Tremella fuciformis strain and preparation and application of selenium polysaccharide of tremella fuciformis strain

Abstract

The invention discloses a tremella fuciformis strain and preparation and application of selenium polysaccharide of the tremella fuciformis strain, and belongs to the technical field of biology. The tremella fuciformis TJN-0926 provided by the invention is preserved in the China Center for Type Culture Collection, and the preservation number of the tremella fuciformis TJN-0926 is CCTCC (China Center for Type Culture Collection): M 20241096. The tremella fuciformis strain TJN-0926 belongs to a safe strain capable of being used for food and has high tolerance and enrichment conversion capacity on inorganic selenium, the yield of the tremella fuciformis selenium polysaccharide prepared through the method is 10-15 g / L, the selenium content in the tremella fuciformis selenium polysaccharide is 500-600 micrograms / g, and the tremella fuciformis selenium polysaccharide has an excellent immunoregulation function and an excellent anti-oxidation characteristic. Therefore, the tremella fuciformis strain TJN-0926 and the tremella fuciformis selenium polysaccharide product thereof can be widely applied to food, medicines, health care products or cosmetics.

Description

Technical Field

[0001] The invention relates to a Tremella fuciformis strain and the preparation and application of selenium polysaccharide thereof, belonging to the technical field of biomedicine. Background Art

[0002] Polysaccharides are macromolecular carbohydrates formed by connecting multiple monosaccharide molecules through glycosidic bonds. They are a class of biological macromolecules widely present in nature, which can play a variety of biological activities in organisms, such as immunomodulation, anti-tumor, anti-oxidation, anti-inflammatory, and antibacterial. Tremella fuciformis, as a good medicinal and edible fungus, has a high development and utilization value; Tremella fuciformis polysaccharide is an acidic heteropolysaccharide existing in Tremella fuciformis fruiting bodies, mycelium and spore fermentation broth. Studies have shown that Tremella fuciformis polysaccharide has moisturizing, anti-inflammatory, anti-oxidation, anti-aging, anti-radiation, improving immunity, lowering blood lipids and blood sugar, etc., and is widely used in the fields of beauty, medicine, and health food. At present, the method for obtaining Tremella fuciformis polysaccharide is generally the fruiting body extraction method, which has the problems of limited raw materials, complicated extraction process, high energy consumption, low yield, and high cost.

[0003] Selenium is an essential trace nutrient for the normal functioning of the body's immune system and antioxidant defense system. Selenium can affect the body's important physiological functions by changing the expression of selenoproteins, and has the effects of anti-oxidation, enhancing immunity, and reducing damage caused by metals to the body. Studies have found that selenopolysaccharides, as selenized products of polysaccharides, combine the physiological activities of selenium and polysaccharides, have high medicinal value, and show good application prospects in anti-tumor, anti-oxidation, and immune regulation.

[0004] Compared with inorganic selenium, organic selenium has higher bioavailability, lower toxicity, better stability and higher safety. Therefore, selenium polysaccharides formed by selenium modification of polysaccharides are used as an ideal selenium supplement. They can not only overcome the shortcomings of inorganic selenium in the body, such as short residence time, low bioavailability and high toxicity, but also have higher pharmacological activity than polysaccharides and selenium. They have high biological activity and safety. While supplementing selenium and exerting the biological activity of polysaccharides, they are easier for the human body to absorb and have higher bioavailability. However, the yield of organic selenium polysaccharides in natural resources is low. The research progress on the synthesis method, structural characteristics and biological activity of selenium polysaccharides (Jing Yongshuai, Zhang Yuwei, Li Jiaying, et al. Research progress on the synthesis method, structural characteristics and biological activity of selenium polysaccharides [J]. Food Industry Science and Technology, 2021, 42(7): 8. DOI: 10.13386 / j.issn1002-0306.2020050188.) mentioned that chemical synthesis, microbial transformation and plant transformation are now the main methods of selenium polysaccharide synthesis. Among them, the selenium polysaccharides prepared by microbial transformation and plant transformation are more natural, but the plant transformation method is more difficult to implement in industry. Therefore, in actual production, edible fungi are often used for the enrichment and production of organic selenium polysaccharides, including Ganoderma lucidum selenium-enriched polysaccharides, Pleurotus eryngii selenium polysaccharides, Cordyceps militaris selenium polysaccharides and Hericium erinaceus selenium polysaccharides, such as Biosynthesis of selenium-containing polysaccharides with antioxidant activity in liquid culture of Hericium erinaceum (Malinowska E, Krzyczkowski W, Herold F, et al. Biosynthesis of selenium-containing polysaccharides with antioxidant activity in liquid culture of Hericium erinaceum[J]. Enzyme & Microbial Technology, 2009, 44(5): 334-343. DOI: 10.1016 / j.enzmictec.2008.12.003.) mentioned that by adding sodium selenite and selenite triglyceride to the culture medium for culturing Hericium erinaceus, organic Hericium erinaceus selenium polysaccharides with antioxidant properties can be obtained; however, the existing organic selenium polysaccharides are not rich enough to meet the current increasing demand for diversified organic selenium polysaccharides. Summary of the invention

[0005] In view of the above technical problems, the first object of the present invention is to provide a new Tremella sp. TJN-0926, which is deposited in the China Center for Type Culture Collection (CCTCC) with the deposit number of CCTCC NO: M 20241096, and is taxonomically named Tremella sp. TJN-0926; the deposit address is Wuhan University, Wuhan, China, and the deposit time is May 30, 2024.

[0006] The tremella strain TJN-0926 of the present invention is selected from a large number of tremella strains by screening the inorganic selenium resistance on a PDA plate containing 100, 200, 400 and 600 mg / L sodium selenite, and by fermenting and culturing in a culture medium containing 0, 10, 20, 50, 100 and 200 mg / L Na2SeO3 to investigate the enrichment and conversion ability of the inorganic selenium, and finally screening out the tremella strain TJN-0926 with selenium resistance and / or selenium-enrichment performance and good tremella spore growth and polysaccharide synthesis.

[0007] The Tremella fuciformis strain TJN-0926 of the present invention has the following morphological characteristics:

[0008] The colonies on PDA medium are round and smooth: when young, the colonies are milky white, thick, moist and opaque, and when old, the colonies are milky yellow and viscous; individual cells are oval, about 3.5-5.0μm×5.0-25.0μm in size, and proliferate by budding.

[0009] The strain of the present invention has selenium resistance and / or selenium-rich properties, and can produce tremella selenium polysaccharide with immunomodulatory activity.

[0010] The second object of the present invention is to provide a Tremella fuciformis selenium polysaccharide, wherein the Tremella fuciformis selenium polysaccharide is obtained by adding inorganic selenium during the liquid submerged fermentation process of Tremella fuciformis strain TJN-0926.

[0011] In one embodiment, the inorganic selenium can be any one of K2SeO3 and Na2SeO3, or a mixture of the two inorganic selenium.

[0012] The third object of the present invention is to provide a product, which contains the above-mentioned Tremella fuciformis strain TJN-0926, and / or an extract of the Tremella fuciformis strain TJN-0926, and / or a fermentation broth of the Tremella fuciformis strain TJN-0926.

[0013] In one embodiment of the present invention, the product is food, medicine, health care product or cosmetics.

[0014] In one embodiment of the present invention, the food includes but is not limited to: functional food, beverages, flour products, meal replacement biscuits, yogurt, and jelly.

[0015] In one embodiment of the present invention, the drugs include but are not limited to: anti-tumor drugs, immunomodulators, blood sugar and lipid lowering products, anticoagulant and antithrombotic drugs, antioxidant drugs, drugs that promote nerve cell growth, antiviral drugs, anti-ulcer drugs and sustained-release gel dressings.

[0016] In one embodiment of the present invention, the health care products include but are not limited to: immune regulating health care products, antioxidant health care products, blood sugar regulating health care products, cardiovascular and cerebrovascular health care products, beauty and skin care health care products, anti-aging health care products, functional food additives or other health care products.

[0017] In one embodiment of the present invention, the health care product is prepared by taking Tremella fuciformis strain TJN-0926 and / or its extract as active ingredients and adding acceptable excipients.

[0018] In one embodiment of the present invention, the cosmetics include but are not limited to: moisturizing products, anti-aging products, anti-allergic soothing products, facial masks, hair care products, beauty serums and essences, creams and lotions, and washing and care products.

[0019] In one embodiment of the present invention, the cosmetics are prepared by taking Tremella fuciformis strain TJN-0926 and / or its extract as active ingredients and adding acceptable excipients.

[0020] In one embodiment of the present invention, the extract includes but is not limited to Tremella selenium polysaccharide.

[0021] In one embodiment of the present invention, the preparation method of the extract Tremella fuciformis selenium polysaccharide is: fermenting and culturing spores of Tremella fuciformis strain TJN-0926 to prepare Tremella fuciformis fermentation broth, and extracting the extract from the prepared Tremella fuciformis fermentation broth.

[0022] In one embodiment of the present invention, the selenium content in Tremella selenium polysaccharide is 512 μg / g.

[0023] In one embodiment of the present invention, the Tremella selenium polysaccharide has significant cell proliferation ability, can promote cell NO secretion, has immunomodulatory function, and can enhance the body's immunity.

[0024] In one embodiment of the present invention, the molecular weight of the Tremella selenium polysaccharide is 1.17×10 4 Da.

[0025] The fourth object of the present invention is to provide an application of Tremella fuciformis selenium polysaccharide with immunomodulatory function in Tremella fuciformis products.

[0026] In one embodiment, the Tremella fuciformis selenium polysaccharide is obtained by adding inorganic selenium during the liquid submerged fermentation of Tremella fuciformis strain TJN-0926, wherein the inorganic selenium can be any one of K2SeO3 and Na2SeO3, or a mixture of the two inorganic seleniums.

[0027] In one embodiment, the Tremella selenium polysaccharide has significant cell proliferation ability, can promote cell NO secretion, has immunomodulatory function, and can enhance the body's immunity.

[0028] In one embodiment, the molecular weight of the Tremella selenium polysaccharide is 1.17×10 4 Da.

[0029] In one embodiment, the product is food, medicine, health care product or cosmetics.

[0030] In one embodiment, the food, medicine, health product or cosmetic is prepared with Tremella selenium polysaccharide as an active ingredient and acceptable auxiliary materials.

[0031] A fifth object of the present invention is to provide a method for preparing Tremella fuciformis selenium polysaccharide with immunomodulatory function, the method comprising fermenting and culturing the Tremella fuciformis strain TJN-0926 as claimed in claim 1 in a culture medium containing 10-200 mg / L of inorganic selenium to obtain Tremella fuciformis selenium polysaccharide, the method further comprising the step of extracting Tremella fuciformis selenium polysaccharide from the fermentation broth obtained by fermenting and culturing the Tremella fuciformis strain TJN-0926;

[0032] The specific steps of the method are as follows:

[0033] 1. Activation of Tremella fuciformis: Inoculate Tremella fuciformis strain TJN-0926 into a slant culture medium, culture at 26°C for 2-3 days, and store for later use;

[0034] 2. Liquid submerged fermentation: The activated Tremella fuciformis strain TJN-0926 obtained in step 1 is inoculated into a seed culture medium at a 10% (v / v) inoculation amount, the liquid volume is 100mL / 250mL, the fermentation temperature is 30°C, and the culture is carried out at 150r / min for 3d to obtain Tremella fuciformis seed liquid; the seed liquid is connected to a 5L fermentor at a 8% (v / v) inoculation amount, the liquid submerged fermentation culture medium liquid volume is 4L, and the liquid submerged fermentation is started at a speed of 800r / min, a ventilation volume of 4L / min, and a temperature of 30°C; 40mgNa2SeO3 is added every day on the 2nd and 3rd days of fermentation, and the pH value is controlled to be 5.0-7.0 during the fermentation process; the fermentation pH curve is observed during the fermentation period, and when the pH of the fermentation liquid rises to 7.5, glucose is added to the fermentation liquid through the feed line flow to a concentration of 50g / L, and the fermentation is terminated when there is no residual sugar, to obtain Tremella fuciformis spore fermentation liquid;

[0035] 3. Extraction of Tremella fuciformis selenium polysaccharide: centrifuge the Tremella fuciformis fermentation liquid obtained in step 2 at a speed of 8000 rpm for 15 min to obtain the supernatant, repeat twice, collect the supernatant, and adopt a heating and reduced pressure concentration method, set the concentration temperature to 55° C., and the vacuum degree to 0.70 MPa, and concentrate the obtained supernatant to 1 / 5 of the original volume to obtain a concentrated solution; add 4 times the volume of anhydrous ethanol to the concentrated solution, let it stand overnight, discard the supernatant, and freeze-dry and concentrate the remaining precipitate to obtain crude Tremella fuciformis selenium polysaccharide;

[0036] 4. Impurity removal of Tremella fuciformis selenium polysaccharide: dissolve the crude Tremella fuciformis selenium polysaccharide obtained in step 3 in deionized water to prepare a polysaccharide solution; add Sevage reagent with a volume of one-fourth of the polysaccharide solution, shake it thoroughly and mix it, shake it for 20 minutes; centrifuge at 4000r / min for 15 minutes to remove the chloroform layer below and the protein layer in the middle; take the supernatant and continue to repeat the above protein removal operation until no denatured protein layer appears; combine the supernatants, concentrate the polysaccharide solution to half the volume and freeze-dry it to obtain a polysaccharide powder; wash the obtained polysaccharide powder with acetone and anhydrous ethanol in turn, let it stand for 16 hours after precipitation, replace the organic solution, repeat three times, and then freeze-dry the precipitate to finally obtain deproteinized selenium polysaccharide.

[0037] Deposit of biological materials:

[0038] The Tremella strain TJN-0926 is deposited in the China Center for Type Culture Collection (CCTCC) with the deposit number CCTCCNO: M 20241096; the taxonomic name is Tremella sp. TJN-0926, the deposit address is Wuhan University, Wuhan, China, and the deposit time is May 30, 2024.

[0039] Beneficial Effects

[0040] (1) The present invention screened out a Tremella fuciformis strain TJN-0926 with selenium resistance, which can grow at a maximum selenium concentration of 600 mg / L and maintain a good morphology.

[0041] (2) The Tremella fuciformis strain TJN-0926 screened by the present invention has good selenium enrichment and conversion capabilities, can organically combine Tremella fuciformis polysaccharides with selenium, and the prepared Tremella fuciformis selenium polysaccharide has both biological functions, has significant cell proliferation ability, can promote cell NO secretion, has immunoregulatory function, and can enhance the body's immunity.

[0042] (3) The present invention adjusts the composition and content of the liquid fermentation medium and adds an inorganic selenium solution of appropriate concentration, thereby ensuring the normal growth of the Tremella fuciformis strain TJN-0926 and improving the yield of Tremella fuciformis selenium polysaccharide and the selenium content in the selenium polysaccharide. The yield of the Tremella fuciformis selenium polysaccharide prepared by the present invention is 10-15 g / L, and the selenium content in the Tremella fuciformis selenium polysaccharide is 500-600 μg / g.

[0043] (4) The Tremella selenium polysaccharide prepared by the present invention can promote the proliferation of RAW 264.7 cells and promote the release of NO.

[0044] (5) The Tremella selenium polysaccharide prepared by the present invention has certain scavenging activity against DPPH· and superoxide anion free radicals, and has significant immunomodulatory function, and can be used in immune enhancement products, including but not limited to food, health products or medicines.

[0045] (6) The present invention uses Tremella fuciformis spores for deep fermentation to produce organic selenium Tremella fuciformis polysaccharide, which not only ensures the active ingredients and quality of the organic selenium Tremella fuciformis polysaccharide, but also can be industrially produced, with the advantages of short cycle, easy control of conditions, low cost, and saving resources and manpower. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The present invention is a colony morphology diagram of spores of Tremella fuciformis strain TJN-0926 on selenium-containing (600 mg / L) / selenium-free plates, wherein (A) is a selenium-containing plate, and (B) is a selenium-free plate.

[0047] Figure 2 The present invention discloses an electron microscope image of spores of Tremella fuciformis strain TJN-0926.

[0048] Figure 3 The figures are the effects of different selenium concentrations on the growth and polysaccharide yield of Tremella fuciformis strain TJN-0926, wherein (A) is a liquid fermentation growth diagram showing the effects of different selenium concentrations on the growth and polysaccharide yield of Tremella fuciformis strain (NCK: control medium without Tremella fuciformis strain, CK: control medium with Tremella fuciformis strain, 10-200 mg / L: growth of Tremella fuciformis strain under different selenium contents, I / II: parallel control), and (B) is a line graph showing the effects of different selenium concentrations on the growth and polysaccharide yield of Tremella fuciformis strain TJN-0926.

[0049] Figure 4 It is a graph of the antioxidant performance of Tremella selenium polysaccharide of the present invention, wherein (A) is the DPPH free radical scavenging rate, and (B) is the superoxide anion free radical scavenging rate.

[0050] Figure 5 This is a graph showing the effect of the Tremella selenium polysaccharide of the present invention on the proliferation activity of RAW 264.7 cells.

[0051] Figure 6 This is a graph showing the effect of the Tremella selenium polysaccharide of the present invention on the NO generating ability of RAW 264.7 cells.

[0052] Figure 7 Graph showing the effect of Tremella fuciformis selenium polysaccharide of the present invention on the differentiation ability of RAW 264.7 cells, (A) is a normal group, (B) is a positive control group (1 μg / mL LPS), and (C) is a Tremella fuciformis selenium polysaccharide group (5 μg / mL). DETAILED DESCRIPTION

[0053] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0054] The test methods involved in the following embodiments are as follows:

[0055] 1. Method for determination of bacterial biomass

[0056] The cell dry weight method was used to determine the bacterial biomass: after the fermentation was completed, the fermentation liquid was centrifuged at 8000r / min for 10min, the bacterial cells were collected, the culture medium remaining on the cell surface was washed with distilled water, the supernatant was discarded, the bacterial precipitate was dried at 105°C, and its dry weight was measured to obtain the bacterial biomass.

[0057] 2. Determination of extracellular polysaccharide content

[0058] The phenol-sulfuric acid method was used to determine the extracellular polysaccharide production: 10 mL of fermentation broth was taken, diluted with water to a certain multiple (according to viscosity), centrifuged at 8000 rpm for 15 min, 1 mL of supernatant was taken in a test tube, 0.1 mL of 0.1% liquid saccharifying enzyme solution was added, and the mixture was reacted in a constant temperature water bath at 58°C for 45 min, followed by adding 3 times the volume of 95% ethanol, oscillating and flocculating, and placed in a 4°C refrigerator for overnight precipitation, then centrifuged at 10000 rpm for 5 min, the supernatant was discarded, the flocculated Tremella polysaccharide was dissolved in 5 mL of distilled water, and the insoluble matter was removed by centrifugation. After the supernatant was diluted to a certain multiple, the total sugar concentration was determined by the phenol-sulfuric acid method, and the reducing sugar concentration was determined by the DNS method.

[0059] Polysaccharide content (g / L) = total sugar concentration (g / L) - reducing sugar concentration (g / L)

[0060] 4. Determination of Selenium Content in Tremella Selenium Polysaccharide

[0061] The selenium content in selenium polysaccharides was determined by atomic fluorescence spectrometry: reference method GB5009.93-2017 first method.

[0062] 5. Method for determining colony growth

[0063] The growth of colony plates (Table 1) was evaluated based on colony morphology and texture, size and quantity. "+" means that there are few colonies with normal colony morphology, "++" means that the colony growth is good, the colony size and quantity are medium, and "+++" means that there are a large number of colonies, the diameter of individual colonies is large, and a large amount of colloid is secreted.

[0064] The growth of the strains in Table 2 represents the growth rate of the strains and the number of colonies per unit volume at the same culture time during liquid fermentation. "+" represents strain growth, "++" represents good strain growth, and "+++" represents vigorous strain growth.

[0065] The raw materials involved in the embodiments:

[0066] The fermentation medium involved in the following examples is composed of: 3.0 g / L corn gluten meal, 1.5 g / L dipotassium hydrogen phosphate, 1.5 g / L magnesium sulfate, 40 g / L sucrose, and a pH value of 6.0.

[0067] The composition of the slant culture medium involved in the following examples is: 200 g / L potato, 20 g / L glucose, 20 g / L agar, 0.5 g / L MgSO4·7H2O, 0.5 g / L K2HPO4, and 5 g / L peptone.

[0068] The seed culture medium involved in the following examples is composed of: 10 g / L peptone, 20 g / L sucrose, 1.5 g / L dipotassium hydrogen phosphate, 1.5 g / L magnesium sulfate and 15 mg / L VB1, and the pH value is 6.0.

[0069] The liquid submerged fermentation medium involved in the following examples is composed of: 3.0 g / L corn gluten meal, 1.5 g / L dipotassium hydrogen phosphate, 1.5 g / L magnesium sulfate, 40 g / L sucrose, and a pH value of 6.0.

[0070] The Sevage reagent involved in the following examples is prepared by mixing chloroform and n-butanol in a ratio of 5:1 (V / V).

[0071] Embodiment 1:

[0072] Isolation and Screening of Selenium-Resistant Tremella Strain

[0073] 1. Select seed ears: Collect and select fresh Tremella fuciformis with large, white, thick flesh, normal spreading, no bacteria and pests, and 80-90% maturity from all over the country as seed ears. Disinfect the seed ears with sterile water, remove the bacteria on the spores, and wipe the surface of the ear pieces with sterile gauze or absorbent paper;

[0074] 2. Ejecting spores: Take a small piece of the selected seed ear and suspend it in the middle of the triangular flask without touching the flask wall and culture medium. The seed ear should be 4-5 cm away from the surface of the culture medium. The triangular flask was placed in an incubator and cultured at 26°C for 2 days. When foggy spores and small colonies grew on the surface of the culture medium, the seed ear was taken out. After continuing to culture for 2-3 days, milky white, translucent, slightly shiny, sticky surface, smooth edges, and raised colonies in the middle could be seen on the surface of the culture medium. These were Tremella fuciformis spores. The isolated and purified Tremella fuciformis strains were numbered respectively, and spread on a PDA plate containing 200 mg / L sodium selenite. Tremella fuciformis GTN-0812, GTN-0815, GTN-0911, TJN-0809, TJN-0811, TJN-0812, TJN-0813, TJN-0905, TJN-0912, and TJN-0926 were screened and their selenium resistance was tested respectively.

[0075] The selenium resistance results of the Tremella strains GTN-0812, GTN-0815, GTN-0911, TJN-0809, TJN-0811, TJN-0812, TJN-0813, TJN-0905, TJN-0912, and TJN-0926 obtained by the above screening are shown in Table 1 below:

[0076] Table 1 Selenium resistance results of Tremella fuciformis strains screened

[0077]

[0078]

[0079] As can be seen from Table 1, different Tremella strains have different tolerance to sodium selenite. The colony morphology and color of different bacteria on the 200 mg / L sodium selenite plate showed differences. According to the results in Table 1, strains TJN-0905, TJN-0912 and TJN-0926 with fast growth rate, larger colony size and better inorganic selenium tolerance were selected from the above strains.

[0080] Example 2: Growth and synthesis of selenium polysaccharides of selenium-resistant Tremella fuciformis strains under different concentrations of inorganic selenium

[0081] The amount of Na2SeO3 added in the fermentation medium was adjusted to 0, 10, 20, 50, 100, and 200 mg / L, respectively, and the fermentation medium liquid volume was 80 mL / 250 mL. The Tremella fuciformis strains TJN-0905, TJN-0912, and TJN-0926 with good selenium resistance obtained in Example 1 were cultured at a fermentation temperature of 30°C and 150 r / min for five days, and then their bacterial biomass, extracellular polysaccharide production, and polysaccharide selenium content were determined.

[0082] The results are as follows:

[0083] Compared with the experimental groups with Na2SeO3 added in an amount of 10, 20, 50, 100, and 200 mg / L, when the amount of Na2SeO3 added was 0 mg / L, the selenium content of Tremella polysaccharide was almost 0; after adding different concentrations of Na2SeO3, different contents of selenium were detected in Tremella polysaccharide, respectively, which indicates that adding Na2SeO3 in the method of the present invention can produce Tremella selenium polysaccharide;

[0084] The results of the determination of bacterial biomass, extracellular polysaccharide production and polysaccharide selenium content of Tremella fuciformis strains TJN-0905, TJN-0912 and TJN-0926 are shown in Table 2;

[0085] Table 2 Determination results of bacterial biomass, extracellular polysaccharide production and polysaccharide selenium content of selenium-resistant strains

[0086]

[0087]

[0088] (1) The results are shown in Table 2. It can be seen from the table that during liquid fermentation, the added concentration of sodium selenite in the culture medium has a greater effect on the growth and polysaccharide synthesis of Tremella fuciformis. When the addition amount of Na2SeO3 is 50 mg / L, the growth and polysaccharide synthesis of Tremella fuciformis spores of Tremella fuciformis strains TJN-0905 and TJN-0912 are significantly inhibited, while Tremella fuciformis strain TJN-0926 shows good growth and synthesis of selenium polysaccharides. Therefore, Tremella fuciformis strain TJN-0926 was finally screened as a selenium-resistant / selenium-rich strain.

[0089] (2) The results of the synthesis of selenium polysaccharides by Tremella fuciformis strain TJN-0926 are as follows Figure 3 As shown in the figure, it can be seen that when the concentration of Na2SeO3 is less than 50mg / L, inorganic selenium has little effect on the growth and polysaccharide synthesis of Tremella fuciformis; when the concentration of Na2SeO3 is 20mg / L, the extracellular polysaccharide production is 11.5g / L, and the polysaccharide selenium content is 527μg / g; when the Na2SeO3 addition concentration is greater than 100mg / L, the extracellular polysaccharide production is 3.50-9.62g / L, and the polysaccharide selenium content can reach up to 535μg / g, but this concentration has a significant inhibitory effect on the growth and polysaccharide synthesis of Tremella fuciformis spores. Therefore, when conducting further amplification experiments, it is selected to add a low concentration of 20-50mg / L of sodium selenite.

[0090] Example 3: Identification and morphological analysis of Tremella fuciformis strain TJN-0926

[0091] The CTAB method was used to extract the genomic DNA of Tremella yeast strains, and the ITS sequence was amplified using the fungal ITS universal primers ITS1 (5'-TCCGTAGGTGAACGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'); the PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the obtained gene sequence was compared with the sequence in the GenBank database using the BLAST program for molecular biological identification.

[0092] The results are as follows:

[0093] The PCR amplification product of Tremella fuciformis strain TJN-0926 has 481 bases, and the sequence alignment results show that the homology with Tremella fuciformis strain TJN-0926 is as high as 99.9%. Figure 1 As shown, the colonies of Tremella fuciformis strain TJN-0926 on the PDA plate are round and smooth. When young, the Tremella fuciformis strain TJN-0926 is milky white, thick, moist, and opaque, while when old, the Tremella fuciformis strain TJN-0926 is milky yellow and viscous.

[0094] The microscopic observation results of Tremella fuciformis strain TJN-0926 are as follows Figure 2 As shown, individual cells of Tremella fuciformis strain TJN-0926 are oval in shape, approximately (3.5-5.0) μm×(5.0-25.0) μm in size, and proliferate by budding.

[0095] The Tremella strain TJN-0926 was deposited in the China Center for Type Culture Collection (CCTCC) with the deposit number CCTCCNO: M 20241096; the taxonomic name is Tremella sp. TJN-0926; the deposit address is Wuhan University, Wuhan, China, and the deposit time is May 30, 2024.

[0096] Example 4: Fermentation preparation of Tremella selenium polysaccharide

[0097] 1. Activation of Tremella fuciformis: Inoculate Tremella fuciformis strain TJN-0926 into a slant culture medium, culture at 26°C for 2-3 days, and store for later use;

[0098] 2. Liquid submerged fermentation: The activated Tremella fuciformis strain TJN-0926 obtained in step 1 is inoculated into a seed culture medium at a 10% (v / v) inoculation amount, the liquid volume is 100mL / 250mL, the fermentation temperature is 30°C, and the culture is carried out at 150r / min for 3d to obtain Tremella fuciformis seed liquid; the seed liquid is connected to a 5L fermentor at a 8% (v / v) inoculation amount, the liquid submerged fermentation culture medium liquid volume is 4L, and the liquid submerged fermentation is started at a speed of 800r / min, a ventilation volume of 4L / min, and a temperature of 30°C; 40mgNa2SeO3 is added every day on the second and third days of fermentation, and the pH value is controlled to be 5.0-7.0 during the fermentation process; the fermentation pH curve is observed during the fermentation period, and when the pH of the fermentation liquid rises to 7.5, glucose is added to the fermentation liquid through the feed line flow to a concentration of 50g / L, and the fermentation is terminated when there is no residual sugar, to obtain Tremella fuciformis fermentation liquid;

[0099] 3. Extraction of Tremella fuciformis selenium polysaccharide: centrifuge the Tremella fuciformis fermentation liquid obtained in step 2 at 8000 rpm for 15 min to obtain the supernatant, repeat twice, collect the supernatant, and adopt a heating and reduced pressure concentration method, set the concentration temperature to 55° C., and the vacuum degree to 0.70 MPa, and concentrate the obtained supernatant to 1 / 5 of the original volume to obtain a concentrated solution; add 4 times the volume of anhydrous ethanol to the concentrated solution, let it stand overnight, discard the supernatant, and freeze-dry and concentrate the remaining precipitate to obtain crude Tremella fuciformis selenium polysaccharide;

[0100] 4. Impurity removal of Tremella fuciformis selenium polysaccharide: dissolve the crude Tremella fuciformis selenium polysaccharide obtained in step 3 in deionized water to prepare a polysaccharide solution, add Sevage reagent with a volume of one-fourth of the polysaccharide solution, shake and mix it thoroughly, and shake the reaction for 20 minutes; centrifuge at 4000r / min for 15 minutes, remove the chloroform layer below and the protein layer in the middle, take the supernatant and continue to repeat the protein removal operation until no denatured protein layer appears; combine the supernatants, concentrate the polysaccharide solution to half the volume and freeze-dry it to obtain a polysaccharide powder; wash the obtained polysaccharide powder with acetone and anhydrous ethanol in turn, let it stand for 16 hours after precipitation, replace the organic solution, repeat three times, and then freeze-dry the precipitate to finally obtain deproteinized selenium polysaccharide.

[0101] The results showed that the selenium content in the selenopolysaccharide was determined by atomic fluorescence spectrometry. The deproteinized selenopolysaccharide prepared in this example had a selenium content of 11.56 g / L, and the selenium content in the Tremella fuciformis selenopolysaccharide was 512 μg / g.

[0102] Example 5: Evaluation of the Antioxidant Capacity of Tremella Selenium Polysaccharide

[0103] On the basis of Example 4, Tremella selenium polysaccharide was prepared.

[0104] In this example, the antioxidant capacity of Tremella selenium polysaccharide was evaluated using DPPH· free radical scavenging rate and superoxide anion free radical scavenging rate.

[0105] 1. Determination of DPPH free radicals: Selenium-rich polysaccharides and Vc were dissolved in distilled water to obtain solutions with a concentration gradient of 0.0625-2 mg / mL; 1 mL of solutions with different concentration gradients were added with 2 mL of DPPH-ethanol reagent and 2 mL of 95% ethanol, and reacted in a dark place at 25°C for 30 min. The absorbance at 570 nm was measured by a UV spectrophotometer; Vc was set as a positive control in the experiment, and the DPPH scavenging rate was calculated according to the following formula:

[0106] DPPH·scavenging rate %=[1-(A1-A2) / A C ]×100%

[0107] In the formula, A1 is the absorbance of the sample group; A2 is the absorbance of the blank group; A C is the absorbance of the control group.

[0108] 2. Superoxide anion free radical scavenging ability: Gallic acid was dissolved in distilled water to prepare a 25mmol / L solution; hydrochloric acid was added to 25mL 0.1mol / L tris(hydroxymethyl)aminomethane (Tris) solution, the pH value was adjusted to 8, and the volume was fixed to 50mL; distilled water was used instead of polysaccharide solution as blank control, and Vc was used as positive control; 180μL buffer and 40μL selenium polysaccharide solution were added to a 96-well plate in sequence, mixed, and reacted at 37℃ for 10min; 20μL gallic acid solution was added, mixed, reacted for 4min, and the absorbance was measured at 325nm; the superoxide anion free radical scavenging rate was calculated according to the following formula:

[0109] Superoxide anion free radical scavenging rate = [A0-(AA x )] / A0×100%

[0110] Where: A0 represents the absorbance of the sample replaced by distilled water; A represents the absorbance of the sample solution added; A x Represents the absorbance of the polysaccharide solution itself.

[0111] The results are as follows:

[0112] The Tremella selenium polysaccharide prepared in Examples 2 and 3 was tested, and the results were as follows: Figure 4 As shown, the results show that although the DPPH·scavenging rate and superoxide anion free radical scavenging rate of the Tremella fuciformis selenium polysaccharide prepared in Examples 2 and 3 of the present invention are lower than those of the positive control sample, they still have a certain DPPH·scavenging rate and superoxide anion free radical scavenging rate, that is, the Tremella fuciformis selenium polysaccharide prepared in Example 4 of the present invention has a certain antioxidant capacity.

[0113] Example 6: Evaluation of immunomodulatory activity of Tremella selenium polysaccharide

[0114] On the basis of Example 4, Tremella selenium polysaccharide was prepared.

[0115] 1. In this example, to evaluate the immunomodulatory effect of Tremella selenium polysaccharide, RAW 264.7 cells were cultured at 1×10 5 / mL density was inoculated in 96-well plates and cultured overnight; Tremella selenium polysaccharide was prepared into 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000μg / mL solution with cell culture medium; lipopolysaccharide (LPS) was used as the positive control group and prepared into 1μg / mL solution with cell culture medium; after the cells adhered to the wall, the original culture medium was removed and the cells were treated according to the experimental groups, namely blank group (complete culture medium), positive group (1μg / mL LPS), selenium polysaccharide experimental group (culture medium with selenium polysaccharide concentration of 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000μg / mL respectively); the experiment was set up with 3 replicates, and each group was placed in an incubator for 24h after administration; 10μL of After incubation with CCK-8 reagent for a certain period of time, the absorbance (OD) at 450 nm was measured with an enzyme reader, and the cell viability was calculated as follows: cell viability = (OD 实验组 / OD 空白组 )×100%.

[0116] 2. RAW 264.7 cells in the logarithmic growth phase were cultured at 1×10 5 The cells were inoculated into a 12-well plate at a concentration of 1 μg / mL and then cultured with 1 μg / mL LPS or 5 μg / mL Tremella selenium polysaccharide for 24 h. The cell morphology of each group was observed and recorded using a bright field microscope. At the same time, 50 μL of the cell culture supernatant from each group was added to a new 96-well plate, and 50 μL of Griess Reagent I and Griess Reagent II were added respectively according to the instructions of the kit (Shanghai Beyotime Biotechnology Co., Ltd. S0021S). The absorbance of each well was detected at 540 nm at room temperature, and the effect of different concentrations of Tremella selenium polysaccharide on the level of NO secreted by RAW 264.7 cells was calculated based on the NO standard curve.

[0117] The result is as follows:

[0118] (1) The results are as follows Figure 5It can be seen that Tremella fuciformis selenium polysaccharide promotes the growth of RAW264.7 cells in a concentration-dependent manner at low dose concentrations. When the concentration of Tremella fuciformis selenium polysaccharide reaches 5 μg / mL, it has the strongest ability to promote the proliferation of RAW 264.7 cells. When the concentration of Tremella fuciformis selenium polysaccharide reaches above 200 μg / mL, it will affect cell viability and cause growth inhibition. Therefore, 5 μg / mL is selected as the optimal concentration of Tremella fuciformis selenium polysaccharide. At this time, Tremella fuciformis selenium polysaccharide has the strongest ability to promote the proliferation of RAW 264.7 cells.

[0119] (2) Results of cellular NO production capacity Figure 6 As shown in the figure, after 24 hours of treatment with Tremella selenium polysaccharide, there was the same trend as the LPS positive control, which promoted the production of cellular NO. This indicates that selenium polysaccharide may improve cellular immunity and enhance cellular immune regulation activity by promoting NO synthesis in cells and activating macrophages.

[0120] (3) Macrophage morphology Figure 7 As shown in the figure, it can be seen that normal RAW 264.7 cells are mononuclear cells with uniform size and round or spindle shape; after LPS activation, compared with normal cells, most RAW 264.7 cells become polymorphic, the cell volume becomes larger, the number increases, and pseudopodia grow, and the number of pseudopodia is large; after selenium polysaccharide stimulation, compared with normal cells, RAW 264.7 cells have changes in morphology and quantity, cells have pseudopodia, and the number is large, most cells are irregular shapes such as rhombus and polygon, and the volume is also increased, which shows that Tremella selenium polysaccharide can activate RAW 264.7 cells and promote cell differentiation.

[0121] The present invention relates to the sequence:

[0122] Amplification of ITS sequences using universal primers for fungal ITS

[0123] SEQ ID NO.1(ITS1): 5'-TCCGTAGGTGAACGG-3'

[0124] SEQ ID NO.2(ITS4): 5'-TCCTCCGCTTATTGATATGC-3'

[0125] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A Tremella sp. TJN-0926, characterized in that: The Tremella sp. TJN-0926 is deposited in the China Center for Type Culture Collection with the deposit number of CCTCCNO: M 20241096, and its taxonomic name is Tremella sp. TJN-0926. The deposit date is May 30, 2024.

2. A Tremella rich in Tremella selenium polysaccharide, characterized in that: The Tremella is obtained by adding inorganic selenium during the cultivation of Tremella fuciformis strain TJN-0926 described in claim 1.

3. The Tremella fuciformis according to claim 2, characterized in that The inorganic selenium can be any one of K2SeO3 and Na2SeO3 or a mixture of the two inorganic selenium.

4. A Tremella selenium polysaccharide, characterized in that The Tremella selenium polysaccharide is obtained by fermenting the Tremella strain TJN-0926 described in claim 1 in a culture medium containing 10-200 mg / L of inorganic selenium.

5. The Tremella selenium polysaccharide according to claim 4, characterized in that The inorganic selenium can be any one of K2SeO3 and Na2SeO3 or a mixture of the two inorganic selenium.

6. A method for preparing Tremella selenium polysaccharide, characterized in that: The method comprises the step of using the Tremella fuciformis strain TJN-0926 as claimed in claim 1 to carry out fermentation culture in a culture medium containing 10-200 mg / L of inorganic selenium to obtain Tremella fuciformis selenium polysaccharide, and the method further comprises the step of extracting Tremella fuciformis selenium polysaccharide from the fermentation liquid obtained by fermentation culture of the Tremella fuciformis strain TJN-0926.

7. A Tremella selenium polysaccharide product, characterized in that: The Tremella fuciformis selenium polysaccharide product is prepared by fermenting the Tremella fuciformis selenium polysaccharide produced by the Tremella fuciformis strain TJN-0926 described in claim 1.

8. The Tremella selenium polysaccharide product according to claim 7, characterized in that: The tremella selenium polysaccharide product is food, medicine, health care product or cosmetic.

9. The Tremella selenium polysaccharide product according to claim 7 or 8, characterized in that: The tremella selenium polysaccharide product is prepared by taking tremella selenium polysaccharide as an active ingredient and adding acceptable auxiliary materials.

10. Use of the Tremella fuciformis strain TJN-0926 as claimed in claim 1 in the preparation of food, medicine, health care products or cosmetics.

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