A strain, method and application of producing extracellular polysaccharide Grifola frondosa

Abstract

The present invention belongs to the field of microbial technology, and discloses a strain of Grifola frondosa with high extracellular polysaccharide production. The strain is named G5922, classified as Grifola frondosa, deposited on June 6, 2023, and deposited at the General Microbiological Center of China National Committee for the Preservation of Microorganisms, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The present invention combines the strain selection technology of mutagenesis breeding and protoplast fusion to rearrange the genome of the Grifola frondosa strain, and screens out a strain with an extracellular polysaccharide production of 8.54 g / L, a biomass of 3.78 g / L, and a growth rate of 2.79 mm·d ‑1 The Grifola frondosa strains were increased by 67.75%, 44.27% and 8.56% respectively compared with the original strain.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to obtaining a Grifola frondosa fusion strain by utilizing genome rearrangement technology, in particular to a Grifola frondosa strain with high extracellular polysaccharide production, a method and an application thereof. Background Art

[0002] Grifola frondosa belongs to the phylum Fungi, the family Polyporaceae, and the genus Grifola. In the early 1980s, my country began to cultivate Grifola frondosa on a large scale, with the main production areas concentrated in Changbai Mountain area of ​​Jilin Province, Anhui, Fujian and Zhejiang. Grifola frondosa is a medicinal and edible fungus with rich nutritional value, including protein, amino acids required by the human body, carbohydrates and multiple vitamins. It is recorded in traditional Chinese herbal medicine books that Grifola frondosa is non-toxic and has the effects of strengthening the spleen and qi and relieving heat.

[0003] As early as the 20th century, researchers reported that Grifola frondosa polysaccharide had significant anti-tumor activity. As the research gradually deepened, researchers discovered more pharmacological values ​​of Grifola frondosa polysaccharide, such as antiviral, immunomodulatory, antioxidant activity and regulation of blood sugar and blood lipid levels. The medical effect of Grifola frondosa polysaccharide is significantly higher than that of other edible and medicinal fungal polysaccharides, and it also has the advantage of convenient medication. For this reason, there are continuous reports on the extraction of Grifola frondosa polysaccharide, its biological activity and pharmacological function related research. However, the current output of Grifola frondosa polysaccharide cannot meet its high demand in the food and pharmaceutical industries, and the importance of increasing polysaccharide production is highlighted. Therefore, it is necessary to screen a Grifola frondosa strain with high polysaccharide production to meet market demand.

[0004] Grifola frondosa is an edible fungus. The commonly used breeding methods for edible fungi are mainly domestication breeding, hybrid breeding, mutation breeding and protoplast fusion breeding. However, wild domesticated Grifola frondosa strains have problems such as low bioconversion rate and poor adaptability. Mutation breeding technology also has the disadvantages of blind screening and very large screening workload. Combined, the traditional screening method takes a long time and the effect is not obvious, so there is an urgent need for a method to accelerate the screening work, study Grifola frondosa breeding and improve the strains. Genome rearrangement technology is a strain breeding technology that combines mutation breeding and protoplast fusion technology. It is to rearrange multiple positive mutant strains after mutagenesis, prepare the strains in the strain mutant library as protoplasts, and perform multiple rounds of recursive genome fusion to finally obtain strains with significantly improved performance. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a high-yield extracellular polysaccharide Grifola frondosa strain and a breeding method thereof.

[0006] The technical solution adopted by the present invention to solve its technical problem is:

[0007] A high-yield extracellular polysaccharide Grifola frondosa strain (Grifola frondosa), the name of the strain is: G5922, the classification name is: Grifola frondosa, the preservation number is: CGMCC NO.40600, the preservation date is: June 6, 2023, the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0008] Furthermore, the strain is transformed from the original strain of Grifola frondosa numbered 5.404 through genome rearrangement technology. The Chinese name of the original strain of Grifola frondosa is Grifola frondosa, the Latin name is Grifola frondosa, the preservation number is: CGMCCNO.5.404, and the preservation date is: December 5, 1987.

[0009] The above-mentioned method for breeding a high-yield extracellular polysaccharide Grifola frondosa strain has the following specific steps:

[0010] (1) Cultivation of Grifola frondosa strains: The original Grifola frondosa strains were provided by China General Microbiological Culture Collection Center;

[0011] The original mycelium of Grifola frondosa is cut and activated in a plate culture medium, then transferred to a seed culture medium for shaking culture until the exponential phase to obtain a seed solution, and then 10% of the seed solution is transferred to a fermentation medium for shaking culture until the end of the fermentation phase to collect the mycelium;

[0012] (2) Optimization of protoplast preparation: The seed liquid obtained in step (1) is used to collect the mycelia of Grifola frondosa, which is filtered and then rinsed with 0.6 M mannitol to suspend the mycelia of Grifola frondosa in an enzymatic hydrolysis solution, and then subjected to an enzymatic hydrolysis reaction. The mycelia of Grifola frondosa is then rinsed with 0.6 M mannitol and then resuspended in an STC buffer to obtain a protoplast suspension; wherein the ratio of Grifola frondosa mycelia to the enzymatic hydrolysis solution is 100:1 in mg:mL;

[0013] (3) Optimization of ultraviolet and normal pressure room temperature plasma mutagenesis: The protoplast suspension obtained in step (2) is induced to undergo mutagenesis under ultraviolet light and normal pressure room temperature plasma, respectively, and the mutagenesis time is optimized as a variable. The protoplast suspension is then spread on a regeneration medium for culture, and a Grifola frondosa strain with high extracellular polysaccharide content, i.e., a Grifola frondosa strain with high polysaccharide production, is selected.

[0014] (4) Genome rearrangement: heat-inactivate and ultraviolet-inactivate the protoplasts of the high-yield polysaccharide Grifola frondosa strain selected in step (3), optimize the time of heat-inactivation and ultraviolet-inactivation of the protoplasts, and then mix the heat-inactivated and ultraviolet-inactivated protoplasts at a mass ratio of 1:1 for fusion, centrifuge for 15 minutes to obtain a precipitate, add 0.6 M mannitol solution to suspend the protoplasts, add PEG fusion agent and then water bath and optimize the fusion conditions, centrifuge to obtain a precipitate, wash it twice with a mannitol solution, and then apply it on a regeneration medium to obtain a first-generation strain F1;

[0015] (5) The F1 strains were screened for growth rate, biomass, and extracellular polysaccharides, and then another round of genome rearrangement was performed to screen out excellent strains with higher extracellular polysaccharides for identification, thereby obtaining a high-yield extracellular polysaccharide Grifola frondosa strain.

[0016] Furthermore, the step (1) is specifically as follows:

[0017] The original mycelium of Grifola frondosa was cut from the inclined surface, inoculated into a plate culture medium, inverted and placed in a 28°C incubator for activation for 15 days, transferred to a seed culture medium, and cultured in a shaking incubator at 28°C and 160 r / min until the exponential phase to obtain a seed solution, and then 10% of the seed solution was transferred to a fermentation medium, and cultured in a shaking incubator at 28°C and 160 r / min until the end of the fermentation phase to collect the mycelium;

[0018] The preparation method of the plate culture medium is as follows: 20% potato boiled extract, 2% glucose, 0.5% peptone, 0.2% KH2PO4, 0.1% MgSO4·7H2O, 0.002% VB1, and 2% agar powder; wherein the potato boiled extract is: the filtrate obtained by boiling potatoes with water for 30 minutes; the percentages are by mass;

[0019] Preparation method of seed culture medium: glucose 4%, peptone 0.5%, KH2PO4 0.1%, MgSO4·7H2O 0.05%, VB1 0.002%; the percentages are mass percentages;

[0020] The preparation method of the fermentation medium is as follows: glucose 2.2%, peptone 0.3%, KH2PO4 0.12%, MgSO4·7H2O 0.08%, VB1 0.012%; the percentages are by mass.

[0021] Furthermore, in the step (2), the enzymatic hydrolysis conditions are: enzymatic hydrolysis temperature 32°C, enzymatic hydrolysis in a shaker at 100 r / min for 4 hours;

[0022] Alternatively, the enzymatic solution is prepared as a 2% lytic enzyme solution, and the specific steps are: using 0.6 mol / L mannitol solution to dissolve 0.02 g lytic enzyme, the volume is fixed to 1 mL, and a 0.22 μm sterile organic filter membrane is used for filtration and sterilization, and the preparation is used immediately;

[0023] Alternatively, filter through a 300 mesh nylon filter.

[0024] Furthermore, in the step (3), the UV mutagenesis time is 50-70 s, and the ARTP mutagenesis time is 25-35 s.

[0025] Furthermore, the regeneration medium is a mannitol regeneration medium, and the preparation method of the mannitol regeneration medium is as follows: 20% potato boiled extract, 2% glucose, 0.5% peptone, 0.2% KH2PO4, 0.1% MgSO4·7H2O, 0.002% VB1, 2% agar powder, dissolved in 0.6M mannitol;

[0026] The potato boiling extract is the filtrate obtained by boiling potatoes with water for 30 minutes.

[0027] Furthermore, in step (4), the heat inactivation conditions are 55°C inactivation for 12 minutes and the UV inactivation time is 160 seconds; the fusion conditions are: PEG fusion agent concentration is 35%, pH is 7.5, temperature is 23°C, and fusion time is 15 minutes.

[0028] Furthermore, in step (5), the first generation strain F1 is screened for growth rate, biomass and extracellular polysaccharides to obtain the second generation strain F2, and after another round of the above screening, the strain is continuously transferred and cultured for 5-10 generations to obtain a high-yield extracellular polysaccharide Grifola frondosa strain.

[0029] The use of the above-mentioned high-exopolysaccharide Grifola frondosa strain in high-exopolysaccharide production.

[0030] The advantages and positive effects achieved by the present invention are:

[0031] 1. The method of the present invention solves the problem of low yield of Grifola frondosa polysaccharide, improves the existing screening method of Grifola frondosa strains, and accelerates the transformation process of Grifola frondosa strains. The present invention combines the strain selection technology of mutagenesis breeding and protoplast fusion to rearrange the genome of Grifola frondosa strains, and screens out a strain with an extracellular polysaccharide yield of 8.54 g / L, a biomass of 3.78 g / L, and a growth rate of 2.79 mm·d -1 The Grifola frondosa strains were increased by 67.75%, 44.27% and 8.56% respectively compared with the original strain.

[0032] 2. The present invention breeds strains with high polysaccharide production from Grifola frondosa, good genetic stability, fast plate growth rate, dense mycelium growth, and strong in vitro antioxidant capacity of extracellular polysaccharides. The existing screening method for Grifola frondosa strains is improved, and the genome rearrangement technology is applied to Grifola frondosa to accelerate the breeding speed.

[0033] 3. The method of the present invention uses growth rate, biomass and extracellular polysaccharide production as screening conditions to obtain a genome rearranged strain G5922 with good growth performance, dense mycelium, fast growth rate and genetic stability. The genetic similarity coefficient of the G5922 strain and the original Grifola frondosa strain was subjected to UPGMA cluster analysis, and the similarity coefficient between G5922 and the original strain was 0.4. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is a flow chart of genome rearrangement in the method of the present invention;

[0035] Figure 2 This is a diagram showing the effect of enzymolysis temperature on protoplast yield in the present invention;

[0036] Figure 3 This is a diagram showing the effect of enzymatic hydrolysis time on the yield of Grifola frondosa protoplasts in the present invention;

[0037] Figure 4 The figure is a graph of the lethality of the ultraviolet mutagenesis of Grifola frondosa protoplasts in the present invention;

[0038] Figure 5 The figure is a graph of the mutagenic lethality of Grifola frondosa spore ARTP in the present invention;

[0039] Figure 6 This is a diagram showing the effect of heat inactivation on Grifola frondosa protoplasts in the present invention;

[0040] Figure 7 This is a diagram showing the effect of ultraviolet inactivation on Grifola frondosa protoplasts in the present invention;

[0041] Figure 8 This is a graph showing the effect of PEG concentration on the fusion rate of Grifola frondosa protoplasts in the present invention;

[0042] Fig. 9 This is a graph showing the effect of pH on the fusion rate of Grifola frondosa protoplasts in the present invention;

[0043] Fig.10 This is a graph showing the effect of fusion temperature on the fusion rate of Grifola frondosa protoplasts in the present invention;

[0044] Fig.11 This is a graph showing the effect of fusion time on the fusion rate of Grifola frondosa protoplasts in the present invention;

[0045] Fig.12This is a plate culture diagram of the original Grifola frondosa strain and the extracellular polysaccharide-enhanced strain G5922 of the present invention;

[0046] Fig.13 This is a cluster diagram of the SRAP analysis of the Grifola frondosa strains in the present invention. DETAILED DESCRIPTION

[0047] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0048] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0049] A high-yield extracellular polysaccharide Grifola frondosa strain (Grifola frondosa), the name of the strain is: G5922, the classification name is: Grifola frondosa, the preservation number is: CGMCC NO.40600, the preservation date is: June 6, 2023, the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0050] Preferably, the strain is transformed from the original strain of Grifola frondosa numbered 5.404 by genome rearrangement technology. The Chinese name of the original strain of Grifola frondosa is Grifola frondosa, the Latin name is Grifola frondosa, the preservation number is: CGMCCNO.5.404, and the preservation date is: December 5, 1987.

[0051] The above-mentioned method for breeding a high-yield extracellular polysaccharide Grifola frondosa strain has the following specific steps:

[0052] (1) Cultivation of Grifola frondosa strains: The original Grifola frondosa strains were provided by China General Microbiological Culture Collection Center;

[0053] The original mycelium of Grifola frondosa is cut and activated in a plate culture medium, then transferred to a seed culture medium for shaking culture until the exponential phase to obtain a seed solution, and then 10% of the seed solution is transferred to a fermentation medium for shaking culture until the end of the fermentation phase to collect the mycelium;

[0054] (2) Optimization of protoplast preparation: The seed liquid obtained in step (1) is used to collect the mycelia of Grifola frondosa, which is filtered and then rinsed with 0.6 M mannitol to suspend the mycelia of Grifola frondosa in an enzymatic hydrolysis solution, and then subjected to an enzymatic hydrolysis reaction. The mycelia of Grifola frondosa is then rinsed with 0.6 M mannitol and then resuspended in an STC buffer to obtain a protoplast suspension; wherein the ratio of Grifola frondosa mycelia to the enzymatic hydrolysis solution is 100:1 in mg:mL;

[0055] (3) Optimization of ultraviolet and normal pressure room temperature plasma mutagenesis: The protoplast suspension obtained in step (2) is induced to undergo mutagenesis under ultraviolet light and normal pressure room temperature plasma, respectively, and the mutagenesis time is optimized as a variable. The protoplast suspension is then spread on a regeneration medium for culture, and a Grifola frondosa strain with high extracellular polysaccharide content, i.e., a Grifola frondosa strain with high polysaccharide production, is selected.

[0056] (4) Genome rearrangement: heat-inactivate and ultraviolet-inactivate the protoplasts of the high-yield polysaccharide Grifola frondosa strain selected in step (3), optimize the time of heat-inactivation and ultraviolet-inactivation of the protoplasts, and then mix the heat-inactivated and ultraviolet-inactivated protoplasts at a mass ratio of 1:1 for fusion, centrifuge for 15 minutes to obtain a precipitate, add 0.6 M mannitol solution to suspend the protoplasts, add PEG fusion agent and then water bath and optimize the fusion conditions, centrifuge to obtain a precipitate, wash it twice with a mannitol solution, and then apply it on a regeneration medium to obtain a first-generation strain F1;

[0057] (5) The F1 strains were screened for growth rate, biomass, and extracellular polysaccharides, and then another round of genome rearrangement was performed to screen out excellent strains with higher extracellular polysaccharides for identification, thereby obtaining a high-yield extracellular polysaccharide Grifola frondosa strain.

[0058] Preferably, the step (1) is specifically:

[0059] The original mycelium of Grifola frondosa was cut from the inclined surface, inoculated into a plate culture medium, inverted and placed in a 28°C incubator for activation for 15 days, transferred to a seed culture medium, and cultured in a shaking incubator at 28°C and 160 r / min until the exponential phase to obtain a seed solution, and then 10% of the seed solution was transferred to a fermentation medium, and cultured in a shaking incubator at 28°C and 160 r / min until the end of the fermentation phase to collect the mycelium;

[0060] The preparation method of the plate culture medium is as follows: 20% potato boiled extract, 2% glucose, 0.5% peptone, 0.2% KH2PO4, 0.1% MgSO4·7H2O, 0.002% VB1, and 2% agar powder; wherein the potato boiled extract is: the filtrate obtained by boiling potatoes with water for 30 minutes; the percentages are by mass;

[0061] Preparation method of seed culture medium: glucose 4%, peptone 0.5%, KH2PO4 0.1%, MgSO4·7H2O 0.05%, VB1 0.002%; the percentages are mass percentages;

[0062] The preparation method of the fermentation medium is as follows: glucose 2.2%, peptone 0.3%, KH2PO4 0.12%, MgSO4·7H2O 0.08%, VB1 0.012%; the percentages are by mass.

[0063] Preferably, in step (2), the enzymatic hydrolysis conditions are: enzymatic hydrolysis temperature 32° C., enzymatic hydrolysis in a shaker at 100 r / min for 4 h;

[0064] Alternatively, the enzymatic solution is prepared as a 2% lytic enzyme solution, and the specific steps are: using 0.6 mol / L mannitol solution to dissolve 0.02 g lytic enzyme, the volume is fixed to 1 mL, and a 0.22 μm sterile organic filter membrane is used for filtration and sterilization, and the preparation is used immediately;

[0065] Alternatively, filter through a 300 mesh nylon filter.

[0066] Preferably, in step (3), the UV mutagenesis time is 50-70 s, and the ARTP mutagenesis time is 25-35 s.

[0067] Preferably, the regeneration medium is a mannitol regeneration medium, and the preparation method of the mannitol regeneration medium is as follows: 20% potato boiled extract, 2% glucose, 0.5% peptone, 0.2% KH2PO4, 0.1% MgSO4·7H2O, 0.002% VB1, 2% agar powder, dissolved in 0.6M mannitol;

[0068] The potato boiling extract is the filtrate obtained by boiling potatoes with water for 30 minutes.

[0069] Preferably, in step (4), the heat inactivation conditions are 55°C inactivation for 12 min, and the UV inactivation time is 160 s; the fusion conditions are: PEG fusion agent concentration is 35%, pH is 7.5, temperature is 23°C, and fusion time is 15 min.

[0070] Preferably, in step (5), the first generation strain F1 is screened for growth rate, biomass and extracellular polysaccharides to obtain the second generation strain F2, and after another round of the above screening, the strain is continuously transferred and cultured for 5-10 generations to obtain a high-yield extracellular polysaccharide Grifola frondosa strain.

[0071] The use of the above-mentioned high-exopolysaccharide Grifola frondosa strain in high-exopolysaccharide production.

[0072] The specific preparation and testing are as follows:

[0073] A high-yield extracellular polysaccharide Grifola sp. strain, characterized in that: the name of the strain is G5922, the classification name is Grifola sp., the preservation number is CGMCC NO.40600, the preservation date is June 6, 2023, and the preservation unit is General Microbiology Center of China Microbiological Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0074] Specifically, the strain is transformed from the original strain of Grifola frondosa numbered 5.404 through genome rearrangement technology. The Chinese name of the original strain of Grifola frondosa is Grifola frondosa, the Latin name is Grifola frondosa, the preservation number is: CGMCCNO.5.404, and the preservation date is: December 5, 1987.

[0075] The above method for breeding the high-yield extracellular polysaccharide Grifola frondosa strain has the following specific steps:

[0076] 1. Cultivation method of Grifola frondosa strains:

[0077] The original mycelium of Grifola frondosa was cut from the inclined surface, inoculated in a plate culture medium, inverted and placed in a 28°C incubator for activation for 15 days, cut into small pieces of 1×1 cm after the plate was fully grown, and 4 pieces were transferred to a seed culture medium and cultured in a shaking incubator at 28°C and 160 r / min until the exponential phase (the 6th day) to obtain a seed solution, and then 10% of the seed solution was transferred to a fermentation medium and cultured in a shaking incubator at 28°C and 160 r / min until the end of the fermentation phase (the 8th day) to collect the mycelium;

[0078] 2. Culture medium preparation method:

[0079] Plate culture medium: 20% potato boiled extract, glucose 2%, peptone 0.5%, KH2PO4 0.2%, MgSO4·7H2O 0.1%, VB1 0.002%, agar powder 2%;

[0080] Seed culture medium: glucose 4%, peptone 0.5%, KH2PO4 0.1%, MgSO4·7H2O 0.05%, VB1 0.002%;

[0081] Fermentation medium: glucose 2.2%, peptone 0.3%, KH2PO4 0.12%, MgSO4·7H2O 0.08%, VB1 0.012%;

[0082] 3. Solution preparation method:

[0083] Mannitol regeneration medium: 20% potato boiled extract, 2% glucose, 0.5% peptone, 0.2% KH2PO4, 0.1% MgSO4·7H2O, 0.002% VB1, 2% agar powder, dissolved in 0.6M mannitol;

[0084] STC buffer: 1 M sorbitol, 10 mM Tris-HCl (pH = 7.5), 25 mM CaCl2, pH natural;

[0085] Preparation of 2% Lywallzyme: Dissolve 0.02 g of Lywallzyme in 0.6 mol / L mannitol solution, adjust the volume to 1 mL, and filter and sterilize with a sterile organic filter membrane (0.22 μm) (prepare and use immediately).

[0086] 4. Methods for genome rearrangement breeding of Grifola frondosa strains, such as Figure 1 As shown, the following steps are included:

[0087] (1) Preparation of protoplasts:

[0088] The cultured seed solution was filtered through a 300-mesh nylon filter to collect the mycelium of Grifola frondosa. After rinsing with 0.6M mannitol, appropriate amounts of mycelium were suspended in different prepared enzymatic solutions (Table 1). The enzymatic solution ratio was 100mg of mycelium suspended in 1mL of enzymatic solution, and enzymatic hydrolysis was performed in a shaker at 26-35°C and 100r / min for 2-5h. The excess mycelium that was not enzymatically hydrolyzed was removed with absorbent cotton, rinsed with 0.6M mannitol, and resuspended in STC buffer. The mycelium was counted using a hemocytometer. The most suitable enzymatic hydrolysis conditions were obtained when the most protoplasts were obtained.

[0089] from Figure 2 , Figure 3 It can be seen that the most suitable enzymatic hydrolysis conditions are: the enzymatic solution is prepared with 2% lytic enzyme, the enzymatic hydrolysis temperature is 32°C, and the enzymatic hydrolysis is carried out in a shaker at 100 r / min for 4 hours.

[0090] Table 1

[0091]

[0092] (2) UV mutagenesis and regeneration

[0093] Add 1 mL of protoplast suspension diluted to a concentration of 106 cells / mL to the culture dish and irradiate under UV light for 0, 10, 30, 60, 90, and 120 seconds. Take 200 μL from each dish and spread it on mannitol regeneration medium. Culture it at 28±0.5℃ in the dark for about 15 days, record the number of colonies, and calculate the lethality. Figure 5As shown, strains with a lethality rate of 70%-80% are selected and transferred to plate culture medium for preservation, which are the mutagenesis regeneration strains;

[0094]

[0095] Wherein: A is the number of regenerated colonies of Grifola frondosa protoplasts after mutagenesis, and B is the number of regenerated colonies of Grifola frondosa protoplasts before mutagenesis.

[0096] Statistics show that the optimal UV mutagenesis time is 60s.

[0097] (3) Ambient temperature and pressure plasma mutation (ARTP) and its regeneration

[0098] ARTP mutagenesis is carried out using Grifola frondosa spores. An appropriate amount of sterile saline is added to the Grifola frondosa strain that has been cultured on a plate for 50-60 days and has vigorous mycelium. The mycelium surface is scraped with a cotton swab to prepare a spore suspension, and the spore concentration is kept at 10 5 / mL range. Take 10μL of bacterial suspension and spread it on the slide. Set the mutagenesis time to 0, 10, 20, 30, 40, and 50s respectively. Set up 5 groups in parallel. After the mutagenesis treatment, mix the bacterial suspension on the surface of the slide with 1mL of sterile saline. Take 200μL and spread it on the plate culture medium. Incubate it in the dark at 28℃ incubator. Record the number of colonies. Calculate the lethality under each mutagenesis gradient according to the formula in (2), as follows: Figure 6 As shown; strains with a lethality rate of 80%-90% are transferred to plate culture medium for preservation, which are mutagenesis regeneration strains;

[0099] Statistics show that the optimal ARTP mutagenesis time is 30s.

[0100] (4) Screening of mutagenic strains

[0101] According to the lethality of different mutagenesis methods, the optimal mutagenesis time was selected to carry out a large number of mutagenesis on the Grifola frondosa strains. After the mutagenized Grifola frondosa strains were regenerated, they were cultured at 28±0.5℃ and antagonistic experiments were carried out with the original strains on the plate culture medium. The mutagenized strains with antagonistic reactions were inoculated on the plate culture medium again, and the single colonies with fast growth rate were selected as the primary screening strains. The primary screening strains were inoculated with seed culture medium, cultured at 28℃ and 160r / min for 5 days, and then transferred to the fermentation medium and cultured under the same conditions for 7 days. After the fermentation, the biomass of the strains, the production of intracellular polysaccharides and extracellular polysaccharides were measured. The mutagenized strains with higher biomass and polysaccharide production than the original strains were selected as the final selected variant strains for protoplast fusion.

[0102] (5) Heat inactivation of protoplasts

[0103] Transfer 1 mL of 106 cells / mL protoplast suspension of Grifola frondosa into a 1.5 mL Eppendorf tube and place it in a 55°C water bath for 5, 6, 7, 8, 9, 10, 11, and 12 min, then draw 200 μL and spread it on mannitol regeneration medium and culture it in the dark at 28°C for 15 days.

[0104]

[0105] Wherein: A is the number of colonies on the mannitol regeneration medium after inactivation; B is the number of colonies on the mannitol regeneration medium before inactivation.

[0106] like Figure 7 As shown, by observing the number of colonies, it was determined that the optimal time for heat inactivation of Grifola frondosa protoplasts was 12 minutes.

[0107] (6) UV inactivation of protoplasts

[0108] 1 mL of 106 cells / mL protoplast suspension of Grifola frondosa was placed in a sterile culture dish, inactivated by ultraviolet light, and irradiated for 120, 130, 140, 150, and 160 seconds, respectively. 0.2 mL of each plate was spread on mannitol regeneration medium, and the plate was wrapped with tin foil and kept in the dark and kept at 28°C for about 15 days.

[0109] Take the plate out of the tin foil and observe and record the number of colonies. Figure 8 As shown, it was determined that the optimal time for UV inactivation of Grifola frondosa protoplasts was 160s.

[0110] (7) Protoplast fusion

[0111] Protoplasts were prepared from the high-yield polysaccharide Grifola frondosa strain obtained by mutagenesis, and the protoplasts were equally divided into two groups, one group was subjected to heat inactivation treatment, and the other group was subjected to ultraviolet irradiation inactivation treatment, and then the two groups were mixed in a ratio of 1:1, 2 mL was taken out and centrifuged at 4°C and 4000 r / min for 15 min, precipitated, 0.2 mL of a 0.6 M mannitol solution was added to suspend the protoplasts, and then 1.8 mL of a PEG fusion agent (concentration 20%-40%, pH 6.5-8.5) was added, and after fusing at different water bath temperatures (21-29°C) and different times (10-35 min), the precipitate was centrifuged and washed twice with a mannitol solution, then resuspended in a mannitol solution, and coated on a mannitol regeneration medium;

[0112]

[0113] Wherein: A is the colony number of fusion protoplasts in mannitol regeneration medium; B is the colony number of inactivated parent in mannitol regeneration medium; C is the colony number of fusion daughter in protoplasts in mannitol regeneration medium.

[0114] like Figure 9-12 As shown, by observing the number of colonies, it was found that the optimal fusion conditions for the single-factor experiment were 35% PEG fusion agent concentration, 7.5 pH, 23°C temperature, and 15 min fusion time.

[0115] (8) Determination of growth performance

[0116] Cut the strain from the plate culture medium and inoculate it on a new plate culture medium. Culture it at 28℃ in the dark for about 15 days. Record the colony radius at the end of growth and calculate the average growth rate of mycelium.

[0117]

[0118] Where: A is the colony radius (mm); B is the number of days of mycelium growth (d).

[0119] After the fermentation was completed, the mycelium in the fermentation broth was collected on filter paper, dried at 60°C to constant weight, and the dry weight of the mycelium, i.e., biomass, was calculated.

[0120] After the fermentation is completed, a certain volume of fermentation liquid is taken, centrifuged to obtain the supernatant, 4 times the volume of 95% ethanol is added, and after alcohol precipitation at 4°C for 12 hours, the precipitate is centrifuged and added with 5 times the volume of distilled water to dissolve it, and then the polysaccharide content in each sample is measured by the phenol-sulfuric acid method.

[0121] (9) Screening and identification of genome rearrangement strains

[0122] The original strain of Grifola frondosa was used as the control group. The first-generation Grifola frondosa fusion strain with fast growth rate and high polysaccharide yield was selected as the parent of the first round of genome rearrangement. Four strains with better performance were selected for the second round of genome rearrangement. Finally, the G5922 strain with improved growth rate and polysaccharide yield was selected. As shown in Table 2, the growth rate of G5922 increased from 2.57 mm·d -1 Improved to 2.79mm·d -1 The biomass of G5922 increased by 44.27% compared with 2.62 g / L of the original strain, and the polysaccharide yield increased to 8.54 g / L, which was 67.75% higher than the original strain.

[0123] Table 2

[0124] strain <![CDATA[Growth rate / (mm·d -1 )]]> Biomass (g / L) Extracellular polysaccharide (g / L) WT 2.57±0.05 2.62 5.09 G5922 2.79±0.14 3.78 8.54

[0125] The colony diagram of the high-yield extracellular polysaccharide Grifola frondosa strain of the present invention is as follows: Fig.12 As shown in the figure, it can be seen that Fig.12This is a comparison of the plate culture medium of the original strain and the G5922 strain. The hyphae of the original strain are relatively thick, while the hyphae of the genome rearranged strain are relatively fluffy, extending in all directions, and growing faster than the original strain. The genetic similarity coefficients of the original strain of Grifola frondosa and the G5922 strain were analyzed by UPGMA cluster analysis using sequence-related Amplified Polymorphism (SRAP), as shown in the following figure: Fig.13 As shown, the similarity coefficient between strain G5922 and the original strain is 0.4, which shows that genome rearrangement further enriched the genetic basis of the original strain on the basis of mutagenesis.

[0126] The extracellular polysaccharides of the original strain and G5922 were extracted and purified. The two crude extracellular polysaccharide samples were loaded onto a DEAE-650 cellulose ion exchange column. They were first eluted with distilled water to obtain neutral extracellular polysaccharides WT-0M and G5922-0M, and then 0.2 M NaCl was used to continue elution to obtain acidic extracellular polysaccharides WT-0.2M and G5922-0.2M.

[0127] As shown in Table 3 , the molecular weight of the exopolysaccharide of the rearranged strain G5922 was lower than that of the original strain WT.

[0128] Table 3

[0129] sample Retention time (min) Molecular weight (kDa) WT-0M 10.245 <![CDATA[1.16×10 3 ]]> WT-0.2M 9.968 <![CDATA[1.38×10 3 ]]> G5922-0M 10.729 <![CDATA[0.86×10 3 ]]> G5922-0.2M 10.267 <![CDATA[1.15×10 3 ]]>

[0130] The ability of antioxidants to scavenge ABTS free radicals was evaluated by measuring the absorbance at 734 nm. When the polysaccharide concentration reached 2.5 mg / mL, the scavenging abilities of WT-0M, WT-0.2M, G5922-0M and G5922-0.2M on ABTS free radicals were 70.57%, 81.91%, 53.55% and 83.69%, respectively. In comparison, the scavenging ability of G5922 acidic polysaccharide was stronger than that of the original strain. When the polysaccharide concentration reached 2.5 mg / mL, the scavenging rates of WT-0M, WT-0.2M, G5922-0M and G5922-0.2M on hydroxyl radicals were 78.25%, 94.72%, 84.21% and 92.63%, respectively. The scavenging ability of G5922 neutral polysaccharide was stronger than that of the original strain. The scavenging rates of WT-0M, WT-0.2M, G5922-0M and G5922-0.2M on DPPH free radicals at a concentration of 2.5 mg / mL were 84.04%, 94.68%, 94.33% and 96.10%, respectively. The scavenging ability of the two purified polysaccharides of G5922 on DPPH free radicals was stronger than that of the original strain. The experiment showed that the scavenging ability of Grifola frondosa polysaccharide on DPPH free radicals was the best, and the genome rearranged strain showed better antioxidant capacity.

[0131] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A strain of Grifola frondosa producing extracellular polysaccharide ( Grifola frondosa )G5922, Features: The deposit number is: CGMCC NO.40600, the deposit date is: June 6, 2023, and the deposit unit is: General Microbiology Center of China Microbiological Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. Use of the exopolysaccharide-producing Grifola frondosa strain according to claim 1 in producing exopolysaccharides.

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