High-yield and high-activity bacterial strain for polyphenols and polysaccharides of oudemansiella mucilaginosa as well as preparation method and application of bacterial strain

Abstract

The invention discloses an oudemansiella mucidaea polyphenol polysaccharide high-yield and high-activity bacterial strain as well as a preparation method and application thereof, the bacterial strain is an oudemansiella mucidaea bacterial strain Du20211005, the bacterial strain is preserved in the China General Microbiological Culture Collection Center on November 20, 2024, and the preservation number is CGMCC NO.41665. The invention further discloses a preparation method of the oudemansiella mucidaea polyphenol polysaccharide high-yield and high-activity bacterial strain for the oudemansiella mucidaea polyphenol polysaccharide high-yield and high-activity bacterial strain for the oudemansiella mucidaea polyphenol polysaccharide. The bacterial strain has the advantages of high content of polyphenol, polysaccharide and other bioactive components, high activity, strong antioxidant ability, good antibacterial effect and high medicinal value, can be used for preparing drugs and bacteriostatic agents for antioxidant food or health care products, and provides a theoretical basis for the application of oudemansiella mucilaginosa in the aspects of drugs, health care products and biopesticides.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a strain of Agaricus glutinosa polyphenol polysaccharide with high yield and high activity, and a preparation method and application thereof. Background Art

[0002] Oudemansiella mucida belongs to the class Basidiomycetes, the order Agaricales, the family Physalacria, and the genus Oudemansiella. Also known as sticky long root mushroom, white ring mushroom, and white sticky honey fungus, it is an important edible and medicinal fungus, mainly distributed in Hebei, Shanxi, Heilongjiang, Fujian, Guangxi, Sichuan, etc. Oudemansiella mucida is rich in nutrients such as amino acids, proteins, polysaccharides, dietary fiber, and mineral elements. It also contains a variety of active ingredients, such as polysaccharides, polyphenols, strobilurins, odermex, and myxomycin, and has biological activities such as anti-oxidation, liver protection, intestinal flora adjustment, anti-tumor, and inhibition of pathogens. It has extremely high edible value and health value.

[0003] Studies have found that edible fungi have a wide range of medicinal values ​​such as antioxidant, anti-cancer, antibacterial, hypoglycemic and cholesterol-lowering. Through the study of its unique antioxidant activity, it plays an important role in delaying aging, removing age spots, and preventing and treating diseases. At present, the research on active substances in edible fungi is mostly focused on polyphenols. For example, Du Ping et al. extracted polyphenol content of Agaricus oosporeus fruiting body between 3.59 and 4.19 mg / g (Agrocybe oosporeus domestication, nutritional components and antioxidant activity [J]); Fu Shenglin extracted polyphenol content of 16.23 mg / g from wild Tiger Milk Mushroom using the best formula (Study on domestication and cultivation of wild Tiger Milk Mushroom and antioxidant activity and structural characterization of its polysaccharides [D]) and An Yifeng et al. extracted polyphenol content of only 3.642 mg / g from Hericium erinaceus by optimizing the pomegranate branch formula, and the polyphenol extract had the highest OH free radical scavenging rate of 76.1% (Effect of pomegranate branches on total phenol content and antioxidant activity of Hericium erinaceus [J]). Ye Shucai et al. studied the antioxidant activity of polyphenols in Rhizoma Odonata, and the results showed that the scavenging rate of polyphenols in Rhizoma Odonata for DPPH free radicals was 60.56%, and the scavenging rate for OH free radicals was 65.68% (ultrasound-assisted extraction of polyphenols in Rhizoma Odonata and its antioxidant activity [J]). However, the existing edible fungi generally have low polyphenol content and low activity, which limits their practical application in medicines.

[0004] Plant antibacterial agents are new antibacterial agents that are directly or indirectly processed and synthesized by extracting active substances with antibacterial effects from plants. Since this type of agent has the advantages of rapid biodegradation in the environment, low toxicity to humans, animals and non-target organisms, and it is not easy for fungi and insects to develop resistance, the research, development and application of this type of agent has become a hot topic in current research. Studies have found that edible fungus fermentation liquid has a significant inhibitory effect on common plant pathogenic fungi and has a good application prospect for plant disease and insect control, but the active ingredients are not clear. Previous studies have shown that Mucidin produced by Odette mushroom can significantly inhibit most plant pathogens, but there are no relevant reports on the antibacterial activity of Odette mushroom polyphenols / polysaccharides. Summary of the invention

[0005] In view of the deficiencies of the above-mentioned prior art, the technical problem to be solved by the present invention is: how to provide a strain of Odetteria glutinosa polyphenols and polysaccharides with high yield and high activity, and a preparation method and application thereof, so as to efficiently obtain strains with high activity of polysaccharides and polyphenols, enrich the Odetteria glutinosa germplasm resource bank, and also lay an important foundation for the pharmacological research related to the fungus and the development and utilization of medicines, health products, and biocontrol agents.

[0006] In order to solve the above technical problems, the present invention adopts the following technical scheme: a strain with high yield and high activity of polyphenols and polysaccharides of Oudemansiella mucida, the strain being Oudemansiella mucida strain Du20211005, which was deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee on November 20, 2024, with a deposit number of CGMCCNO.41665; the extraction amount of polyphenols in the fruiting body of the strain Du20211005 is 38.80 mg / g, and the extraction amount of polysaccharides in the fruiting body of the strain Du20211005 is 33.192 mg / g.

[0007] Another object of the present invention is to provide a method for preparing polyphenols of Odetteria glutinosa, comprising the following steps:

[0008] 1) inoculating the mycelium of the above-mentioned strain into a seed culture medium, placing it in a constant temperature shaking incubator, and culturing it at a speed of 210 to 240 r / min and 25 to 29° C. for 7 to 10 days to obtain a strain;

[0009] 2) inoculating the strain obtained in step 1) into a cultivation medium, culturing at 25-29° C. for 25-32 days until the mycelium fills the cultivation bag, and then inducing at low temperature at 10-21° C. until the mushrooms are fruited to obtain fruiting bodies; the cultivation medium comprises the following components by mass fraction: 30% sawdust, 16% wheat bran, 5% corn kernels, 1% lime, 1% gypsum, and the balance is corn cobs, or the cultivation medium comprises the following components by mass fraction: 30% sawdust, 8% wheat bran, 3% corn kernels, 1% lime, 1% gypsum, and the balance is corn cobs;

[0010] 3) drying and grinding the fruiting bodies obtained in step 2), placing them in anhydrous ethanol solution to obtain an extract, ultrasonically treating them, and centrifuging them to obtain the supernatant to obtain the Odenia glutinosus polyphenols.

[0011] Preferably, the volume fraction of ethanol in the extract is 70% to 75%, and the mass volume ratio of the fruiting body to the ethanol solution is 1:50 to 1:55 g / mL; the power during the ultrasonic treatment is 200 to 400 W, the temperature is 40 to 60° C., and the ultrasonic time is 30 min.

[0012] Another object of the present invention is to provide a method for preparing Odenia glutinosae polysaccharide, comprising the following steps:

[0013] S1: inoculating the mycelium of the above-mentioned strain into a seed culture medium, placing it in a constant temperature shaking incubator, and culturing it at a speed of 210-240 r / min and 25-29° C. for 7-10 days to obtain a strain;

[0014] S2: inoculating the strain obtained in step 1) into a cultivation medium, culturing at 25-29° C. for 25-32 days until the mycelium fills the cultivation bag, and then inducing at low temperature at 10-21° C. until the mushrooms are fruited to obtain fruiting bodies; the cultivation medium comprises the following components by mass fraction: 30% sawdust, 16% wheat bran, 5% corn kernels, 1% lime, 1% gypsum, and the balance is 47% corn cobs, or the cultivation medium comprises the following components by mass fraction: 30% sawdust, 8% wheat bran, 3% corn kernels, 1% lime, 1% gypsum, and the balance is corn cobs;

[0015] S3: drying and grinding the fruiting bodies obtained in step 2), placing them in anhydrous ethanol solution to obtain an extract, extracting at high temperature, keeping at 4°C overnight, and centrifuging to obtain a precipitate to obtain the Agaricus glutinosa polysaccharide.

[0016] Preferably, the mass volume ratio of the fruiting body to the anhydrous ethanol solution is 1:35 g / mL, the volume fraction of ethanol in the extract is 85% to 95%, the extraction time is 3 to 4.5 hours, and the extraction temperature is 90°C.

[0017] Preferably, the seed culture medium comprises the following components: 20 g / L xylose, 2 g / L yeast extract, 0.5 g / L potassium dihydrogen phosphate, 0.75 g / L magnesium sulfate, 200 g / L potato, and pH 5.5.

[0018] Another object of the present invention is to provide the use of the above strain or the polyphenols / polysaccharides prepared by the above method in the preparation of antioxidant drugs or health products.

[0019] Preferably, the anti-oxidation is achieved by reducing the content of active oxygen, which includes superoxide radicals, hydrogen peroxide and / or hydroxyl radicals.

[0020] Another object of the present invention is to provide the use of the above strain or the polyphenol / polysaccharide prepared by the above method in the preparation of an antibacterial agent.

[0021] Preferably, the antibacterial agent is used to control black spot pathogen of Eucommia ulmoides, Polytrichomoniasis palmi, Gibberella zeae or Cladosporium cladosporium.

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

[0023] 1. The yield of polyphenols and polysaccharides of the sticky small Ode mushroom strain Du20211005 provided by the present invention is significantly higher than that of other strains. At the same time, biological characteristics are studied, and a medium and a culture method suitable for mycelial growth are screened out by single factor and orthogonal test. Under the same culture conditions, by optimizing the cultivation medium and extraction conditions, the yield of polysaccharides and polyphenols is significantly improved. And the present invention obtains high activity of polysaccharides and polyphenols, strong antioxidant capacity, good antibacterial effect, high medicinal value, and can be used to prepare drugs and antibacterial agents for the effect of antioxidant foods or health products, providing a theoretical basis for the application of sticky small Ode mushrooms in medicines, health products and biological pesticides. The present invention screens out strains with high polysaccharide and polyphenol yields and excellent quality, which can enrich the germplasm resource bank of small Ode mushrooms and have good application prospects.

[0024] 2. The preparation method of the present invention is simple, easy to operate, has low production and processing costs, high yields of active products such as polysaccharides and polyphenols, and is convenient for commercial large-scale production. At the same time, it can also be prepared into medicines or plant antibacterial agents, which are non-toxic, free of residue and pollution, and not easy to produce drug resistance. Environmental friendliness broadens the application field. At the same time, the present invention fills the research gap on the polysaccharides and polyphenols of Mushroom Glue, and also provides a theoretical basis for the research on the polysaccharides and polyphenols of Mushroom Glue. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a colony morphology diagram of strain Du20211005 of the present invention.

[0026] Figure 2This is a morphological diagram of the wild fruiting body of strain Du20211005 of the present invention.

[0027] Figure 3 Polyphenol content in the fruiting bodies of strain Du20211005 cultured on different cultivation media.

[0028] Figure 4 is the polyphenol content of fruiting bodies of different strains.

[0029] Figure 5 Polysaccharide content in the fruiting bodies of strain Du20211005 cultured on different cultivation media.

[0030] Figure 6 is the polysaccharide content of the fruiting bodies of different strains.

[0031] Figure 7 The antibacterial property of the fruiting body polysaccharide of strain Du20211005 of the present invention. DETAILED DESCRIPTION

[0032] The following will be combined with specific embodiments of the present invention to clearly and completely describe the technical solution of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. The experimental methods used in the present invention described below are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0033] The palm pseudodiscus, wheat fusarium, eucommia black spot and cladosporium used in the following examples were isolated from wild powder gills, tobacco pipe mushrooms, Alternaria albuminosa and Agaricus blazei fruiting bodies, respectively. Ode mushroom Du20210899 (abbreviated as Du-1), Ode mushroom Du20210900 (abbreviated as Du-2), Ode mushroom Du20210901 (abbreviated as Du-3), Ode mushroom Du20210902 (abbreviated as Du-4), Ode mushroom Du20210939 (abbreviated as Du-6) and Ode mushroom Du20210949 (abbreviated as Du-7) were all collected from Simianshan, Jiangjin District, Chongqing, and are now stored in the Edible Fungus Cultivation Laboratory of Yangtze Normal University.

[0034] Example 1 Isolation of strains

[0035] The specimens of the Odette mushroom strain were collected from dead standing trees or fallen trees of Simianshan broad-leaved trees in Jiangjin District, Chongqing on October 23, 2021. The fresh, mildew-free, and intact fruiting bodies were used as the material for strain isolation. In the clean bench, the surface of the fruiting body was first wiped with a 75% alcohol cotton ball for disinfection, and then the fruiting body was cut in half with a sterile scalpel, and 0.5 cm 2The internal tissue blocks on the left and right sides were inoculated into PDA plates, three blocks were cross-inoculated on each plate, and cultured in the dark at 25°C for 5 days. When white fluffy hyphae grew around the tissue blocks, the pure strain mycelium was inoculated into the PDA test tube slant culture medium according to the sterile operation procedures, and cultured at 25°C for 9 to 12 days. The test tubes with well-developed mycelium and no contamination from other bacteria were selected and stored in a 4°C refrigerator to obtain the purified active strain, which was named Du20211005 (abbreviated as Du-5).

[0036] Example 2 Identification of strains

[0037] 1. Morphological characteristics

[0038] 1. Mycelium morphological characteristics

[0039] The mycelium of strain Du20211005 of the present invention is white and dense, grows vigorously, has well-developed aerial mycelium, and has neatly grown edges. Figure 1 As shown, the average daily growth rate of mycelium in the optimal culture medium (xylose 25g / L, yeast extract 3g / L, potassium dihydrogen phosphate 0.5g / L, magnesium sulfate 0.75g / L, potato 200g / L, agar powder 20g / L, pH 5) is 0.6325cm / d; under the following culture medium formula and culture conditions: xylose 20g / L, yeast extract 2g / L, potassium dihydrogen phosphate 0.5g / L, magnesium sulfate 0.75g / L, potato 200g / L, liquid volume 120mL, rotation speed 220rpm, pH 5.5, culture 8d, the liquid fermentation mycelium biomass is 82.88mg / d.

[0040] 2. Fruiting body morphology characteristics

[0041] The wild fruiting body morphology of strain Du20211005 of the present invention is as follows Figure 2 As shown. Its fruiting body is white, with the middle of the cap light yellow or light brown, the cap diameter is 3-7cm, hemispherical when young, gradually flattened after maturity, water-soaked, sticky or gummy on the surface, white flesh; white gills, 0.3-0.7cm wide, sparse, straight to curved; stipe is medium, 1.5-4cm long, 0.2-0.6cm thick, cylindrical, slightly swollen at the base, not rhizoid-like, light yellow-brown, gradually turning white upwards, fibrous, solid inside; the ring grows in the middle and upper part of the stipe, white membranous, and the spore print is white. The fruiting bodies grow in groups on dead standing trees, causing wood decay.

[0042] 2. Molecular Biological Characteristics

[0043] The present invention performs DNA extraction and ITS and LSU amplification sequencing on the mycelium of the obtained strain Du20211005. The total length of the ITS sequence is 775 bp, and the sequence measured by the sequencing result is SEQ ID NO.1, which has a genetic consistency of 99.61% with the ITS (Internal Transcribed Spacer) of Oudemansiella mucida in the Genebank database. The total length of the LSU (large ribosomal subunits) sequence is 1328 bp, and the sequence measured by the sequencing result is SEQ ID NO.2, which has a genetic consistency of 99.77% with Oudemansiella mucida.

[0044] In view of the above morphological characteristics and the classification and identification research of ITS and LSU sequences, it is determined that the strain Du20211005 of the present invention belongs to the Basidiomycetes, Agaricales, Physalacria, Oudemansiella, and is identified as a new strain of Oudemansiellamucida. The strain has been deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on November 20, 2024, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No: 41665.

[0045] Example 3 Preparation method of Odette mushroom polyphenols

[0046] The specific steps include:

[0047] 1) Inoculating the mycelium of the Du20211005 strain of Agaricus glutinosa into a seed culture medium, placing the culture medium in a constant temperature shaking incubator, and culturing the culture medium at a speed of 220 r / min and 25-29° C. for 7-10 days to obtain a strain; the seed culture medium comprises the following components: 20 g / L xylose, 2 g / L yeast extract, 0.5 g / L potassium dihydrogen phosphate, 0.75 g / L magnesium sulfate, 200 g / L potato, pH 5.5, and a 250 mL triangular bottle having a liquid volume of 120 mL.

[0048] 2) inoculating the fungus obtained in step 1) into a cultivation medium, culturing at 25-29° C. for 25-32 days until the mycelium fills the cultivation bag, and then transferring the culture medium to a fruiting room at about 15° C. for low-temperature induction, and fruiting of mushrooms and fruiting bodies can be obtained in about one month; the cultivation medium comprises the following components by mass fraction: 30% sawdust, 8% wheat bran, 3% corn kernels, 1% lime, 1% gypsum, and 57% corn cobs, with a natural pH value, which is recorded as formula 1.

[0049] 3) drying and grinding the fruiting bodies obtained in step 2), filtering and screening with a 40-mesh screen, weighing 0.100 g of the fruiting body powder and placing it in a 15 mL centrifuge tube, adding anhydrous ethanol solution at a solid-liquid ratio of 1:55, ultrasonically extracting polyphenols at a power of 200 W, a temperature of 40° C., and a time of 40 min, centrifuging at 5500 r / min for 15 min, and taking the supernatant to obtain the polyphenol extract of the Odette mushroom.

[0050] Meanwhile, the cultivation medium formula 1 is replaced by formulas 2 to 9 in Table 1, respectively, and the other steps are the same to obtain the polyphenol extract of Agaricus glutinosa.

[0051] Table 1

[0052]

[0053]

[0054] 2. The Du20211005 strain of Odetteria glutinosa and 6 comparison strains of Odetteria glutinosa (numbered Du20210899, Du20210900, Du20210901, Du20210902, Du20210939, and Du20210949) were taken as research objects. The above-mentioned culture method and cultivation medium formula 3 were used to obtain 7 Odetteria glutinosa polyphenol extracts after culture and extraction.

[0055] Example 4 Preparation method of Odette polysaccharide

[0056] The specific steps include:

[0057] 1) Inoculating the mycelium of the Du20211005 strain of Agaricus glutinosa into a seed culture medium, placing the culture medium in a constant temperature shaking incubator, and culturing the culture medium at a speed of 220 r / min and 25-29° C. for 7-10 days to obtain a strain; the seed culture medium comprises the following components: 20 g / L xylose, 2 g / L yeast extract, 0.5 g / L potassium dihydrogen phosphate, 0.75 g / L magnesium sulfate, 200 g / L potato, pH 5.5, and a 250 mL triangular bottle having a liquid volume of 120 mL;

[0058] 2) inoculating the strain obtained in step 1) into a cultivation medium, culturing at 25-29° C. for 25-32 days until the mycelium fills the cultivation bag, and then transferring to a fruiting room at about 15° C. for low-temperature induction, and fruiting can be achieved in about one month to obtain fruiting bodies; the cultivation medium comprises the following components by mass fraction: 30% sawdust, 8% wheat bran, 3% corn kernels, 1% lime, 1% gypsum, and 57% corn cobs, with a natural pH value, recorded as formula 1;

[0059] 3) The fruiting bodies obtained in step 2) are dried and ground, filtered and screened with a 40-mesh sieve, 0.100 g of the fruiting body powder is weighed and placed in a 15 mL centrifuge tube, anhydrous ethanol solution is added at a solid-liquid ratio of 1:30 so that the solution system contains 80% anhydrous ethanol, and the polysaccharide crude extract is obtained under the extraction conditions of 90° C. for 3 hours and 3 extraction times, anhydrous ethanol is added, the mixture is kept at 4° C. overnight, and then centrifuged at 4500 rpm for 15 minutes to obtain a precipitate, thereby obtaining the Odenia muscaria polysaccharide.

[0060] Meanwhile, the cultivation medium formula 1 is replaced by formulas 2 to 9 in Table 1, respectively, and the other steps are the same to obtain the Odenia glutinosae polysaccharide.

[0061] 2. The Du20211005 strain of Odetteria glutinosa and 6 comparison strains of Odetteria glutinosa (numbered Du20210899, Du20210900, Du20210901, Du20210902, Du20210939, and Du20210949) were taken as research objects. The above-mentioned culture method and cultivation medium formula 3 were used to obtain 7 Odetteria glutinosa polysaccharides after culture and extraction.

[0062] Example 5 Product Verification

[0063] 1. Determination of polyphenol content

[0064] Take 200 μL of the polyphenol extract obtained in Example 3, add water to 1 mL, add 0.2 mL of Folin phenol reagent, shake well, stand for 6 min in dark, then add 2 mL of 10% Na2CO3 solution, stand for 90 min in dark, use pure water as blank control, measure absorbance at 760 nm with UV-visible spectrophotometer, and measure three times for each sample. Calculate the polyphenol content according to the following formula 1.

[0065]

[0066] In the formula: ω-- is the polyphenol content of O. stickyra, mg / g; ρ-- is the polyphenol mass concentration of the extract, ug / mL; V-- is the total volume of the extract, mL; m-- is the mass of the fruiting body dry powder, g; n-- is the dilution multiple of the extract.

[0067] Effects of different cultivation formulas on the polyphenol content of Agaricus glutinosa fruiting bodies. Figure 3 As shown, there are certain differences in the polyphenol extraction amounts of Agaricus glutinosa fruiting bodies cultured in 9 different cultivation media, among which the highest extraction amount is in recipe 3, which is 38.80 mg / g, which is significantly different from the other recipes, followed by recipe 2, which has a polyphenol extraction amount of 32.35 mg / g; the lowest polyphenol extraction amount is in recipe 4, which is 21.02 mg / g, which is significantly different from the other recipes.

[0068] The effects of different strains on the polyphenol content of Agaricus glutinosa fruiting bodies are shown in the following table. Figure 4 As shown, the polyphenol extraction amount of Du20211005 was the highest, which was 36.20 mg / g, and the difference with the other 6 strains was significant; the polyphenol extraction amount of strain Du20210949 was lower, which was 30.00 mg / g, and there was no significant difference with Du20210939 and Du20210902, but there was a significant difference with the other 4 strains.

[0069] 2. Determination of polysaccharide content

[0070] Put the polysaccharide obtained in Example 4 into a 250mL conical flask, add 50mL water and 15mL concentrated hydrochloric acid. Connect a condensing reflux device and place in a water bath at about 90°C for 3h. Then filter, wash the filtrate and make up the volume to a 250mL volumetric flask. This liquid is the test solution. Pipette 1mL of the test solution into a 10mL test tube, then add 1mL of 5% phenol solution and 5mL of concentrated sulfuric acid. Let the reaction solution stand for 10min, then use a vortex oscillator to fully mix the reaction solution, then put the test tube into a 30°C water bath for 20min. Take an appropriate amount of the reaction solution and measure the absorbance at a wavelength of 490nm, with the glucose mass concentration as the horizontal coordinate and the absorbance value as the vertical coordinate. Each sample is measured three times in parallel, and the polysaccharide content is calculated according to the following formula 2:

[0071]

[0072] Wherein, X--the content of total sugar in the sample, in grams per hundred grams (g / 100g); V1--the fixed volume of the example, in milliliters (mL); V2--the volume of the example measurement solution transferred during colorimetric determination, in milliliters (mL); m1--the sugar content in the example measurement solution obtained from the standard curve, in micrograms (μg); m2--the mass of the example, in grams (g); ω--the water content of the example, %, 10000--conversion coefficient.

[0073] Effects of different cultivation formulas on the polysaccharide content of Agaricus glutinosa fruiting bodies. Figure 5 As shown, the final polysaccharide content of the cultivation medium selected from Formula 3 was the highest, at 33.192 mg / g, and was significantly different from the polysaccharide content of the fruiting bodies cultivated from the other formulas.

[0074] The effects of different strains on the polysaccharide content of Agaricus glutinosa fruiting bodies are shown in the following table. Figure 6 As shown, the polysaccharide extraction amount of Du-2 was the highest, which was 30.708 mg / g. The difference between it and Du-5 (Du20211005, 28.606 mg / g) and Du-4, Du-7 was not significant, but the differences between it and the other three strains were significant.

[0075] 3. Antioxidant performance test

[0076] The polysaccharide and polyphenol extracts obtained by culturing different strains in Example 3 and Example 4 were subjected to DPPH free radical scavenging ability assay, hydroxyl free radical (·OH) scavenging ability assay and ABTS free radical scavenging ability assay, respectively.

[0077] DPPH free radical scavenging ability determination specific operation: take 0.8mL of polyphenol / polysaccharide extracts from different Odea glutinosae fruiting bodies, add 0.2mL of 0.1mmol / L DPPH free radical ethanol solution in sequence, mix well, and stand at room temperature and away from light for 30min. Measure the absorbance value A1 at 517nm. Use anhydrous ethanol solution instead of DPPH solution as the control group, and measure the absorbance value A2. Use pure water instead of polyphenol solution as the blank group, and measure the absorbance value A0. The scavenging rate is calculated according to the following formula 3.

[0078]

[0079] The specific operation of the hydroxyl radical (·OH) scavenging ability determination is as follows: 0.4 mL of polyphenol / polysaccharide extracts from different fruiting bodies were taken, and 0.2 mL of 9 mmol / L FeSO4 solution, 0.2 mL of 9 mmol / L salicylic acid-ethanol solution, and 0.2 mL of 8.8 mmol / L H2O2 were added, mixed, reacted in a 37°C water bath for 30 min, and the absorbance value A1 was measured at 510 nm. Pure water was used to replace the polyphenol / polysaccharide solution as the blank group, and the absorbance value was measured as A0. Pure water was used to replace H2O2 as the control group, and the absorbance value was measured as A2. The scavenging rate was calculated according to the following formula 4.

[0080]

[0081] ABTS free radical scavenging ability determination specific operation: take the same volume of ABTS (7.4mmol / L) and K2S2O8 (2.6mmol / L) solution and mix them, place them at room temperature away from light for 12 to 16 hours, after sufficient reaction, dilute it 25 to 30 times with anhydrous ethanol to make its absorbance at 734nm 0.70±0.02, and obtain ABTS + Working solution. Take 0.1mL of polyphenol / polysaccharide extracts from different fruiting bodies and add 1mL ABTS + The working solution was mixed evenly and allowed to stand at room temperature in the dark for 6 min. The absorbance value A1 was measured at 734 nm. Anhydrous ethanol was used as the control group and the absorbance value A0 was measured. The clearance rate was calculated according to the following formula 5.

[0082]

[0083] The antioxidant activity of polyphenols from the fruiting bodies of different Odetteria glutinosa strains is shown in Table 2. It can be seen that the polyphenols from the fruiting bodies of strain Du20211005 have a significant effect on ABTS + , ·OH and DPPH all had high scavenging ability, with the highest scavenging rate for ·OH reaching 99.46%, and the difference between them and other strains reached a significant level; for ABTS + The highest clearance rate was 93.22%, and the difference with other strains also reached a significant level; although the clearance rate of DPPH free radicals was not the highest, it reached 94.09%, second only to Du-6, Du-1 and Du-7 strains, and had good antioxidant effect.

[0084] Table 2

[0085]

[0086] Note: Data are expressed as mean + standard deviation. The lowercase letters in the table indicate significant differences at the 0.05 level, the same below.

[0087] The antioxidant activity of polysaccharides from the fruiting bodies of different strains of Agaricus glutinosa is shown in Table 3. It can be seen that the polysaccharides from the fruiting bodies of strain Du20211005 have good antioxidant activity, and its scavenging rate for ABTS free radicals is the highest, which is 91.267%, and the difference between it and the other 6 strains reaches a significant level; the strain with the highest scavenging rate for OH-free radicals is Du-6

[0088] (95.83%), but there was no significant difference between it and Du20211005 (92.375%), Du-3 and Du-1, but it was significantly higher than the other three strains. Therefore, the Du20211005 strain has a significant advantage.

[0089] Table 3

[0090]

[0091]

[0092] 4. Antibacterial performance test

[0093] The fruiting body polyphenols and fruiting body polysaccharides obtained by culturing the Du20211005 strain of the sticky mushroom obtained in Example 3 and Example 4 in different culture medium formulas were used to determine the antibacterial activity using the mycelium growth rate method. The extracted polyphenol / polysaccharide solution was diluted to the same mass concentration. Under sterile conditions, 3 mL of the polyphenol solution was taken and added to the sterilized PDA culture medium. After mixing, the plates were poured to prepare the test culture medium with the corresponding mass concentration, and the blank culture medium was used as the control. A neatly grown plant pathogenic bacteria cake was punched out with a 5 mm diameter puncher, and the cake was inoculated into the center of the polyphenol culture medium with the mycelium facing up. It was cultured in an incubator at 25°C for 3 to 5 days. Each experiment was repeated 3 times. The colony diameter (cm) was measured and recorded using the cross method. The antibacterial rate was calculated according to the following formula 6. The results are shown in the figure. Figure 7 And as shown in Table 4.

[0094]

[0095] Table 4

[0096]

[0097] Figure 7 The antibacterial effect of polyphenols of the fruiting bodies of Agaricus glutinosa Du20211005 strain under different formulas was obtained. It can be seen that the antibacterial rate of polyphenols of the fruiting bodies of Agaricus glutinosa Du20211005 strain in formula 1 against Eucommia ulmoides black spot pathogen can reach up to 51.96%, and the difference with other formulas is significant; in formula 2, the highest antibacterial activity of polyphenols of the fruiting bodies against the three tested bacteria, Pseudomonas palmitodiscus, Gibberella tritici and Cladosporium cladosporium, is 42.14%, 44.83% and 30.43%, respectively, which are significantly higher than other formulas.

[0098] Table 4 shows the antibacterial effects of the polysaccharides of the fruiting bodies of the Agrocybe glutinosae Du20211005 strain under different formulas. It can be seen that the polysaccharides of the fruiting bodies of the Agrocybe glutinosae Du20211005 strain in formula 1 have the highest antibacterial rate against wheat fusarium, which is 65.741%, significantly higher than other formulas; the polysaccharides of the fruiting bodies cultivated in formula 2 have the highest antibacterial rate against eucommia black spot, which is 47.699%, and the difference with other formulas has reached a significant level, and the highest antibacterial rate against palm pseudodisc polysaccharides is 35.526%, which is not significantly different from formulas 1, 4 and 7, but significantly higher than other formulas.

[0099] In summary, the polyphenols and polysaccharides of Agaricus glutinosa obtained by using Formula 1 in the present invention have good antibacterial effects on different plant pathogens, can replace pesticides to prevent and treat pathogenic diseases of edible fungi, and will not cause environmental pollution. At the same time, it also provides a theoretical basis for the further development and utilization of polyphenols and polysaccharides of Agaricus glutinosa.

[0100] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain of Odette mushroom with high yield and high activity of polyphenol polysaccharides, characterized in that: The strain is the Oudemansiella mucida strain Du20211005, which was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on November 20, 2024, with the deposit number CGMCC NO.41665.

2. A method for preparing polyphenols of Odette mushroom, characterized in that: The following steps are involved: 1) inoculating the mycelium of the strain according to claim 1 into a seed culture medium, placing it in a constant temperature shaking incubator, and culturing it at a speed of 210 to 240 r / min and 25 to 29° C. for 7 to 10 days to obtain a strain; 2) inoculating the strain obtained in step 1) into a cultivation medium, culturing at 25-29° C. for 25-32 days until the mycelium fills the cultivation bag, and then inducing at low temperature at 10-21° C. until the mushrooms are fruited to obtain fruiting bodies; the cultivation medium comprises the following components by mass fraction: 30% sawdust, 16% wheat bran, 5% corn kernels, 1% lime, 1% gypsum, and the balance is corn cobs, or the cultivation medium comprises the following components by mass fraction: 30% sawdust, 8% wheat bran, 3% corn kernels, 1% lime, 1% gypsum, and the balance is corn cobs; 3) drying and grinding the fruiting bodies obtained in step 2), placing them in anhydrous ethanol solution to obtain an extract, ultrasonically treating them, and centrifuging them to obtain the supernatant to obtain the Odenia glutinosus polyphenols.

3. The preparation method according to claim 2, characterized in that: The volume fraction of anhydrous ethanol in the extract is 70% to 75%, and the mass volume ratio of the fruiting body to the anhydrous ethanol solution is 1:50 to 1:55 g / mL; the power during the ultrasonic treatment is 200 to 400 W, the temperature is 40 to 60° C., and the ultrasonic time is 30 minutes.

4. A method for preparing polysaccharide of Odenia glutinosae, characterized in that: The following steps are involved: S1: inoculating the mycelium of the strain according to claim 1 into a seed culture medium, placing it in a constant temperature shaking incubator, and culturing it at a speed of 210 to 240 r / min and 25 to 29° C. for 7 to 10 days to obtain a strain; S2: inoculating the strain obtained in step 1) into a cultivation medium, culturing at 25-29° C. for 25-32 days until the mycelium grows all over the cultivation bag, and then inducing at low temperature at 10-21° C. until the mushrooms are fruiting to obtain fruiting bodies; the cultivation medium comprises the following components by mass fraction: 30% sawdust, 16% wheat bran, 5% corn kernels, 1% lime, 1% gypsum, and the balance is corn cobs, or the cultivation medium comprises the following components by mass fraction: 30% sawdust, 8% wheat bran, 3% corn kernels, 1% lime, 1% gypsum, and the balance is corn cobs; S3: drying and grinding the fruiting bodies obtained in step 2), placing them in anhydrous ethanol solution to obtain an extract, extracting at high temperature, keeping at 4°C overnight, and centrifuging to obtain a precipitate to obtain the Agaricus glutinosa polysaccharide.

5. The preparation method according to claim 4, characterized in that: The mass volume ratio of the fruiting body to the anhydrous ethanol solution is 1:35 g / mL, the volume fraction of ethanol in the extract is 85% to 95%, the extraction time is 3 to 4.5 hours, and the extraction temperature is 90°C.

6. The preparation method according to any one of claims 2 to 4, characterized in that: The seed culture medium comprises the following components: 20 g / L xylose, 2 g / L yeast extract, 0.5 g / L potassium dihydrogen phosphate, 0.75 g / L magnesium sulfate, 200 g / L potato, and pH 5.

5.

7. Use of the strain according to claim 1 or the polyphenols / polysaccharides prepared by the method according to any one of claims 2 to 6 in the preparation of antioxidant drugs or health products.

8. The use according to claim 7, characterized in that: The anti-oxidation is achieved by reducing the content of active oxygen, which includes superoxide radicals, hydrogen peroxide and / or hydroxyl radicals.

9. Use of the strain according to claim 1 or the polyphenol / polysaccharide prepared by the method according to any one of claims 2 to 6 in the preparation of an antibacterial agent.

10. The use according to claim 9, characterized in that: The antibacterial agent is used for preventing and controlling black spot pathogen of Eucommia ulmoides, Pseudomonas palmitodischis, Gibberella fusca of wheat or Cladosporium cladosporium.

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