Phallus impudicus strain preservation method

Abstract

The invention provides a phallus impudicus strain preservation method. The specific method comprises the following steps: (1) preparing an allogenic substance plate culture medium; (2) activating and culturing phallus impudicus strains; (3) punching hyphae, transferring the hyphae to a cryopreservation tube, and standing; and (4) cooling, and preserving in liquid nitrogen. According to the invention, influence factors such as preserved allogenic substances, a preservative, standing conditions, a cooling mode and thawing temperature are investigated through a single factor experiment and a response surface experiment, and the growth speed of the preserved phallus impudicus strain Qianzhuang No.1 reaches (1.18 + / -0.14) mm.d <-1 >. The preservation method is simple, convenient, stable and reliable to operate, can preserve the phallus impudicus strains for a long time, and lays a foundation for edible fungus germplasm resource protection and seed industry chip.

Description

Technical Field

[0001] The invention belongs to the technical field of edible fungus strain preservation, and particularly relates to a method for preserving winter fungus strains. Background Art

[0002] Phallus dongsun, also known as white ghost pen, belongs to the Basidiomycotina, Gasteromycetes, Phallales, Phallaceae, and genus Phallus (Ting et al., 2020; Lu et al., 2023; Li et al., 2024). Dongsun is a valuable edible and medicinal fungus. Its fruiting bodies are rich in polysaccharides, crude fiber, and various amino acids. Its stipes are used as medicine, exhibiting dehumidifying and analgesic properties, antioxidant, anticoagulant, anti-glycation, and anticancer properties (Kikuchi et al., 1984; Song Di, 2021). It also regulates the soil environment (Wang YH et al., 2023).

[0003] Culture preservation (culture collection) refers to the technology used to maintain the vitality and genetic traits of microbial strains. The goal is to preserve wild strains isolated from nature or pure strains derived through artificial selection, ensuring their survival, preventing loss, contamination, and minimal or no mutation, while maintaining the strain's original characteristics and physiological activity. Edible mushroom strains are important resources for scientific research and production. Common methods for their preservation include subculture, paraffin oil, and aqueous solution storage, but all of these methods suffer from slow growth.

[0004] The present invention team has been committed to the research of edible fungus strain preservation methods. For example, the invention team applied for a patent (CN202310144913.X, a liquid nitrogen preservation method for solid blocks of winter fungus strains). The technology discloses that the protective agent is 20% to 30% D-trehalose and the cooling program is 1°C min. -1 The growth rate of the winter iris fungus preserved at -50°C is 0.75 mm / day. The winter iris fungus preserved by this technology has the problem of slow growth rate.

[0005] To address these issues, the inventive team used the "Qianzhuang 1" strain of the winter iris fungus as their research target. Using response surface methodology, they systematically investigated the effects of preservatives, cooling methods, thawing temperatures, exogenous substances, resting temperatures, and resting times on the preservation of the fungus, optimizing its preservation methods. The optimized liquid nitrogen preservation process for the winter iris fungus strain developed in this invention provides technical support for the long-term preservation of fungus strains, laying the foundation for the conservation of edible fungus germplasm resources and the "chip" nature of the seed industry. Summary of the Invention

[0006] The present invention aims to provide a method for preserving winter iris fungus species.

[0007] The present invention is achieved through the following technical solutions:

[0008] The present invention provides a method for preserving spores of the winter iris, comprising the following steps:

[0009] (1) Preparation of exogenous material plate culture medium: 200 g potatoes, 80 g sawdust, 20 g glucose, 5 g peptone, 8 g agar, 1000 mL water, pH natural, weigh the corresponding reagents, heat and stir, dissolve to volume, and then dispense into 500 mL conical flasks. After sterilization in an autoclave at 121°C for 20 min, place in a clean bench, add 5 g / L L-proline, mix well, pour into plates, and wait for the plates to solidify naturally;

[0010] (2) Take the strain "Qianzhuang No. 1" of Ilex strychnifolia, activate it in a plate culture medium containing exogenous substances, and culture it at a constant temperature of 25°C;

[0011] (3) When the mycelium grows to two-thirds of the 90mm plate, use a 0.5cm 2 Punch a hole 0.5 cm behind the tip of the hyphae with a borer, inoculate the tube into a 2 mL cryotube, add 1.5 mL of 5% to 25% PEG20000, and let it stand at 4 to 25°C for 1 to 9 hours.

[0012] (4) Cryopreservation tubes are used at 1-9°C / min -1 The speed was reduced to 4℃, and then to 1~9℃·min -1 The sample was cooled to -50°C and stored in liquid nitrogen.

[0013] Preferably, the concentration of PEG20000 in step (3) of the present invention is 15% to 25%.

[0014] Further preferably, the concentration of PEG20000 in step (3) of the present invention is 20%.

[0015] Preferably, the standing condition in step (3) of the present invention is: standing at 20-25° C. for 1-5 hours.

[0016] Further preferably, the standing condition in step (3) of the present invention is: standing at 25° C. for 3 hours.

[0017] Preferably, the cooling method in step (4) of the present invention is: 4°C·min -1 or 9℃·min -1 Reduce to 4℃, then increase to 1℃·min -1 Drop to -50℃.

[0018] The thawing temperature of the winter iris strain "Qianzhuang No. 1" preserved by the method of the present invention is 20-40°C.

[0019] Preferably, the thawing temperature of the Ilex sanguinea strain "Qianzhuang No. 1" preserved by the method of the present invention is 20-30°C.

[0020] Further preferably, the thawing temperature of the Ilex strychnifolia strain "Qianzhuang No. 1" preserved by the method of the present invention is 25°C.

[0021] Beneficial effects

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The iris strain "Qianzhuang No. 1" preserved by the preservation method of the present invention has a strong recovery ability and a fast growth rate after recovery. The present invention obtained the main factors affecting the mycelial growth rate after preservation through response surface experiment: standing time, thawing temperature, protective agent and cooling rate. The results of optimizing the first three main factors using response surface method are standing time (h) = 2.940, protective agent concentration (%) = 20.980, thawing temperature (℃) = 24.407, and the predicted mycelial growth rate (mm·d -1 )=1.183. Taking into account the experimental conditions, operability and rationality, the above optimal conditions were corrected to: standing time (h)=3, protective agent concentration (%)=20, thawing temperature (℃)=25. The measured growth rate of mycelium of Winter Sunflower was (1.18±0.14) mm·d -1 , which is 99% of the model prediction value. The response prediction value is basically consistent with the actual experimental value. The mycelium growth rate is 1.5 times that of the existing technology.

[0024] 2. The present invention investigates the exogenous substances for the activation culture of the winter clover strain "Qianzhuang No. 1". The results show that the survival rate and growth rate of the winter clover strain "Qianzhuang No. 1" are optimal when 5g / L L-proline is added as an exogenous substance.

[0025] 3. The present invention investigates the types of protective agents, and the results show that, compared with D-trehalose and L-proline, the survival rate and growth rate of the winter iris strain "Qianzhuang No. 1" are the best when PEG20000 is used as a protective agent.

[0026] 4. The present invention investigates the standing temperature and time of the winter iris strain "Qianzhuang No. 1" after being transferred to a cryopreservation tube. The results show that the survival rate and growth rate of the winter iris strain "Qianzhuang No. 1" are optimal when standing at 25°C for 3 hours.

[0027] 5. The present invention investigated the cooling method of cryopreservation tubes. The results showed that the cooling method of 4℃·min -1 or 9℃·min-1 The speed was reduced from 25℃ to 4℃, and then to 1℃·min -1 The survival rate and growth rate of the winter clover strain "Qianzhuang No. 1" were the best when the temperature was reduced to -50℃.

[0028] 6. The preservation method of the present invention is simple, stable and reliable to operate, and can preserve the winter fungus species for a relatively long period of time, laying the foundation for the protection of edible fungus germplasm resources and the "chip" of the seed industry.

[0029] Strain name: Phallus dongsun Qianzhuang No. 1;

[0030] Depository: China Center for Type Culture Collection (CCTCC);

[0031] Deposit address: Wuhan University, Wuhan, China;

[0032] Deposit date: June 8, 2022;

[0033] The deposit number is CCTCC NO: M 2022840. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Pareto chart (α=0.05)

[0035] Figure 2 Effects of the interaction between standing time and protective agent concentration on the growth rate of mycelium of winter iris (including contour plot (left) and response surface plot (right))

[0036] Figure 3 Effects of the interaction between standing time and thawing temperature on the growth rate of mycelium of Winter Sunflower (including contour plot (left) and response surface plot (right))

[0037] Figure 4 Effect of the interaction between protective agent concentration and thawing temperature on the growth rate of winter fungus mycelium (including contour plot (left) and response surface plot (right))

[0038] Figure 5 Comparison of the growth of mycelium of Ilex strychnifolia after unoptimized and optimized preservation (unoptimized preservation (left), optimized preservation (right)) DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further described in detail below through specific embodiments.

[0040] Example 1

[0041] (1) Preparation of exogenous material plate culture medium: 200 g potatoes, 80 g sawdust, 20 g glucose, 5 g peptone, 8 g agar, 1000 mL water, pH natural, weigh the corresponding reagents, heat and stir, dissolve to volume, and then dispense into 500 mL conical flasks. After sterilization in an autoclave at 121°C for 20 min, place in a clean bench, add 5 g / L L-proline, mix well, pour into plates, and wait for the plates to solidify naturally;

[0042] (2) Take the strain "Qianzhuang No. 1" of Ilex strychnifolia, activate it in a plate culture medium containing exogenous substances, and culture it at a constant temperature of 25°C;

[0043] (3) When the mycelium grows to two-thirds of the 90mm plate, use a 0.5cm 2 Punch a hole 0.5 cm behind the tip of the hyphae with a borer, inoculate into a 2 mL cryotube, add 1.5 mL of 20% PEG20000, and let it stand at 25°C for 3 h;

[0044] (4) Cryopreservation tubes are used at 4℃·min -1 The speed was lowered to 4℃, and then to 1℃·min -1 Cool down to -50°C and store in liquid nitrogen;

[0045] (5) After 30 days of storage, take out the frozen product and thaw it in a constant temperature water bath at 25°C.

[0046] Example 2

[0047] (1) Preparation of exogenous material plate culture medium: 200 g potatoes, 80 g sawdust, 20 g glucose, 5 g peptone, 8 g agar, 1000 mL water, pH natural, weigh the corresponding reagents, heat and stir, dissolve to volume, and then dispense into 500 mL conical flasks. After sterilization in an autoclave at 121°C for 20 min, place in a clean bench, add 5 g / L L-proline, mix well, pour into plates, and wait for the plates to solidify naturally;

[0048] (2) Take the strain of Ilex strychnifolia “Qianzhuang No. 1”, activate it in a plate culture medium containing exogenous substances, and culture it at a constant temperature of 25°C;

[0049] (3) When the mycelium grows to two-thirds of the 90mm plate, use a 0.5cm 2 Punch a hole 0.5 cm behind the tip of the hyphae with a borer, inoculate into a 2 mL cryotube, add 1.5 mL of 20% PEG20000, and let it stand at 25°C for 3 h;

[0050] (4) Cryopreservation tubes are used at 9℃·min -1 The speed was lowered to 4℃, and then to 1℃·min -1 Cool down to -50°C and store in liquid nitrogen;

[0051] (5) After 30 days of storage, take out the frozen product and thaw it in a constant temperature water bath at 25°C.

[0052] Example 3

[0053] (1) Preparation of exogenous material plate culture medium: 200 g potatoes, 80 g sawdust, 20 g glucose, 5 g peptone, 8 g agar, 1000 mL water, pH natural, weigh the corresponding reagents, heat and stir, dissolve to volume, and then dispense into 500 mL conical flasks. After sterilization in an autoclave at 121°C for 20 min, place in a clean bench, add 20 g / L L-proline, mix well, pour into plates, and wait for the plates to solidify naturally;

[0054] (2) Take the strain of Ilex strychnifolia “Qianzhuang No. 1”, activate it in a plate culture medium containing exogenous substances, and culture it at a constant temperature of 25°C;

[0055] (3) When the mycelium grows to two-thirds of the 90mm plate, use a 0.5cm 2 Punch a hole 0.5 cm behind the tip of the hyphae with a borer, inoculate into a 2 mL cryotube, add 1.5 mL of 25% PEG20000, and let it stand at 20°C for 9 h;

[0056] (4) Cryopreservation tubes are used at 9℃·min -1 Reduce to 4℃, then at 4℃·min -1 Cool down to -50°C and store in liquid nitrogen;

[0057] (5) After 30 days of storage, take out the frozen product and thaw it in a constant temperature water bath at 40°C.

[0058] Example 4

[0059] (1) Preparation of exogenous material plate culture medium: 200 g potatoes, 80 g sawdust, 20 g glucose, 5 g peptone, 8 g agar, 1000 mL water, pH natural formula. Weigh the corresponding reagents, heat and stir, dissolve to volume, and then divide into 500 mL conical flasks. After sterilization in an autoclave at 121 °C for 20 min, place in a clean bench, add 15 g / L L-proline, mix well, pour into plates, and wait for the plates to solidify naturally.

[0060] (2) Take the strain of Ilex strychnifolia “Qianzhuang No. 1”, activate it in a plate culture medium containing exogenous substances, and culture it at a constant temperature of 25°C;

[0061] (3) When the mycelium grows to two-thirds of the 90mm plate, use a 0.5cm 2 Punch a hole 0.5 cm behind the tip of the hyphae with a borer, inoculate into a 2 mL cryotube, add 1.5 mL of 15% PEG20000, and let it stand at 20°C for 7 h;

[0062] (4) Cryopreservation tubes are used at 4℃·min -1 Reduce to 4℃, then at 9℃·min -1 Cool down to -50°C and store in liquid nitrogen;

[0063] (5) After 30 days of storage, take out the frozen product and thaw it in a constant temperature water bath at 35°C.

[0064] Example 5

[0065] (1) Preparation of exogenous material plate culture medium: 200 g potatoes, 80 g sawdust, 20 g glucose, 5 g peptone, 8 g agar, 1000 mL water, pH natural, weigh the corresponding reagents, heat and stir, dissolve to volume, and then dispense into 500 mL conical flasks. After sterilization in an autoclave at 121°C for 20 min, place in a clean bench, add 10 g / L L-proline, mix well, pour into plates, and wait for the plates to solidify naturally;

[0066] (2) Take the strain of Ilex strychnifolia “Qianzhuang No. 1”, activate it in a plate culture medium containing exogenous substances, and culture it at a constant temperature of 25°C;

[0067] (3) When the mycelium grows to two-thirds of the 90mm plate, use a 0.5cm 2 Punch a hole 0.5 cm behind the tip of the hyphae with a borer, inoculate into a 2 mL cryotube, add 1.5 mL of 10% PEG20000, and let it stand at 4 °C for 5 h;

[0068] (4) Cryopreservation tubes are used at 1°C·min -1 Reduce to 4℃, then at 9℃·min -1 Cool down to -50°C and store in liquid nitrogen;

[0069] (5) After 30 days of storage, take out the frozen product and thaw it in a constant temperature water bath at 30°C.

[0070] Example 6

[0071] (1) Preparation of exogenous material plate culture medium: 200 g potatoes, 80 g sawdust, 20 g glucose, 5 g peptone, 8 g agar, 1000 mL water, pH natural formula. Weigh the corresponding reagents, heat and stir, dissolve to volume, and then divide into 500 mL conical flasks. After sterilization in an autoclave at 121 °C for 20 min, place in a clean bench, add 15 g / L L-proline, mix well, pour into plates, and wait for the plates to solidify naturally.

[0072] (2) Take the strain of Ilex strychnifolia “Qianzhuang No. 1”, activate it in a plate culture medium containing exogenous substances, and culture it at a constant temperature of 25°C;

[0073] (3) When the mycelium grows to two-thirds of the 90mm plate, use a 0.5cm 2Punch a hole 0.5 cm behind the tip of the hyphae with a borer, inoculate into a 2 mL cryotube, add 1.5 mL of 5% PEG20000, and let it stand at 4°C for 1 h;

[0074] (4) Cryopreservation tubes are used at 1°C·min -1 Reduce to 4℃, then at 4℃·min -1 Cool down to -50°C and store in liquid nitrogen;

[0075] (5) After 30 days of storage, take out the frozen product and thaw it in a constant temperature water bath at 20°C.

[0076] In order to verify the effectiveness of the present invention, the invention team conducted a series of experiments, as follows:

[0077] 1. Materials and Methods

[0078] 1.1 Experimental Materials

[0079] Test strains: The winter spruce strain was provided by the Guizhou Institute of Crop Varieties Resources (No.: Qianzhuang No. 1).

[0080] Reagents used: potato, glucose, potassium dihydrogen phosphate, magnesium sulfate, DMSO (cell grade), D-trehalose (analytical grade), L-proline (analytical grade), and PEG20000 (analytical grade).

[0081] Test instruments: programmed cooling instrument, Thermo; liquid nitrogen tank; BSP-400 biochemical incubator; clean bench; steam sterilizer; reverse osmosis water purifier, etc.

[0082] 1.2 Experimental methods

[0083] 1.2.1 Culture medium preparation

[0084] Plate culture medium: 200g potatoes (sliced, boiled, and filtered), 80g sawdust, 20g glucose, 5g peptone, 8g agar, 1000mL water, natural pH. Weigh the corresponding reagents according to the above recipe, heat and stir, dissolve to volume, and then divide into 500mL Erlenmeyer flasks. Sterilize in an autoclave at 121°C for 20min. After sterilization, remove the plate and place it in a clean bench for pouring into plates. Allow the plate to solidify naturally before use in experiments.

[0085] Exogenous substance plate culture medium: Prepare the plate culture medium, sterilize it in an autoclave at 121°C for 20 min, place it in an ultra-clean workbench, add L-proline, mix well, and pour it into the plate. After the plate solidifies naturally, use it in the experiment.

[0086] 1.2.2 Culture of bacterial strains and preparation of preservation tubes

[0087] Activate the fungus on a plate culture medium and culture it at a constant temperature of 25°C. When the mycelium grows to two-thirds of the 90mm plate, use a 0.5cm 2 Punch a hole 0.5 cm behind the tip of the hyphae with a borer, inoculate into a 2 mL cryotube, add 1.5 mL of protective agent (20% D-trehalose), and let it stand at 4 °C for 9 h. -1 Cool to -50°C and store in liquid nitrogen.

[0088] 1.2.3 Single-factor experiment

[0089] (1) Screening of protective agents

[0090] Change the protective agent and its concentration (Table 1) according to the method in 1.2.2. After 30 days of storage, take out the frozen samples and thaw them quickly in a 35℃ constant temperature water bath. After they are completely thawed, inoculate them on plates for culture. Each treatment is replicated 4 times.

[0091] Table 1 Protective agent concentration design

[0092]

[0093] (2) Cooling method screening

[0094] Change the cooling method according to the method in 1.2.2 (Table 2), take out the samples after 30 days of storage, and quickly thaw them in a constant temperature water bath at 35℃. After they are completely thawed, inoculate them on plates for culture, with 4 replicates for each treatment.

[0095] Table 2 Cooling method design

[0096]

[0097]

[0098] (3) Thawing temperature screening

[0099] Preserve the strains according to the method in 1.2.2, take them out after 30 days, and thaw them in a constant temperature water bath at 20℃, 25℃, 30℃, 35℃, and 40℃ respectively. After they are completely thawed, take out the strains and inoculate them on plates for culture. Repeat 4 times for each treatment.

[0100] (4) Exogenous substance concentration screening

[0101] Take the fungus strain of Ilex serrata, activate it in the exogenous material plate culture medium, and preserve the strain according to the method in 1.2.2. Take it out after 30 days, quickly thaw it in a constant temperature water bath at 35℃, and inoculate it into the plate for culture after it is completely thawed. Each treatment is repeated 4 times.

[0102] (5) Static temperature screening

[0103] According to the method in 1.2.2, the prepared bacteria were placed at 4℃, 20℃ and 25℃ for 1h, and then heated at 1℃·min -1 The cells were cooled to -50°C and stored in liquid nitrogen. After 30 days, the cells were taken out and thawed quickly in a 35°C constant temperature water bath. After complete thawing, the cells were inoculated on plates for culture. Each treatment was replicated four times.

[0104] (6) Screening of standing time

[0105] Prepare the strain according to the method in 1.2.2. Based on the experiment (5), after standing for 1h, 3h, 5h, 7h, and 9h, the strain was heated at 1℃·min -1 The cells were cooled to -50°C and stored in liquid nitrogen. After 30 days, the cells were taken out and thawed quickly in a 35°C constant temperature water bath. After complete thawing, the cells were inoculated on plates for culture. Each treatment was replicated four times.

[0106] 1.2.4 Plackett-Burman experiment

[0107] Based on the single-factor experiment, two high and low levels of the experimental factors were selected to design and screen out factors that have a significant impact on the experimental results. A PB design with N=12 experiments was used, and each factor was set at high (1) and low (-1) levels (Table 3). The response value was Y (growth rate).

[0108] Table 3 Factor levels of Plackett-Burman experimental design

[0109]

[0110] 1.2.5 Response surface optimization design

[0111] Based on single-factor and Plackett-Burman experiments, a Box-Behnken experimental design was used. A three-factor, three-level response surface methodology was selected. The experimental combinations were determined using resting time (A), PEG20000 concentration (B), and thawing temperature (C) as experimental factors and growth rate as the evaluation indicator. The factor levels are shown in Table 4.

[0112] Table 4 Box-Behnken experimental design factor levels

[0113]

[0114] 1.2.6 Data Analysis

[0115] The preserved strains were taken out, thawed, inoculated on plate culture, and cultured at a constant temperature of 25°C. The survival rate and growth rate of the bacterial blocks were recorded, and the preservation effect was analyzed using IBM SPSS Statistics 21.

[0116] 2. Experimental Results

[0117] 2.1 Results of single factor screening for liquid nitrogen preservation of Ilex spp.

[0118] 2.1.1 Preservation effects of different protective agents

[0119] The survival rate and growth rate of the activated bacteria were different in different concentrations of D-trehalose, L-proline and PEG20000 (Table 5): 20% PEG20000 as a protective agent had the best survival rate and growth rate; followed by 10% and 15% PEG20000 as protective agents.

[0120] Table 5 Effects of protective agents on bacterial survival rate and growth rate

[0121]

[0122]

[0123] Note: Different lowercase letters in the same column in the table indicate significant differences (P<0.05), the same as in the following tables.

[0124] 2.1.2 Preservation effects of different cooling methods

[0125] The survival rate and growth rate of the activated bacteria were different among the different cooling treatments (Table 6). Under the different cooling treatments, the survival rate and growth rate of the F and I cooling methods were the best, followed by the G cooling method.

[0126] Table 6 Effects of cooling methods on bacterial survival rate and growth rate

[0127]

[0128] 2.1.3 Preservation effect of different thawing temperatures

[0129] The survival rate and growth rate of the activated bacteria were different among the different thawing temperature treatments (Table 7): the survival rate and growth rate were the best when the thawing temperature was 25℃, followed by 40℃.

[0130] Table 7 Effect of thawing temperature on bacterial survival rate and growth rate

[0131]

[0132] 2.1.4 Preservation effect of different concentrations of L-proline

[0133] The survival rate and growth rate of the activated bacteria were different under different concentrations of exogenous substances (Table 8). The survival rate and growth rate were the best when 5 g / L L-proline was added as an exogenous substance, followed by 15 g / L L-proline.

[0134] Table 8 Effects of exogenous substances on bacterial survival rate and growth rate

[0135]

[0136] 2.1.5 Preservation effect at different standing temperatures

[0137] The survival rate and growth rate of the activated bacteria were different at different static temperatures (Table 9): the survival rate and growth rate were the best at 25°C, followed by 20°C.

[0138] Table 9 Effect of static temperature on bacterial survival rate and growth rate

[0139]

[0140] 2.1.6 Preservation effect of different standing times

[0141] The survival rate and growth rate of the activated bacteria were different at different standing times (Table 10): when the standing time was greater than or equal to 3 h, the survival rate and growth rate were better than those at 1 h.

[0142] Table 10 Effect of standing time on bacterial survival rate and growth rate

[0143]

[0144] 2.2 Plackett-Burman Experimental Results

[0145] Based on the single-factor experiment, an experimental design with N=12 trials was selected, with growth rate as the response value. The experimental design and results are shown in Table 11.

[0146] Table 11Plackett-Burman test design and results

[0147]

[0148] From Table 12 and Figure 1 It can be seen that each factor has a different impact on the response value. The factors with the greatest influence are F (resting time) > C (thawing temperature) > A (protective agent) > B (cooling rate) (P < 0.05). All of them are the main factors affecting the mycelial growth rate after storage. Resting time, thawing temperature, protective agent, and cooling rate all have positive effects on mycelial growth rate. In subsequent optimization experiments, the top three major factors with the greatest influence will be selected for the next step of the experiment.

[0149] Table 12 Plackett-Burman test factors, levels and significance analysis

[0150]

[0151] Note: * indicates significant difference (p<0.05); ** indicates extremely significant difference (p<0.01). Same below.

[0152] 2.3 Response surface experiment results and variance analysis

[0153] 2.3.1 Box-Behnken Experimental Design and Results

[0154] Based on single-factor and Plackett-Burman experiments, optimization was performed using resting time (A), varying concentrations of PEG 20000 (B), and thawing temperature (C). Based on the Box-Behnken central composite design principle, Design-Expert 13 was used to optimize these three factors. The experimental design and results are shown in Table 13.

[0155] Table 13 Box-Behnken test design and results

[0156]

[0157] 2.3.2 Model establishment and regression equation significance analysis

[0158] Using Design-Expert 13, the results of the Box-Behnken experimental design were fitted with binary multiple regression, and the quadratic multiple regression model of growth rate on each factor was obtained as follows:

[0159] Y = 1.18 - 0.0050A + 0.0568B - 0.0456C - 0.0369AB - 0.0019AC - 0.0639BC - 0.1994A2 - 0.1665B2 - 0.2442C2, where Y is the predicted growth rate response value.

[0160] Table 14 shows that the model F value is 88.68, and the P value is 0.0001, indicating that the model is extremely significant (P < 0.01). The correction coefficient R² is 0.9913, indicating a good linear relationship between growth rate and all independent variables. The P value for the lack-of-fit term is 0.1014, which is not significant (> 0.05), indicating that the regression model fits well and reflects the reliability of the experimental results. Protectant concentration (B) and thawing temperature (C) are significant, while resting time (A) is not significant. The order of influence on growth rate is: protectant concentration (B) > thawing temperature (C) > resting time (A). A², B², and C² are extremely significant, AB and BC are extremely significant, and AC is not significant.

[0161] The response surface method can be used to obtain a three-dimensional spatial diagram (such as Figures 2-4), where the greater the surface slope, the greater the influence of the independent variable on the response value. In terms of surface slope: protective agent concentration > thawing temperature and standing time, it means that the influence of protective agent concentration on growth rate is greater than that of thawing temperature and standing time (such as Figure 2 、 4 ), which is the same as the result of variance analysis. Figure 3 Left) It can be seen that the interaction between thawing temperature and standing time is not significant.

[0162] The coefficients of the fitted binary multiple regression equation were all negative, indicating that the response value had a maximum. The optimal conditions obtained from the fitted equation were: standing time (A) = 2.940, protective agent concentration (B) = 20.980, thawing temperature (C) = 24.407, and the predicted growth rate (Y) = 1.183. Based on the actual situation, the actual values ​​were adjusted to: standing time (A) = 3, protective agent concentration (B) = 20, thawing temperature (C) = 25. Repeated experiments were performed, and the average mycelial growth rate was (1.18 ± 0.14) mm·d. -1 , which is 99% of the model; this shows that the optimized quadratic model and regression equation are reliable in analyzing and predicting the mycelial growth rate.

[0163] Table 14 Effects of factors and significance evaluation in Box-Behnken design

[0164]

[0165] 2.3.3 Verification Experiment

[0166] Through response surface optimization analysis, the optimal treatment method was obtained according to the fitting formula: standing time (h) = 2.940, protective agent concentration (%) = 20.980, thawing temperature (℃) = 24.407, and the predicted mycelial growth rate (mm·d-1) = 1.183. In order to facilitate practical operation, the above optimal conditions were corrected to: standing time (h) = 3, protective agent concentration (%) = 20, thawing temperature (℃) = 25. Repeated experiments were carried out under the corrected conditions ( Figure 5 ), the mycelial growth rate of Ilex sanguinea was measured to be (1.18±0.14) mm·d⁻¹, which is 99% of the model prediction. Although the present invention has been described in detail above using general instructions, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications or improvements can be made based on the present invention. Therefore, such modifications or improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of the present invention.

Claims

1. A method for preserving winter iris fungus, characterized in that: The winter iris fungus species is the winter iris strain "Qianzhuang No. 1", and the preservation method comprises the following steps: (1) Preparation of exogenous material plate culture medium: 200 g potatoes, 80 g sawdust, 20 g glucose, 5 g peptone, 8 g agar, 1000 mL water, pH natural. Weigh the corresponding reagents according to the above formula, heat and stir, dissolve to volume, and then divide into 500 mL conical flasks. After sterilization in an autoclave at 121°C for 20 min, place in a clean bench, add 5 g / L L-proline, mix well, pour into plates, and wait for the plates to solidify naturally. (2) Take the strain "Qianzhuang No. 1" of Ilex strychnifolia, activate it in a plate culture medium containing exogenous substances, and culture it at a constant temperature of 25°C; (3) When the mycelium grows to two-thirds of the 90mm plate, use a 0.5cm 2 Punch a hole 0.5 cm behind the tip of the hyphae with a borer, inoculate into a 2 mL cryovial, add 1.5 mL of 5% to 25% PEG20000, and let it stand at 4 to 25°C for 1 to 9 h. (4) Cryopreservation tubes are used at 1-9°C / min -1 The speed was reduced to 4℃, and then to 1~9℃·min -1 The sample was cooled to -50°C and stored in liquid nitrogen.

2. The method for preserving the fungus of Ilex serrata according to claim 1, wherein: The concentration of PEG20000 in step (3) is 15% to 25%.

3. The method for preserving Ilex serrata strains according to claim 4, wherein: The concentration of PEG20000 in step (3) is 20%.

4. The method for preserving Ilex serrata strains according to claim 1, wherein: The standing condition in step (3) is: standing at 20-25° C. for 1-5 hours.

5. The method for preserving Ilex serrata strains according to claim 6, wherein: The standing condition in step (3) is: standing at 25° C. for 3 hours.

6. The method for preserving Ilex serrata strains according to claim 1, wherein: The cooling method in step (4) is: 4℃·min -1 or 9℃·min -1 Reduce to 4℃, then increase to 1℃·min -1 Drop to -50℃.

7. The method for preserving Ilex serrata strains according to claim 1, wherein: The thawing temperature of the winter iris strain "Qianzhuang No. 1" preserved according to the method is 20-40°C.

8. The method for preserving Ilex serrata strains according to claim 7, wherein: The thawing temperature of the Ilex strychnifolia strain "Qianzhuang No. 1" preserved according to the method is 20-30°C.

9. The method for preserving Ilex serrata strains according to claim 8, wherein: The thawing temperature of the Ilex strychnifolia strain "Qianzhuang No. 1" stored according to the method is 25°C.

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