Preservation method and unfreezing method of sparassis crispa strain

Abstract

The invention provides a preservation method and an unfreezing method of a sparassis crispa strain, and belongs to the technical field of strain preservation. The preservation method comprises the following steps: (1) inoculating a sparassis crispa strain into a culture medium, and culturing to obtain sparassis crispa hypha blocks; and (2) mixing the sparassis crispa hypha blocks obtained in the step (1) with a protective agent, cooling step by step, and cryopreserving to obtain the cryopreserved sparassis crispa strain. According to the preservation method, the preservation time of the sparassis crispa strain can be remarkably prolonged, and the growth speed and the yield of the sparassis crispa strain can be ensured not to be influenced. The strain preserved in the invention can be used for industrial cultivation after growth recovery, has good stability, is not easy to degrade, and can be stored for 3-10 years.

Description

Technical Field

[0001] The present invention relates to the technical field of strain preservation, and in particular to a preservation method and a thawing method of hydrangea strains. Background Art

[0002] Sparassis latifolia, also known as hydrangea, is rich in bioactive substances with anti-tumor, hematopoietic, and immune-modulating properties. It holds great promise for applications in health supplements, functional foods, and cosmetics. As people pursue a healthier diet and embrace a holistic approach to food, the market demand for fresh hydrangea is increasing year by year.

[0003] In recent years, with the continued expansion of hydrangea production, production problems caused by strain degeneration have frequently occurred during hydrangea factory cultivation. These problems include slower mycelial growth, darker mycelial color, difficulty differentiating primordia, poor consistency, and softening and yellowing of the base of fresh mushrooms, significantly impacting fresh mushroom yield and product quality. Furthermore, during year-round cultivation, periodic difficulty in fruiting can be common, impacting production efficiency. Degeneration of hydrangea strains is often caused by improper strain storage and excessive transfers between in vitro cultures. Therefore, developing a stable and effective strain storage method is crucial.

[0004] Currently, the preservation method for hydrangea is generally to store it in a 4°C refrigerator, on a slant in a test tube, and subculture it regularly. This method has the following disadvantages: (1) short storage time and easy degradation; (2) inability to maintain the strain's characteristics for a long time, resulting in unstable production; and (3) the workload of technicians in subculturing the culture in the test tube is high.

[0005] Ultra-low temperature preservation minimizes strain metabolism and is an effective method for long-term preservation of fungi. Liquid nitrogen preservation is considered the most effective and reliable method for long-term preservation, but due to the high cost of equipment and the need for regular monitoring and addition of liquid nitrogen, it is less commonly used in production. Cryopreservation using -80°C ultra-low temperature freezers requires readily available equipment and is simple to manage, making it suitable for production applications. While ultra-low temperature preservation can provide long-term stability and allow for normal mycelial growth after recovery, for edible fungi that require reproductive differentiation, mycelial growth does not guarantee fruiting. Therefore, maintaining the quality of the fungi and ensuring stable production are paramount. Summary of the Invention

[0006] The object of the present invention is to provide a preservation method and a thawing method for hydrangea strains, wherein the preservation method can significantly extend the preservation time of hydrangea strains and ensure that the growth rate and yield thereof are not affected.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preserving hydrangea strains, comprising the following steps:

[0009] (1) inoculating the hydrangea species into a culture medium, culturing, and obtaining hydrangea mycelium blocks;

[0010] (2) mixing the hydrangea mycelium blocks obtained in step (1) with a protective agent, cooling the mixture in steps, and freezing the mixture to obtain frozen hydrangea strains.

[0011] Preferably, the culture medium in step (1) is PDPA culture medium, and the composition of the PDPA culture medium is: 180-220 g / L of potato, 18-22 g / L of glucose, 1-3 g / L of fish meal peptone, 15-20 g / L of agar powder, and the balance is water per 1 L of culture medium.

[0012] Preferably, the culture temperature in step (1) is 24±1° C., and the culture time is 25 to 30 days.

[0013] Preferably, the protective agent in step (2) is glycerol, and the volume percentage of the glycerol is 10-15%.

[0014] Preferably, the step-by-step cooling method in step (2) is freezing at 3-5°C for 0.5-1.5h, and freezing at -15--25°C for 0.5-1.5h.

[0015] Preferably, the freezing temperature in step (2) is -75 to -85°C.

[0016] The present invention also provides a method for thawing the frozen hydrangea strains obtained by the preservation method, comprising the following steps:

[0017] The frozen hydrangea strains are placed at 35-40° C. to thaw, and the thawed hydrangea strains are placed in a PDA culture medium for culture.

[0018] Preferably, the frozen hydrangea strain is a strain that has been frozen for 1 month, 1 year to 10 years.

[0019] Preferably, the culture temperature is 24±1° C., and the culture time is 25 to 30 days.

[0020] The beneficial effects of the present invention compared with the prior art are:

[0021] The cryopreserved strains preserved by the method provided by the present invention can be directly inoculated onto PDPA culture medium for cultivation, exhibiting excellent stability, high uniformity, and simple operation. Furthermore, the preservation method provided by the present invention can effectively maintain the properties of the strains, resulting in excellent stability, uniform fruiting, and ready for industrial cultivation after growth recovery. Furthermore, within the same batch of cryopreserved strains, the hydrangea strains exhibit high consistency, stable properties, minimal variation, and are not susceptible to degradation. The strains can be stored for 3 to 10 years, ensuring their safe use.

[0022] The present invention uses a simple step-by-step cooling method (freezing the cryovial at 3-5°C for 0.5-1.5 hours and then at -15-25°C for 0.5-1.5 hours) to reduce the impact of crystallization on cells during gradient cooling. Compared with programmed cooling, this method does not require complex equipment and cumbersome operations, nor does it require the regular use and maintenance of nitrogen tanks used with programmed cooling equipment, thus reducing production safety risks. Furthermore, by ultra-low-temperature preservation of Hydrangea, the present invention can obtain excellent, stably preserved strains, reducing the uncertainty of subculture preservation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 This is a picture of the recovery growth of Hydrangea species on PDPA plates;

[0025] Figure 2 The differentiation of primordium of Hydrangea species;

[0026] Figure 3 This is the appearance of fresh mushrooms of the hydrangea species when they are fruiting. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] The present invention provides a method for preserving hydrangea strains, comprising the following steps:

[0033] (1) inoculating the hydrangea species into a culture medium, culturing, and obtaining hydrangea mycelium blocks;

[0034] (2) mixing the hydrangea mycelium blocks obtained in step (1) with a protective agent, cooling the mixture in steps, and freezing the mixture to obtain frozen hydrangea strains.

[0035] In the present invention, the culture medium in step (1) is preferably PDPA culture medium, and the composition of the PDPA culture medium is preferably: 180-220 g / L of potato, 18-22 g / L of glucose, 1-3 g / L of fish meal peptone, 15-20 g / L of agar powder, and the balance is water per 1 L of culture medium. It is further preferred that 190-210 g / L of potato, 19-21 g / L of glucose, 1.5-2.5 g / L of fish meal peptone, and the balance is water per 1 L of culture medium. g / L, agar powder 16-18g / L, the balance is water, and more preferably, each 1L of culture medium contains 200g / L of potato, 20g / L of glucose, 2g / L of fish meal peptone, 17g / L of agar powder, and the balance is water; the PDPA culture medium is preferably obtained after high-temperature sterilization and cooling treatment; the culture temperature is preferably 24±1°C, more preferably 24±0.5°C, and even more preferably 24°C; the culture time is preferably 2 5 to 30 days, more preferably 27 to 29 days, and more preferably 28 days; the protective agent in step (2) is preferably glycerol, and the volume percentage of the glycerol is preferably 10 to 15%, more preferably 12 to 14%, and more preferably 13%; the mixing is preferably carried out in a cryopreservation tube, and the capacity of the cryopreservation tube is preferably 2 to 5 ml; the cryopreservation tube is preferably sterilized at 121°C and then the protective agent is added and mixed with the hydrangea mycelium block; the step-by-step cooling method is preferably 3 to 5°C freezing for 0.5 to 1.5 hours, -15 to -25°C freezing for 0.5 to 1.5 hours; more preferably 3.5 to 4.5°C freezing for 0.8 to 1.2 hours, -18 to -22°C freezing for 0.8 to 1.2 hours; more preferably 4°C freezing for 1.0 hours, -20°C freezing for 1.0 hours; the freezing temperature is preferably -75 to -85°C, more preferably -78 to -82°C, and more preferably -80°C.

[0036] The present invention also provides a method for thawing the frozen hydrangea strains obtained by the preservation method, comprising the following steps:

[0037] The frozen hydrangea strains are placed at 35-40° C. to thaw, and the thawed hydrangea strains are placed in a PDA culture medium for culture.

[0038] In the present invention, the frozen hydrangea strain is preferably a strain that has been frozen for 1 month, 1 year to 10 years; the thawing temperature is preferably 36-38°C, and more preferably 37°C; the culture temperature is preferably 24±1°C, and more preferably 24°C; the culture time is preferably 25-30 days, and more preferably 27-29 days, and even more preferably 28 days.

[0039] Example 1

[0040] A method for preserving hydrangea strains, comprising the following steps:

[0041] (1) Cut a piece of Hydrangea spp. (Minxiu No. 1, provided by the Institute of Edible Fungi, Fujian Academy of Agricultural Sciences) and inoculate it into PDPA medium (composed of 200 g / L potato, 20 g / L glucose, 2 g / L fish meal peptone, 18 g / L agar powder, and the balance water per 1 L of culture medium; sterilized at 121°C for 15 min and cooled) with an inoculating needle. Culture at 24 ± 1°C for 28 days. Punch the Hydrangea spp. mycelial plate into uniform-sized mycelial blocks using a 5 mm borer to obtain Hydrangea spp. mycelial blocks.

[0042] (2) Sterilize 2 ml cryovials at 121°C for 15 min, add 10% by volume of glycerol, place the mycelial blocks obtained in step (1) in 2 ml cryovials containing glycerol, place 5 blocks in each tube, freeze at 4°C for 1.0 h, freeze at -20°C for 1.0 h, and freeze at -80°C to obtain frozen mycelial strains.

[0043] Example 2

[0044] A method for preserving hydrangea strains, comprising the following steps:

[0045] (1) Cut a piece of Hydrangea latifolia and inoculate it into PDPA medium (composition: 180 g / L potato, 18 g / L glucose, 1 g / L fish meal peptone, 15 g / L agar powder, and the balance water per 1 L of culture medium; sterilized at 121°C for 15 min and cooled) with an inoculating needle. Culture at 24 ± 0.5°C for 25 days. Punch the Hydrangea latifolia mycelial plate into uniform mycelial blocks using a 5 mm borer to obtain Hydrangea latifolia mycelial blocks.

[0046] (2) Sterilize 5 ml cryovials at 121°C for 15 min, add 15% by volume of glycerol, place the mycelial blocks obtained in step (1) in 2 ml cryovials containing glycerol, place 10 blocks in each tube, freeze at 3°C for 1.5 h, freeze at -25°C for 0.5 h, and freeze at -75°C to obtain frozen mycelial blocks.

[0047] Example 3

[0048] A method for preserving hydrangea strains, comprising the following steps:

[0049] (1) Cut a piece of Hydrangea latifolia and inoculate it into PDPA medium (composed of 220 g / L potato, 22 g / L glucose, 3 g / L fish meal peptone, 20 g / L agar powder, and the balance water per 1 L of culture medium; sterilized at 121°C for 15 min and cooled) using an inoculating needle. Culture at 24°C for 30 days, cut the Hydrangea latifolia mycelium plate into mycelium blocks of uniform size, and obtain Hydrangea latifolia mycelium blocks.

[0050] (2) Sterilize a 2 ml cryopreservation tube at 121°C for 15 min, add 12% by volume of glycerol, place the mycelial block of Hydrangea obtained in step (1) in a 2 ml cryopreservation tube containing glycerol, freeze at 5°C for 0.5 h, freeze at -15°C for 1.5 h, and freeze at -85°C to obtain the frozen Hydrangea strain.

[0051] Example 4

[0052] A thawing method comprises the following steps:

[0053] The cryopreservation tube of the Hydrangea species obtained in Example 1, which had been frozen for one month, was placed at 37° C. to thaw, and a mycelium block was taken from the tube and placed in PDPA medium at 24±1° C. for 25 days.

[0054] Example 5

[0055] A thawing method, the steps are as follows:

[0056] The frozen tube of the Hydrangea species obtained in Example 1 that had been frozen for one year was placed at 35° C. to thaw, and a mycelium block was taken from the tube and placed in PDPA medium at 24° C. to culture for 30 days.

[0057] Example 6

[0058] A thawing method comprises the following steps:

[0059] The frozen tube of the Hydrangea species obtained in Example 1 that had been frozen for 10 years was placed at 37° C. to thaw, and a mycelium block was taken from the tube and placed in PDPA medium at 24±0.5° C. to culture for 28 days.

[0060] Comparative Example 1

[0061] The conventional 4°C slant preservation method was used to preserve the Hydrangea species as follows:

[0062] Prepare PDPA culture medium: Each 1L of culture medium contains: 200g / L potato, 20g / L glucose, 2g / L fish meal peptone, 18g / L agar powder, and the balance is water. Mix the ingredients according to the above formula, dispense into test tubes, sterilize at high temperature, and after cooling, arrange them into inclined surfaces to obtain PDPA test tubes.

[0063] The broad-leaved hydrangea strain was inoculated into PDPA culture medium and cultured at 24±1°C for 28 days. The hydrangea mycelium plate was punched into mycelium blocks of uniform size with a 5mm puncher to obtain hydrangea mycelium blocks; a piece of the above-obtained hydrangea mycelium block was inoculated in the middle of the slant of the PDPA test tube and cultured at a constant temperature of 24±1°C for 25 to 30 days to obtain well-grown hydrangea mycelium; the test tube slant with well-grown mycelium was stored at 4°C. After the storage was completed, the hydrangea strain was placed in PDA culture medium and cultured at 24±1°C for 25 to 30 days to obtain the preserved hydrangea strain.

[0064] Test Example 1 Determination of mycelium growth performance

[0065] The preservation was carried out according to the method of Example 1 to obtain hydrangea strains after being frozen for 1 month, 1 year, 2 years and 3 years, respectively. The frozen hydrangea strains were thawed according to the method of Example 4 to obtain thawed hydrangea strains.

[0066] According to the method in Comparative Example 1, hydrangea strains were obtained after 1 month, 1 year, 2 years and 3 years of storage, respectively.

[0067] After 28 days of culture, the colony diameters of the Hydrangea species obtained above on PDPA plates were measured using a vernier caliper, and the mycelial growth rate (mm / d) was calculated as: mycelial growth rate (mm / d) = colony diameter (mm) / number of days in culture (d). The results are shown in Table 1.

[0068] Table 1 Mycelial growth rate after revival of preserved strains

[0069]

[0070] As shown in Table 1, the growth rate of mycelia preserved by the preservation method provided by the present invention remains basically the same at different storage times. After the slant storage time at 4°C exceeds one year, the mycelial activity decreases and the mycelial growth state is poor.

[0071] Test Example 2: Mushroom Fruiting Test

[0072] The preservation was carried out according to the method of Example 1 to obtain hydrangea strains after being frozen for 1 month, 1 year, 2 years and 3 years, respectively. The frozen hydrangea strains were thawed according to the method of Example 4 to obtain thawed hydrangea strains.

[0073] According to the method in Comparative Example 1, hydrangea strains were obtained after 1 month, 1 year, 2 years and 3 years of storage, respectively.

[0074] The bag cultivation method was used, using 330mm x 170mm x 0.05mm polypropylene bags filled with 0.85kg of wet material (composition: 75% sawdust, 25% complex nitrogen source), perforated in the center, autoclaved (126°C, 120 minutes), and cooled for later use. Thawed and preserved hydrangea strains were inoculated into the bags, respectively, and the mycelial culture temperature was 20°C. Ventilation was maintained during the incubation period. Once the primordia continued to grow and protrusions appeared, the bags were moved to a fruiting room and incubated at 16°C-20°C and 90%-95% relative humidity. Harvesting began when the fruiting bodies expanded and the edges became wavy. Fresh mushrooms were harvested by cutting the mushroom bodies above the cultivation medium and weighing the weight (g / bag). The average value for each batch was calculated. The results are shown in Table 2.

[0075] Table 2 Fruiting test results

[0076]

[0077] As shown in Table 2, the yield of Hydrangea species preserved by the preservation method provided by the present invention is higher than that when preserved at 4°C slant.

[0078] As can be seen from the above examples, the present invention provides a preservation method and a thawing method for Hydrangea species. The preservation method provided by the present invention can significantly extend the preservation time of Hydrangea species and ensure that their growth rate and yield are not affected.

[0079] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preserving hydrangea species, characterized in that: The steps include: (1) Inoculating the hydrangea strain into the culture medium, culturing, and obtaining hydrangea mycelium blocks; (2) Mixing the hydrangea mycelium blocks obtained in step (1) with a protective agent, cooling the mixture in steps, and freezing the mixture to obtain frozen hydrangea strains.

2. The preservation method according to claim 1, wherein: The culture medium in step (1) is PDPA culture medium, and the composition of the PDPA culture medium is as follows: each 1L of culture medium contains 180-220g / L of potato, 18-22g / L of glucose, 1-3g / L of fish meal peptone, 15-20g / L of agar powder, and the balance is water.

3. The preservation method according to claim 1, wherein: The culture temperature in step (1) is 24±1°C, and the culture time is 25-30 days.

4. The preservation method according to claim 1, wherein: The protective agent in step (2) is glycerol, and the volume percentage of the glycerol is 10-15%.

5. The preservation method according to claim 1, wherein: The step-by-step cooling method in step (2) is freezing at 3-5°C for 0.5-1.5h and freezing at -15--25°C for 0.5-1.5h.

6. The preservation method according to claim 1, wherein: The freezing temperature in step (2) is -75~-85°C.

7. A method for thawing frozen Hydrangea species obtained by the preservation method according to any one of claims 1 to 6, characterized in that: The steps include: The frozen hydrangea strains are placed at 35-40° C. to thaw, and the thawed hydrangea strains are placed in a PDA culture medium for culture.

8. The thawing method according to claim 7, characterized in that: The frozen hydrangea strains are strains that have been frozen for 1 month, 1 year to 10 years.

9. The thawing method according to claim 7, characterized in that: The culture temperature is 24±1° C., and the culture time is 25 to 30 days.

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