verification
[0078] Using the group where the sclerotia obtained in the sclerotium culture were directly inoculated into the test tube of the mother culture medium for propagation culture without preservation as the control group, the strains after propagation of the control group and the groups of Examples 1-3 and Comparative Examples 1-3 were inoculated into the bagged substrate for artificial cultivation, and 100 groups were set up in parallel respectively. The growth conditions of the strains in the control group, Examples 1-3 and Comparative Examples 1-3 were counted daily on time, and the specific conditions are shown in Table 1.
[0079] Table 1 Growth conditions of strains in each group
[0080]
[0081] It can be seen from Table 1 that after the sclerotia preserved in Examples 1-3 were activated and cultured, compared with the first-generation sclerotia (control group), there were no significant differences in the mycelium germination rate, growth rate, mycelium growth potential and single mushroom weight, indicating that the sclerotia using the preservation method of the present invention have the same mycelium vitality and strain cultivation characteristics as the original sclerotia. However, due to the adjustment of the preservation medium in Comparative Examples 1-2, the sclerotia vitality and mycelium growth potential obtained were inferior to those of the present invention; in Comparative Example 3, low-temperature preservation was used, resulting in a decrease in sclerotia vitality, difficult germination, and affecting subsequent mycelium growth.
[0082] The above-described embodiments are only used to describe the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.
Claims
1. A method for preserving the strain of Boletus aereus, characterized in that, It includes the steps of inoculating the Boletus aereus strain into a Boletus aereus sclerotium preservation medium for cultivation to produce sclerotia, and then storing them in the dark at 15°C with the humidity controlled at 50%-60%; the Boletus aereus sclerotium preservation medium is made from the following raw materials: by weight percentage, wheat grains 48%, miscellaneous sawdust 16%, pine sawdust 16%, wheat bran 18%, gypsum 1% and white sugar 1%; The preparation method of the Boletus aereus sclerotium preservation medium includes the following steps: (1) Boil the wheat grains until they are saturated with water absorption and the skin does not break, and then filter and dry them; (2) Mix the remaining raw materials, add lemon water to them, and finally add the filtered and dried wheat grains and stir evenly to obtain the Boletus aereus sclerotium preservation medium; The lemon water in step (2) has a concentration of 20 mg / L, and the mass ratio of it to the remaining raw materials is 1:1.
4.
2. A method for preserving the strain of Boletus aereus, characterized in that, It includes the steps of inoculating the Boletus aereus strain into a Boletus aereus sclerotium preservation medium for cultivation to produce sclerotia, and then storing them in the dark at 16°C with the humidity controlled at 50%-60%; the Boletus aereus sclerotium preservation medium is made from the following raw materials: by weight percentage, wheat grains 45%, miscellaneous sawdust 18%, pine sawdust 15%, wheat bran 19%, gypsum 2% and white sugar 1%; The preparation method of the Boletus aereus sclerotium preservation medium includes the following steps: (1) Boil the wheat grains until they are saturated with water absorption and the skin does not break, and then filter and dry them; (2) Mix the remaining raw materials, add lemon water to them, and finally add the filtered and dried wheat grains and stir evenly to obtain the Boletus aereus sclerotium preservation medium; The lemon water in step (2) has a concentration of 20 mg / L, and the mass ratio of it to the remaining raw materials is 1:1.
4.
3. A method for preserving the strain of Boletus aereus, characterized in that, It includes the steps of inoculating the Boletus aereus strain into a Boletus aereus sclerotium preservation medium for cultivation to produce sclerotia, and then storing them in the dark at 18°C with the humidity controlled at 50%-60%; the Boletus aereus sclerotium preservation medium is made from the following raw materials: by weight percentage, wheat grains 47%, miscellaneous sawdust 15%, pine sawdust 17%, wheat bran 18%, gypsum 1% and white sugar 2%; The preparation method of the Boletus aereus sclerotium preservation medium includes the following steps: (1) Boil the wheat grains until they are saturated with water absorption and the skin does not break, and then filter and dry them; (2) Mix the remaining raw materials, add lemon water to them, and finally add the filtered and dried wheat grains and stir evenly to obtain the Boletus aereus sclerotium preservation medium; The lemon water in step (2) has a concentration of 20 mg / L, and the mass ratio of it to the remaining raw materials is 1:1.
4.
4. The method for preserving the Boletus aereus fungal strain according to any one of claims 1-3, characterized in that, The Boletus aereus strain is the mother strain obtained by culturing Boletus aereus in a mother culture medium.
5. The method for preserving the Boletus aereus strain according to any one of claims 1 to 3, characterized in that, The cultivation conditions are as follows: first, cultivate in the dark at 28-30°C with the humidity controlled at 60%-70% for 30 d, then adjust the temperature to 25-27°C, and continue to cultivate for 20-30 d with the humidity controlled at 55%-65% and the light intensity at 50-100 lx.