Preservation medium and method for preventing morchella sp. degeneration

Abstract

The application belongs to the field of agricultural microorganism technology, and particularly relates to a preservation medium and a preservation method for preventing Morel strain degeneration. The formula of 1L of the preservation medium is as follows: 12g of sucrose, 6.0-6.2g of ammonium sulfate, 1g of potassium dihydrogen phosphate, 0.5-0.6g of magnesium sulfate, 18-20g of agar, 1000mL of distilled water, and natural pH. The application also adopts a method for preserving Morel strain at-80 DEG C ultra-low temperature. The method can preserve the good strain with fast mycelium growth and high yield during strain activation, and the good strain can be preserved to the second year for use, so that it is unnecessary to prepare the strain every year, and the economic cost and time cost are reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of agricultural microorganism technology, and particularly relates to a preservation medium and a preservation method for preventing Morchella spp. from degeneration. BACKGROUND

[0002] Morchella spp. is named because its appearance is very similar to a sheep's stomach. Morchella spp. is crisp and delicious, and contains various amino acids, rich mineral elements and vitamins necessary for human body. According to the report of Emlie Jacguetantd, there are 28 species of Morchella spp. in total. According to the known report, there are 20 species of Morchella spp. in China at present, which are mainly distributed in Hebei, Henan, Xinjiang, Sichuan, Hunan, Gansu, Qinghai, Heilongjiang and Liaoning, and have many local names due to different regions, such as sheep's stomach vegetable, son fungus, honeycomb fungus, sheep's stomach, sheep's stomach mushroom and sheep's fungus.

[0003] Artificial cultivation is an essential link for Morchella spp. due to its small quantity, although the artificial cultivation of Morchella spp. has been successful, the problems of unstable yield and quality are still difficult to break through. The Morchella spp. cultivation technology invented by D. Ower in 1986 obtained a patent, was adopted by multiple companies for commercialized patent cultivation, and operated for more than ten years in the United States. In 2008, the production was finally stopped due to the degeneration of the production strain, which not only affects the quality and yield of the product itself, but also seriously affects the efficiency of the enterprise. How to preserve the strain to keep its original properties unchanged has an important influence on the normal operation of factory production and scientific research.

[0004] The existing technology has the problem that the conventional preservation method of Morchella spp. is 4 DEG C test tube slant cold storage, but the mycelium grows slowly when the strain is activated after the preservation by the method, the strain is difficult to be preserved to the second year for continuous use, and the strain needs to be re-produced every year, which leads to an increase in economic cost and time cost. SUMMARY

[0005] The preservation medium and the preservation method for preventing Morchella spp. from degeneration provided by the application can make the mycelium grow fast when the strain is activated after the preservation, the strain can be preserved to the second year for continuous use, the strain does not need to be re-produced every year, and the economic cost and the time cost are reduced.

[0006] The purpose of the application is to provide a preservation medium for preventing Morchella spp. from degeneration: 1L of the preservation medium has the following formula: 12g of sucrose, 6.0-6.2g of ammonium sulfate, 1g of potassium dihydrogen phosphate, 0.5-0.6g of magnesium sulfate, 18-20g of agar and 1000mL of distilled water, and the natural pH.

[0007] 1L of the preservation medium is prepared as follows: sucrose 12g, ammonium sulfate 6.2g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.5g, agar 18g, distilled water 1000mL, natural pH.

[0008] The application also provides a preservation method for preventing the degradation of the Morel spore by using the preservation medium, comprising the following steps:

[0009] (1) the mycelium block is obtained by culturing according to the following method:

[0010] The mycelium to be preserved is inoculated into a test tube slant containing the preservation medium, and the mycelium block is obtained after the mycelium grows on the slant;

[0011] (2) the mycelium block is preserved by paraffin oil preservation method, ultra-low temperature preservation method or physiological saline preservation method;

[0012] The ultra-low temperature preservation method is as follows: a prepared sterile glycerol solution with a volume fraction of 30% is used as a protective liquid and added into a cryogenic tube, the mycelium-containing mycelium block is added into the cryogenic tube, and the sterile glycerol solution completely immerses the mycelium block, and the cryogenic tube is placed at-80℃ for preservation; after one day of preservation at-80℃, the cryogenic tube is taken out for inspection, and the surviving spores are selected for long-term preservation at-80℃.

[0013] Preferably, in the above preservation method, the sterile glycerol solution with a volume fraction of 30% is prepared by the following method: 30mL of glycerol is added into distilled water, and the volume is made up to 100mL, and then the mixture is autoclaved at 121℃ for 30min.

[0014] Preferably, in the above preservation method, the paraffin oil preservation method is as follows: sterile liquid paraffin is injected into the test tube in which the mycelium grows on the slant to cover the whole slant, and then the test tube is vertically placed in 4-6℃ for preservation.

[0015] Preferably, in the above preservation method, the physiological saline preservation method is as follows: the above preservation medium without agar is used as a liquid preservation medium for the mycelium; 0.7-0.9g of sodium chloride is dissolved in 100mL of distilled water, and then the mixture is autoclaved at 121℃ for 30min to obtain sterile physiological saline, which is prepared for use; the spores to be preserved are inoculated into the liquid preservation medium, and the mixture is cultured at room temperature with shaking at 220r / min for 5-7 days; then the cultured liquid is injected into a sterile cryogenic tube containing the physiological saline, the cryogenic tube cover is sealed with paraffin, and the mixture is preserved at 4℃.

[0016] Compared with the prior art, the preservation medium and the preservation method for preventing the degradation of the Morel spore provided by the application have the following beneficial effects:

[0017] The present application adopts special preservation medium and preservation method, after -80℃ ultra-low temperature preservation, the mycelium grows fast when the strain is activated, the mycelium degradation degree is smaller, the mycelium biomass is higher, the mycelium extracellular laccase activity is high, the influence on the morella esculenta strain degradation is minimum, the high yield good strain can be preserved to the second year and continue to use, without needing to prepare the strain every year, reduces the economic cost and time cost, can be used in large scale industrialization. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Fig. 1 is the mycelium growth situation of the morella esculenta under different preservation methods (preservation 90d);

[0019] Figure 1 Fig. 1 is the mycelium growth situation of the morella esculenta under different preservation methods (preservation 90d); Figure 1 Fig. 1 is the mycelium growth situation of the morella esculenta under different preservation methods (preservation 90d); DETAILED DESCRIPTION

[0020] The present application will be described in detail below in combination with specific embodiments, but should not be understood as limiting the present application. The test method is not specified in the following examples, and is usually operated according to the conventional conditions, and since it does not involve the inventive point, the steps are not described in detail.

[0021] The present application provides a preservation medium for preventing the morella esculenta strain from degrading, and the formula of 1L of the preservation medium is as follows: sucrose 12g, ammonium sulfate 6.0-6.2g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.5-0.6g, agar 18-20g, distilled water 1000mL, natural pH, 121℃ high-pressure sterilization for 20min for standby.

[0022] Example 1

[0023] The present application provides a preservation medium for preventing the morella esculenta strain from degrading, and the formula of 1L of the preservation medium is as follows: sucrose 12g, ammonium sulfate 6.2g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.5g, agar 18g, distilled water 1000mL, natural pH, 121℃ high-pressure sterilization for 20min for standby.

[0024] Example 2

[0025] The present application provides a preservation medium for preventing the morella esculenta strain from degrading, and the formula of 1L of the preservation medium is as follows: sucrose 12g, ammonium sulfate 6.0g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.6g, agar 20g, distilled water 1000mL, natural pH, 121℃ high-pressure sterilization for 20min for standby.

[0026] Example 3

[0027] A preservation medium for preventing the degeneration of Morel spores, the formula of 1L of the preservation medium is as follows: sucrose 12g, ammonium sulfate 6.0g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.55g, agar 18g, distilled water 1000mL, natural pH, 121℃ high pressure sterilization for 20min for standby.

[0028] Based on the same inventive concept, the application further provides a preservation method for preventing the degeneration of Morel spores, comprising the following steps:

[0029] (1) The mycelium is cultured according to the following method to obtain the mycelial block:

[0030] The mycelium to be preserved is transferred to a test tube slant containing the culture medium, and the test tube is sealed with a white rubber plug, and the outside of the test tube is wrapped with newspaper. After the mycelium grows on the slant (4d), the mycelial block is obtained.

[0031] The culture medium used is PDA culture medium: potato 200g, glucose 20g, agar 18g, distilled water 1000mL, pH natural, 121℃ high pressure sterilization for 20min;

[0032] Or wheat bran culture medium: wheat bran 150g, glucose 20g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.5g, agar 18g, distilled water 1000mL, pH natural; It should be noted that the wheat bran is boiled with water for 30min, then filtered with four layers of gauze, and then the other substances of the wheat bran culture medium are added to the filtrate, and 121℃ high pressure sterilization for 20min;

[0033] Or sucrose culture medium (corresponding to the preservation medium of Example 1).

[0034] (2) The mycelial block is preserved by paraffin oil preservation method, ultra-low temperature preservation method or physiological saline preservation method.

[0035] Preferably, a preservation method for preventing the degeneration of Morel spores comprises the following examples.

[0036] Example 4

[0037] A preservation method for preventing the degeneration of Morel spores, comprising the following steps:

[0038] (1) The mycelium is cultured according to the following method to obtain the mycelial block:

[0039] The mycelium to be preserved is transferred to a test tube slant containing the culture medium, and the test tube is sealed with a white rubber plug, and the outside of the test tube is wrapped with newspaper. After the mycelium grows on the slant (4d), the mycelial block is obtained.

[0040] The used culture medium is sucrose culture medium, i.e. the preservation culture medium of Example 1.

[0041] (2) The mycelium blocks are preserved by the method of paraffin oil preservation.

[0042] The method of paraffin oil preservation is as follows: in a sterile operation table, sterile liquid paraffin (sterilized by high-pressure sterilization at 121 ℃ for 30 min twice, and cooled to room temperature for standby) is injected into the test tube in which the slant surface is full of mycelium, so as to cover the whole slant surface, and then the rubber plug is continued to seal and placed upright in a 4-6 ℃ refrigerator (i.e. a low-temperature environment) and a room temperature environment for preservation.

[0043] The 30% sterile glycerol is prepared in the following manner: 30 mL of glycerol is accurately measured and added to distilled water, and the volume is made up to 100 mL, and high-pressure sterilization is performed at 121 ℃ for 30 min.

[0044] Example 5

[0045] A preservation method for preventing the degeneration of Morchella spores, comprising the following steps:

[0046] (1) The mycelium blocks are obtained by culture in the following manner:

[0047] The mycelium to be preserved is respectively transferred to a test tube slant surface containing culture medium, and sealed with white glue plug, and the outside of the test tube is wrapped with newspaper, and after the mycelium grows on the slant surface (4 days), the mycelium blocks are obtained.

[0048] The used culture medium is sucrose culture medium, i.e. the preservation culture medium of Example 1.

[0049] (2) The mycelium blocks are preserved by the method of paraffin oil preservation.

[0050] The method of paraffin oil preservation is as follows: in a sterile operation table, sterile liquid paraffin (sterilized by high-pressure sterilization at 121 ℃ for 30 min twice, and cooled to room temperature for standby) is injected into the test tube in which the slant surface is full of mycelium, so as to cover the whole slant surface, and then the rubber plug is continued to seal and placed upright in a 4-6 ℃ refrigerator (i.e. a low-temperature environment) and a room temperature environment for preservation.

[0051] Example 6

[0052] A preservation method for preventing the degeneration of Morchella spores, comprising the following steps:

[0053] (1) The mycelium blocks are obtained by culture in the following manner:

[0054] The mycelium to be preserved is respectively transferred to a test tube slant containing culture medium, and is sealed with a white rubber plug, and the outside of the test tube is wrapped with newspaper, and after the mycelium grows on the slant (4 days), a mycelium block is obtained.

[0055] The culture medium used is sucrose culture medium, i.e., the preservation culture medium of Example 1.

[0056] (2) The mycelium block is preserved by using the ultra-low temperature preservation method.

[0057] The physiological saline preservation method is as follows: a sucrose liquid culture medium (corresponding to the preservation culture medium of Example 1, without agar) is used as the liquid preservation culture medium for the mycelium; 0.7 g of pure sodium chloride is dissolved in 100 ml of distilled water, and after high-pressure sterilization at 121 ℃ for 30 min, sterile physiological saline is obtained and is ready for use; the strain to be preserved is inoculated into the liquid preservation culture medium and is cultured at room temperature with shaking at 220 r / min for 5 days; and a small amount of the cultured liquid is taken out by sterile operation, is injected into a sterile freezing tube containing the physiological saline, and the freezing tube cover is sealed with paraffin wax, and is preserved at a low temperature of 4 ℃.

[0058] Example 7

[0059] A preservation method for preventing the degeneration of Morchella strain, comprising the following steps:

[0060] (1) The mycelium block is obtained by culturing in the following manner:

[0061] The mycelium to be preserved is respectively transferred to a test tube slant containing culture medium, and is sealed with a white rubber plug, and the outside of the test tube is wrapped with newspaper, and after the mycelium grows on the slant (4 days), a mycelium block is obtained.

[0062] The culture medium used is sucrose culture medium, i.e., the preservation culture medium of Example 1.

[0063] (2) The mycelium block is preserved by using the ultra-low temperature preservation method.

[0064] The physiological saline preservation method is as follows: a sucrose liquid culture medium (corresponding to the preservation culture medium of Example 1, without agar) is used as the liquid preservation culture medium for the mycelium; 0.7 g of pure sodium chloride is dissolved in 100 ml of distilled water, and after high-pressure sterilization at 121 ℃ for 30 min, sterile physiological saline is obtained and is ready for use; the strain to be preserved is inoculated into the liquid preservation culture medium and is cultured at room temperature with shaking at 220 r / min for 5 days; and a small amount of the cultured liquid is taken out by sterile operation, is injected into a sterile freezing tube containing the physiological saline, and the freezing tube cover is sealed with paraffin wax, and is preserved at a low temperature of 4 ℃.

[0065] In order to explore the effect of the present application, the following experiment was carried out. The temperature of "room temperature" in the following experiment is 20 ℃.

[0066] 1 Mycelium activity determination after preservation

[0067] 1.1 Mycelium growth rate determination after preservation

[0068] In the following experiments, sheep 1 (purchased from Chengde), sheep 2 (purchased from Shanxi), and sheep 3 (purchased from Hunan) were all commercially available Morchella sextelata.

[0069] The slant cultures of the strains under different media and different preservation methods at different temperatures were activated once. Under sterile conditions, a sterile 9mm puncher was used to punch a hole in the center of the PDA culture plate, and the activated strains were transferred to the center of the plate, with three replicates. The culture was carried out in a 25°C constant temperature incubator, and the diameter of the colony (mm) was measured with a ruler, using the cross method, and the average value was taken. The measurement endpoint was when the mycelium of each strain under a certain preservation method grew full of the culture dish, the growth days were recorded, and the photograph was taken.

[0070] Mycelium daily growth rate (mm / d) = colony diameter (mm) / mycelium growth days (d)

[0071] 1.2 Mycelium biomass determination after preservation

[0072] The slant cultures of the strains under different media and different preservation methods at different temperatures were activated once. The activated strains were transferred to liquid PDA medium and placed in a constant temperature shaking incubator, cultured at 25°C and 120r / min. After four days, the cultured mycelium balls were removed and the inoculum was removed, washed three times with distilled water, and placed in an oven at 45°C for 24h to dry to constant weight.

[0073] 1.3 Extracellular physiological index determination of Morchella

[0074] The extraction of crude enzyme solution and the determination of laccase activity were carried out according to Chen Guoliang (Chen Guoliang, Zhang Xiangqian, He Xiaolong, et al. Study on the changes of extracellular enzyme activity of five Morchella during liquid culture [J]. North Horticulture, 2010, (10): 210-213.) and others. In this determination step, the OD value at 0 minutes after adding the substrate and the OD value after 30 minutes of water bath incubation were also determined; the glucose utilization rate of Morchella was determined using a biosensor analyzer; and the protein content was determined according to the Coomassie Brilliant Blue reagent kit A054-2 (Nanjing Jiancheng).

[0075] 2 Results and analysis

[0076] 2.1 Mycelium growth rate index determination of Morchella under different media and preservation methods

[0077] 2.1.1 Determination of Morchella mycelium growth rate

[0078] Table 1 is the growth rate of mycelium under different preservation methods, wherein the control group is the slope before preservation, the slope 4℃ preservation refers to directly placing the mycelium slope at 4℃ for preservation, the slope room temperature preservation refers to directly placing the mycelium slope at room temperature for preservation, the distilled water 4℃ preservation refers to placing the fungus cake in a cryopreservation tube (the cryopreservation tube contains distilled water) for 4℃ preservation, the distilled water room temperature preservation refers to placing the fungus cake in a cryopreservation tube (the cryopreservation tube contains distilled water) for room temperature preservation, the physiological saline 4℃ preservation refers to placing the fungus cake in a cryopreservation tube (the cryopreservation tube contains 0.9% by mass physiological saline) for 4℃ preservation, the physiological saline room temperature preservation refers to placing the fungus cake in a cryopreservation tube (the cryopreservation tube contains 0.9% by mass physiological saline) for room temperature preservation, the liquid paraffin 4℃ preservation refers to covering the slope with sterile liquid paraffin, and then vertically placing the slope at 4℃ for preservation, the liquid paraffin room temperature preservation refers to covering the slope with sterile liquid paraffin, and then vertically placing the slope at room temperature for preservation, and the ultralow temperature preservation refers to the ultralow temperature preservation method of Example 4.

[0079] The culture medium used in Table 1 is the culture medium of Example 1, and the preservation time is 90 days. The results in Table 1 show that the mycelium growth rates of the three Morchella strains are less degraded before and after preservation by the ultralow temperature preservation, physiological saline 4℃ preservation, physiological saline room temperature preservation, liquid paraffin 4℃ preservation, liquid paraffin room temperature preservation and paraffin oil preservation methods. The mycelium growth rate of Sheep 1 and Sheep 2 increases after ultralow temperature preservation, indicating that the ultralow temperature preservation method not only effectively prevents the degradation of the mycelium growth rate, but also can improve the mycelium growth rate. The mycelium growth rate of Sheep 3 has little difference with the control after ultralow temperature preservation. However, the mycelium growth rate of the three Morchella strains significantly slows down and the degradation degree is obvious after the slope 4℃ preservation without any treatment. Therefore, the ultralow temperature preservation method provided by the present application has the best effect.

[0080] Table 1 is the growth rate of mycelium under different preservation methods

[0081]

[0082] Note: The same letter in the same column represents significant difference at the 5% level, and the same letter represents no significant difference, and different letters represent significant difference.

[0083] Figure 1 is the mycelium growth under different preservation methods of Morchella (preservation for 90 days), Figure 1 Figures A1-A4 in the middle row are the distilled water 4℃ preservation, liquid paraffin room temperature preservation, physiological saline 4℃ preservation and ultralow temperature preservation methods of Sheep 3, respectively; Figure 1Fig. B1-B4 are respectively distilled water 4℃ preservation, liquid paraffin room temperature preservation, physiological saline 4℃ preservation and ultra-low temperature preservation method of Morchella esculenta No. 1; Fig. C1-C4 are respectively distilled water 4℃ preservation, liquid paraffin room temperature preservation, physiological saline 4℃ preservation and ultra-low temperature preservation method of Morchella esculenta No. 2; the results show that the three strains of Morchella esculenta No. 1, Morchella esculenta No. 2 and Morchella esculenta No. 3 all grow well under the ultra-low temperature preservation method.

[0084] Table 2 is the mycelium growth rate under different culture media, the preservation method is the ultra-low temperature preservation method of Example 4, and the preservation time is 90 days. By comparing the mycelium growth rate of Morchella esculenta mycelium before preservation during the mycelium culture process, the mycelium growth rate of the wheat bran medium is the fastest, and the mycelium growth rate of the PDA medium is the slowest. After the mycelium on the three kinds of culture media is preserved by the ultra-low temperature preservation method of Example 4, the mycelium growth rate of the wheat bran medium is significantly slowed down; the mycelium growth rate of the sucrose culture medium (Example 1) is the least degraded, so the preservation effect of the sucrose culture medium (Example 1) is better than that of the conventional PDA medium and the wheat bran medium.

[0085] Table 2 mycelium growth rate under different culture media

[0086]

[0087] Note: The same letter in the same column represents the significant difference at the 5% level, and the same letter represents no significant difference, and the different letters represent significant difference.

[0088] Table 3 mycelium growth rate of Morchella esculenta at different preservation times, the preservation method is the ultra-low temperature preservation method of Example 4, and the preservation medium is the medium of Example 1. The mycelium growth rates of the three Morchella esculenta before preservation and after preservation for 30 days, 60 days and 90 days are compared, and the preservation time is not proportional to the mycelium growth rate. The growth rate of Morchella esculenta No. 1 after preservation for 90 days is significantly higher than that before preservation. After preservation for one year and activation once, the mycelium growth rates of Morchella esculenta No. 1, Morchella esculenta No. 2 and Morchella esculenta No. 3 are similar to those before preservation. The results show that the medium of Example 1 and the preservation method of Example 4 can effectively maintain the growth rate of Morchella esculenta mycelium, and the activity is still effective after 12 months, without the need to re-propagate, thereby reducing the economic cost and time cost.

[0089] Table 3 mycelium growth rate of Morchella esculenta at different preservation times

[0090]

[0091] Note: The same letter in the same column represents the significant difference at the 5% level, and the same letter represents no significant difference, and the different letters represent significant difference.

[0092] 2.1.2 Determination of Morchella esculenta mycelium biomass

[0093] Table 4 is the biomass of the mycelium of Morchella esculenta under different preservation methods, each preservation method in Table 4 is the same as 2.1.1, and the preservation medium is the medium of Example 1. After 90 days of preservation, the mycelium biomass of the three strains after the ultra-low temperature preservation method is 0.74 g, 0.59 g, and 0.51 g, respectively, which is nearly 2 times the mycelium biomass before preservation; it is 1.5 times the mycelium biomass preserved by the slant 4°C preservation method. The results show that the ultra-low temperature preservation method is the best, which is better than the conventional slant 4°C preservation method.

[0094] Table 5 is the biomass of the mycelium of Morchella esculenta under different media, and the preservation medium is the preservation method of Example 4. After 90 days of preservation, the mycelium biomass of the mycelium cultured with sucrose medium is about 1.6 times the mycelium biomass before preservation; the mycelium biomass cultured with PDA medium is increased compared with the mycelium biomass before preservation; and the mycelium biomass cultured with wheat bran medium has no difference with the control. Therefore, the preservation effect of sucrose medium (Example 1) and ultra-low temperature preservation (Example 4) is better than that of conventional PDA medium and wheat bran medium.

[0095] Table 4 is the biomass of the mycelium of Morchella esculenta under different preservation methods

[0096]

[0097] Note: The letters in the same column represent the difference significance at the 5% level, and the same letter means no significant difference, and different letters mean significant difference.

[0098] Table 5 is the biomass of the mycelium of Morchella esculenta under different media

[0099]

[0100] Note: The letters in the same column represent the difference significance at the 5% level, and the same letter means no significant difference, and different letters mean significant difference.

[0101] 2.2 Determination of extracellular physiological indexes of Morchella esculenta mycelium in liquid culture

[0102] 2.2.1 Extracellular laccase content of Morchella esculenta mycelium in liquid culture

[0103] Table 6 is the content of extracellular laccase of Morchella esculenta under different preservation methods, and each preservation method in Table 6 is the same as 2.1.1. The preservation medium is the culture medium of Example 1, and the preservation time is 90 days. The mycelium of Morchella esculenta is cultured after being preserved by different methods, and the content of extracellular laccase is different. The mycelium of Morchella esculenta preserved in physiological saline at 4°C has the highest extracellular laccase activity. The laccase activity is higher under the methods of ultralow temperature preservation and liquid paraffin preservation at 4°C. The laccase activity is lower under the other preservation methods. Laccase is related to the decomposition of lignin aromatic compounds, the higher the enzyme activity, the faster the decomposition speed, the more nutrients provided for the mycelium, and the faster the growth speed of the mycelium.

[0104] Table 6 is the content of extracellular laccase of Morchella esculenta under different preservation methods, and each preservation method in Table 6 is the same as 2.1.1. The preservation medium is the culture medium of Example 1, and the preservation time is 90 days. The mycelium of Morchella esculenta is cultured after being preserved by different methods, and the content of extracellular laccase is different. The mycelium of Morchella esculenta preserved in physiological saline at 4°C has the highest extracellular laccase activity. The laccase activity is higher under the methods of ultralow temperature preservation and liquid paraffin preservation at 4°C. The laccase activity is lower under the other preservation methods. Laccase is related to the decomposition of lignin aromatic compounds, the higher the enzyme activity, the faster the decomposition speed, the more nutrients provided for the mycelium, and the faster the growth speed of the mycelium, and the faster the growth speed of the mycelium.

[0105]

[0106] Note: The same letter in the same column represents the significant difference at the 5% level, and the same letter means no significant difference, and different letters mean significant difference.

[0107] It should be noted that in the above experiment, each parameter test of all preserved strains is measured after activation, so in actual application, it is applied only after activation, so these data can effectively evaluate the mycelium growth speed and other conditions of the strains after being activated again after a long time of preservation.

[0108] It should be noted that when numerical ranges are involved in the present application, it is understood that each intervening value, to the utmost limits of the ranges specified, is also contemplated. The above description is intended to encompass all such variations and modifications within the scope of the present application. Accordingly, the application is not limited to the specific embodiments described in the specification above, but only by the claims below.

[0109] Obviously, various modifications and changes can be made to the present application by those skilled in the art without departing from the spirit and scope of the present application. Thus, it is intended that the present application embrace all such modifications and changes that fall within the scope of the appended claims and their equivalents.

Claims

1. A method for preserving morel mushroom strains to prevent degradation, characterized in that, The formula for 1L of preservation medium is as follows: 12g sucrose, 6.2g ammonium sulfate, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 18g agar, 1000mL distilled water, natural pH; The preservation method for preventing morel strain degradation using the aforementioned preservation culture medium includes the following steps: (1) The mycelium blocks were obtained by culturing them according to the following method: The mycelia to be preserved were transferred to a test tube slant containing the preservation culture medium. After the mycelia covered the slant, mycelial blocks were obtained. (2) The mycelium blocks are preserved by ultra-low temperature preservation. The ultra-low temperature preservation method is as follows: A 30% sterile glycerol solution is prepared and added to a cryovial. Mycelial blocks containing hyphae are added to the cryovial, and the sterile glycerol solution is used to completely submerge the mycelial blocks. The cryovial is then stored at -80°C. After one day of storage at -80°C, the cryovial is taken out for testing, and the surviving strains are selected for long-term storage at -80°C. The morel mushroom in question is the Six Sister Morel.

2. The preservation method according to claim 1, characterized in that, 30% sterile glycerol by volume fraction was prepared as follows: 30 mL of glycerol was added to distilled water and brought to a final volume of 100 mL. The mixture was then autoclaved at 121 °C for 30 min.

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