Lucid ganoderma strain liquid nitrogen preservation method

Abstract

The invention provides a lucid ganoderma strain liquid nitrogen preservation method which comprises the following steps: 1) before liquid nitrogen preservation, taking an activated lucid ganoderma strain, adding a protective agent, freezing for 2-4 hours at-80 to-20 DEG C, and then putting into liquid nitrogen; the protective agent is glycerol; 2) when the ganoderma lucidum strain is taken, unfreezing the ganoderma lucidum strain preserved by liquid nitrogen at 25-35 DEG C until the ganoderma lucidum strain is in a suspension state; according to the liquid nitrogen preservation method, the obtained ganoderma lucidum strain still has good activity after long-time liquid nitrogen low-temperature preservation, and indexes such as the germination rate, the recovery time, the mycelium growth rate and the mycelium dry weight of the strain can be guaranteed. Meanwhile, the ganoderma lucidum strain preservation method is suitable for preservation of various ganoderma lucidum strains, is high in universality, wide in application range and simple and convenient to operate, and has a good application prospect.

Description

Technical Field

[0001] The invention specifically relates to a method for preserving ganoderma lucidum strains in liquid nitrogen. Background Art

[0002] Ganoderma lucidum is a kind of medicinal and edible fungus. The "Shennong's Herbal Classic" points out that Ganoderma lucidum is "bitter, flat, and non-toxic", and its main treatments are "chest knot, heart qi, tonifying the middle, increasing wisdom, and not forgetting", "long-term consumption makes the body light and immortal, and prolongs life". The "Compendium of Materia Medica" records that Ganoderma lucidum has magical effects such as "nourishing and strengthening, prolonging life, benefiting joints, and curing deafness", and lists it as a top-grade medicine. Due to its good medicinal activity, research on Ganoderma lucidum has gradually become a hot topic. However, most researchers focus on the classification and identification, pharmacological effects, effective substances, genome and gene functions of Ganoderma lucidum, but ignore the effective preservation of strains. However, improper preservation will cause the strains to mutate and degenerate, and it will be difficult to continue later research. Therefore, the preservation of strains is very important.

[0003] There are many methods for preserving strains. For edible and medicinal fungi, liquid nitrogen preservation is a recognized and long-term effective preservation method. However, studies have shown (Chen Yanyan. Ultra-low temperature freezing and preservation of basidiomycetes. Journal of Mycology, 1987, 6 (2): 110-117.) that if Ganoderma lucidum is directly placed in a gaseous nitrogen refrigerator at about -150°C, the strain will be significantly inactivated. In order to further improve the liquid nitrogen preservation effect of Ganoderma lucidum, the document "Analysis of Liquid Nitrogen Preservation Process of Ganoderma lucidum Strain" (Xie Hang et al. Zhejiang Agricultural Sciences. 2021, 62 (4): 705-708.) provides a liquid nitrogen preservation process for Ganoderma lucidum strains. The cooling procedure of Ganoderma lucidum strains is to cool down from room temperature to 4°C at a rate of 4°C or 9°C per minute, and then cool down to -40°C at a rate of 1°C per minute; PDA with sawdust extract added is used as the culture medium, and the size of the bacterial cake is 8mm; 10% to 15% glycerol is injected for liquid nitrogen preservation, and the strain has better vitality after rapid thawing in a 35°C water bath. However, the preservation time of this preservation process is only 2 months, and the preservation process is complicated, which is not conducive to industrial promotion and application. It is necessary to establish a simpler and feasible liquid nitrogen preservation method suitable for Ganoderma lucidum strains. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a method for preserving Ganoderma lucidum strains in liquid nitrogen, the method comprising the following steps:

[0005] 1) Before liquid nitrogen preservation, take activated Ganoderma lucidum strains, add protective agent, freeze at -80 to -20°C for 2 to 4 hours, and then put into liquid nitrogen; the protective agent is glycerol;

[0006] 2) When using, the Ganoderma lucidum strains preserved in liquid nitrogen are thawed at 25-35°C to a suspension state.

[0007] Furthermore, the Ganoderma lucidum strains in step 1) are activated using PDA culture medium.

[0008] Furthermore, the activated Ganoderma lucidum strains in step 1) are placed in a preservation tube.

[0009] Furthermore, the activated Ganoderma lucidum strain is a Ganoderma lucidum strain whose mycelium just completely covers the culture medium.

[0010] Furthermore, 1 to 2 ml of protective agent is added to every 3 pieces of the activated Ganoderma lucidum fungi, and each piece is 3 to 10 mm.

[0011] Furthermore, 1 ml of protective agent is added to every 3 pieces of the activated Ganoderma lucidum, and each piece is 8 mm.

[0012] Furthermore, the protective agent is 10% glycerol.

[0013] Furthermore, the freezing temperature is -80°C and the time is 2 hours.

[0014] Furthermore, the thawing temperature is 28±2°C.

[0015] The method for liquid nitrogen preservation of the ganoderma lucidum strains of the present invention has made a detailed study on the freezing method, protective agent and thawing method of the liquid nitrogen preservation of the ganoderma lucidum strains. Through the liquid nitrogen preservation method of the present invention, that is, the ganoderma lucidum strains with the addition of protective agent are frozen at a specific temperature for a specific time, and then placed in liquid nitrogen, even after long-term liquid nitrogen low-temperature preservation, the ganoderma lucidum strains thawed under specific environmental conditions when taken out still have good vitality, and can ensure the germination rate of the strains, recovery time, mycelium growth rate, mycelium dry weight and other indicators. At the same time, the method for liquid nitrogen preservation of the ganoderma lucidum strains is applicable to the preservation of a variety of ganoderma lucidum strains, has strong universality, a wide range of applications, and is easy to operate, and has good application prospects.

[0016] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0017] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. DETAILED DESCRIPTION

[0018] Example 1: Liquid nitrogen storage method for Ganoderma lucidum strains of the present invention

[0019] Take 3 pieces of Ganoderma lucidum strains (8 mm each) activated with PDA flat medium, whose mycelium just completely covers the medium, place them in a 2 ml storage tube, add 1 ml of 10% glycerol, store at -80°C for 2 hours, then store in liquid nitrogen, and thaw at room temperature to a suspension state when using.

[0020] Example 2: Method for liquid nitrogen preservation of Ganoderma lucidum strains of the present invention

[0021] Take 3 pieces of Ganoderma lucidum strains (10 mm each) activated with PDA flat medium, whose mycelium just completely covers the medium, place them in a 2 ml storage tube, add 1 ml of 10% glycerol, store at -50°C for 3 hours, then store in liquid nitrogen, and thaw at 25°C to a suspension state when using.

[0022] Example 3: Liquid nitrogen storage method for Ganoderma lucidum strains of the present invention

[0023] Take 3 pieces of Ganoderma lucidum strains (3 mm each) activated with PDA flat medium, whose mycelium just completely covers the medium, place them in a 2 ml storage tube, add 2 ml of 10% glycerol, store at -20°C for 4 hours, then store in liquid nitrogen, and thaw at 35°C to a suspension state when using.

[0024] The beneficial effects of the present invention are specifically described below through test examples:

[0025] Test Example 1: Method for liquid nitrogen preservation of Ganoderma lucidum strains

[0026] 1. Ganoderma lucidum strains tested

[0027] The Ganoderma lucidum strain “Medicinal Ganoderma Lucidum No. 1” (Sichuan Pharmaceutical No. 2011003) was cryopreserved at 4°C slant surface in the Institute of Fungi and Medicinal Materials, Sichuan Academy of Chinese Medicine.

[0028] 2. Test methods

[0029] Take out the test strain "Ganoderma Lucidum No. 1", activate it with PDA test tube culture medium, and then transfer it to PDA flat culture medium for culture at 26℃. When the mycelium is about to cover the culture medium, use an 8mm puncher to take 3 pieces of the strain 3mm away from the growing edge of the mycelium and place them in a 2ml preservation tube. On the basis of the previous experimental results, according to the design table 1, add 1ml of the corresponding protective agent, and treat it according to the corresponding cooling method. After being preserved in liquid nitrogen for 1 month, take it out, thaw and revive it according to the corresponding method, and measure the relevant indicators.

[0030] Table 1 Experimental parameter level design

[0031]

[0032] Note: Normal temperature is room temperature 28±2℃.

[0033] In Table 1, the preparation method of protective agent 10% glycerol is: prepare 10 ml glycerol and 90 ml water, mix the glycerol and water according to the proportion, and stir evenly; the preparation method of protective agent 10% dimethyl sulfoxide is: prepare 10 ml dimethyl sulfoxide and 90 ml water, mix the dimethyl sulfoxide and water according to the proportion, and stir evenly.

[0034] In Table 1, cooling method ① (4°C) - (-20°C) - (-80°C) - liquid nitrogen means that the Ganoderma lucidum strains are stored in a 4°C refrigerator for 2 hours, then transferred to a -20°C refrigerator for 2 hours, then transferred to a -80°C refrigerator for 2 hours, and finally placed in liquid nitrogen for low-temperature preservation; ② (-80°C) - liquid nitrogen means that the Ganoderma lucidum strains are stored at -80°C for 2 hours, and then placed in liquid nitrogen for low-temperature preservation; ③ liquid nitrogen means that the Ganoderma lucidum strains are directly placed in liquid nitrogen for storage.

[0035] In Table 1, thawing method ① (-80℃)-(-20℃)-(4℃)-room temperature means that the Ganoderma lucidum strains are taken out of liquid nitrogen and placed at -80℃ for thawing for 2 hours, then transferred to -20℃ for thawing for 2 hours, then transferred to 4℃ for thawing for 2 hours, and finally thawed at room temperature; ② room temperature means that the Ganoderma lucidum strains are directly thawed at room temperature to a suspension state; ③ 37℃ water bath means that the Ganoderma lucidum strains are directly thawed in a 37℃ water bath to a suspension state.

[0036] 3. Index determination

[0037] (1) Germination rate of bacterial strains: After the preserved bacterial strains were thawed, the bacterial block was inoculated aseptically in the center of the PDA plate and cultured at 26°C in the dark to observe its germination.

[0038] Germination rate of bacteria = germinated bacteria / inoculated bacteria*100%

[0039] (2) Recovery time: After the preserved strains are thawed, the strain block is inoculated aseptically in the center of the PDA plate and cultured at 26°C in the dark. The germination is observed on the first few days after inoculation.

[0040] (3) Mycelial growth rate: After thawing, the preserved strains were aseptically inoculated in the center of a PDA plate and cultured at 26°C in the dark. After germination, cross lines were drawn with the center of the plate as the midpoint until the mycelium covered the entire plate. The colony diameter was measured daily and the mycelial growth rate was calculated.

[0041] Mycelium growth rate (mm / d) = (average colony diameter – 8) / (mycelium growth days × 2)

[0042] (4) Dry weight of mycelium: The thawed bacterial block was inoculated into 100 ml of PDA liquid culture medium and cultured at 26°C and 120 rpm in the dark for 13 days. The culture was then filtered through 4 layers of gauze, rinsed with ultrapure water three times, dried at 60°C, and weighed to determine the biomass.

[0043] 4. Test results

[0044] After using SPSSAU software to perform type consistency and dimensionless processing on the experimental data, the CRITIC weight coefficients of germination rate, recovery time, mycelium growth rate, and mycelium dry weight were obtained to be 16.83%, 16.81%, 21.66%, and 44.70%, respectively. The experimental data were analyzed using spss.17, and the intuitive analysis results are shown in Table 2. According to Table 2 and the previous experiments, through the rapid pre-freezing stage of Ganoderma lucidum strains before being placed in liquid nitrogen, it was found that temperature control at -80℃ was beneficial to the recovery of strain activity after culture, and the optimal process conditions were determined as: using a cooling method of -80℃-liquid nitrogen, 10% glycerol as a protective agent, and thawing and recovery at room temperature.

[0045] Table 2 Experimental intuitive analysis table

[0046]

[0047] Test Example 2: Verification of the Liquid Nitrogen Preservation Method for Ganoderma Lucidum

[0048] 1. Ganoderma lucidum strains tested

[0049] The verified strains ZL2, ZL6, ZL53, ZL162, ZL144, ZL187, ZL86, ZL81, ZL230 and ZL114, which were significantly different in genetics (ISSR bands), agronomic traits (yield, growth cycle, etc.) and biological activities (polysaccharides, triterpenes, etc.), were selected and cryopreserved on slant surfaces at 4°C in the Institute of Fungi and Medicinal Materials, Sichuan Academy of Chinese Medicine.

[0050] 2. Test methods

[0051] The verified strain was taken out, activated with PDA test tube culture medium, and then transferred to PDA flat culture medium for culture at 26°C. When the mycelium was about to cover the culture medium, 3 pieces of strain were taken out with an 8mm puncher at 3mm away from the growing edge of the mycelium and placed in a 2ml preservation tube. The optimal preservation scheme obtained from the experiment in Experimental Example 1 was adopted: -80°C-liquid nitrogen cooling method, 10% glycerol was used as a protective agent, and thawed and revived at room temperature. Samples were taken for index determination at 1 month, 3 months, 6 months, 9 months, and 12 months of storage.

[0052] 3. Index determination

[0053] (4) Germination rate of bacterial strains: After the preserved bacterial strains were thawed, the bacterial block was inoculated aseptically in the center of the PDA plate and cultured at 26°C in the dark to observe its germination.

[0054] Germination rate of bacteria = germinated bacteria / inoculated bacteria*100%

[0055] (5) Recovery time: After the preserved bacterial strains are thawed, the bacterial block is inoculated aseptically in the center of the PDA plate and cultured at 26°C in the dark. The germination is observed on the first few days after inoculation.

[0056] (6) Mycelial growth rate: After thawing, the preserved strains were aseptically inoculated in the center of a PDA plate and cultured at 26°C in the dark. After germination, cross lines were drawn with the center of the plate as the midpoint until the mycelium covered the entire plate. The colony diameter was measured daily and the mycelial growth rate was calculated.

[0057] Mycelium growth rate (mm / d) = (average colony diameter – 8) / (mycelium growth days × 2)

[0058] (4) Dry weight of mycelium: The thawed bacterial block was inoculated into 100 ml of PDA liquid culture medium and cultured at 26°C and 120 rpm in the dark for 13 days. The culture was then filtered through 4 layers of gauze, rinsed with ultrapure water three times, dried at 60°C, and weighed to determine the biomass.

[0059] 4. Test results

[0060] (1) Germination rate of different Ganoderma lucidum strains after liquid nitrogen preservation method of the present invention

[0061] As can be seen from Table 3, the survival rate of the 10 verified strains preserved according to the liquid nitrogen preservation method of the present invention for 1 month was 100%; the survival rate for 3 months was 100%; the survival rate for 6 months was 100%; the survival rate for 9 months was 100%; after 1 year of preservation, only the germination rate of ZL86 was 66.7%, that is, 1 strain died in three replicates, and the survival rates of the remaining strains were all 100%, which fully guaranteed the germination rate of the preserved strains. Therefore, this preservation method can ensure the mycelium germination rate.

[0062] Table 3 Germination rate of different Ganoderma lucidum strains after liquid nitrogen preservation method of the present invention (%)

[0063]

[0064]

[0065] (2) Recovery time of different Ganoderma lucidum strains after liquid nitrogen preservation method of the present invention

[0066] As can be seen from Table 4, the recovery time of 10 verified strains preserved for 1-12 months according to the liquid nitrogen preservation method of the present invention is between 1-7 days, and there is a trend of first increasing and then decreasing with the increase of preservation time. 70% of the preserved strains have no significant difference after 12 months of preservation compared with before preservation, and 30% of the strains show differences, among which the most significant strain is only 4 days longer after 12 months of preservation than before preservation, which is within the recovery time range accepted by production and scientific research. Therefore, this preservation method can ensure the recovery time of mycelium.

[0067] Table 4 Recovery time of different Ganoderma lucidum strains after preservation by the liquid nitrogen preservation method of the present invention (days)

[0068]

[0069] The data in the table are "mean ± standard deviation"; different lowercase letters after the data in the same column indicate significant differential expression of the same strain at different storage times at the 5% level. The same below.

[0070] (3) Mycelial growth rate of different Ganoderma lucidum strains after liquid nitrogen preservation method of the present invention

[0071] As can be seen from Table 5, the mycelial growth rate of 10 verified strains preserved for 1-12 months according to the liquid nitrogen preservation method of the present invention increases with the increase of preservation time, 80% of the preserved strains have no significant difference after 12 months of preservation compared with before preservation, 20% of the different strains increase significantly, and there is a significant difference in the mycelial growth rate of these two strains after preservation (p≤0.05), however, due to the low amplitude of the change, although it is statistically significant, this change is not enough to support biological changes, therefore, due to uncontrollable variables or strain characteristics, this change is considered to be a biological change of the Ganoderma lucidum strain; therefore, this preservation method can ensure the mycelial growth rate.

[0072] Table 5 Mycelial growth rate of different Ganoderma lucidum strains after preservation by the liquid nitrogen preservation method of the present invention (mm / d)

[0073]

[0074] (4) Dry weight of mycelium of different Ganoderma lucidum strains after liquid nitrogen preservation by the present invention

[0075] As can be seen from Table 6, the dry weight of mycelium of the 10 verified strains preserved for 1-12 months according to the liquid nitrogen preservation method of the present invention shows a trend of first decreasing and then increasing with the increase of preservation time, and 100% of the preserved strains have no significant difference after 12 months of preservation compared with before preservation; therefore, this preservation method can ensure the mycelium growth rate.

[0076] Table 6 Dry weight of mycelium of different Ganoderma species after preservation by the liquid nitrogen preservation method of the present invention (g)

[0077]

[0078]

[0079] In summary, the present invention provides a method for preserving Ganoderma lucidum strains in liquid nitrogen. The method is simple to operate, and the strains still have good vitality after preservation by the method, and can ensure the germination rate, recovery time, mycelial growth rate, mycelial dry weight and other indicators of the strains. At the same time, the method for preserving Ganoderma lucidum strains is suitable for the preservation of various Ganoderma lucidum strains, has strong universality, a wide range of applications, and has good application prospects.

Claims

1. A method for cryopreserving Ganoderma lucidum strains in liquid nitrogen, Characterized in that: The method comprises the following steps: 1) Before cryopreservation in liquid nitrogen, take the activated Ganoderma lucidum strain, add a cryoprotectant, freeze at -80 to -20 °C for 2 to 4 h, and then put it into liquid nitrogen; the cryoprotectant is glycerol; 2) When taking it out, thaw the Ganoderma lucidum strain cryopreserved in liquid nitrogen at 25 to 35 °C until it is in a suspension state.

2. The method according to claim 1, Characterized in that: The Ganoderma lucidum strain in step 1) is activated with a PDA medium.

3. The method according to claim 1, Characterized in that: The activated Ganoderma lucidum strain in step 1) is placed in a preservation tube.

4. The method according to any one of claims 1 to 3, Characterized in that: The activated Ganoderma lucidum strain is a Ganoderma lucidum strain whose mycelium just completely covers the medium.

5. The method according to claim 1, Characterized in that: For every 3 pieces of the activated Ganoderma lucidum strain, add 1 to 2 ml of cryoprotectant, and each piece is 3 to 10 mm.

6. The method according to claim 5, Characterized in that: For every 3 pieces of the activated Ganoderma lucidum strain, add 1 ml of cryoprotectant, and each piece is 8 mm.

7. The method according to claim 1, 5 or 6, Characterized in that: The cryoprotectant is 10% glycerol.

8. The method according to claim 1, Characterized in that: The freezing temperature is -80 °C and the time is 2 h.

9. The method according to claim 1, Characterized in that: The thawing temperature is 28 ± 2 °C.

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