Thelephora ganbajun Zang polysaccharide and application thereof

Abstract

The invention belongs to the technical field of natural product extraction and preparation, and particularly relates to thelephora ganbajun Zang polysaccharide and application thereof. Specifically, thelephora ganbajun Zang mycelium prepared by fermentation is used as a raw material, extraction and purification process conditions are optimized, and finally thelephora ganbajun Zang mycelium zinc polysaccharide TZIP and polysaccharide TIP are obtained. Thelephora ganbajun Zang mycelium polysaccharide has multiple effects of improving learning and memory ability, regulating intestinal flora, protecting degenerative change caused by zinc deficiency and aging and the like, and the Thelephora ganbajun Zang mycelium zinc polysaccharide TZIP is better in performance, so that the Thelephora ganbajun Zang mycelium zinc polysaccharide can be widely applied to various fields of food, medicines, feed and the like, and the comprehensive utilization value of Thelephora ganbajun Zang is effectively improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of extraction and preparation of natural products, and particularly relates to a polysaccharide from Thelephora ganbajun and its applications. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily to be regarded as an admission or any form of implication that this information constitutes prior art already known to those of ordinary skill in the art.

[0003] With the extension of the average lifespan, the aging of the population is a general trend in the development of the world's population. In the challenges of the rapid aging of the population structure, the prevention of brain aging and neurodegenerative diseases is particularly important. Therefore, it is of great significance to find natural components with the ability to improve learning and memory abilities and cognitive functions.

[0004] As an important source of natural active substances, polysaccharides from edible fungi have become a research hotspot in the food and pharmaceutical fields due to their significant biological activities such as immunomodulation, antioxidant and anti-tumor effects. The traditional acquisition of edible fungi polysaccharides mainly relies on fruiting body extraction or solid cultivation techniques, but there are limitations such as long production cycles, low efficiency and large resource consumption. With the development of liquid fermentation technology, its advantages in efficiently obtaining mycelia and extracellular polysaccharides have gradually emerged, providing a new way for the large-scale production of high-purity active polysaccharides. However, the existing technology still lacks in-depth development of polysaccharide resources of specific rare strains.

[0005] Thelephora ganbajun, a rare symbiotic wild edible fungus unique to Yunnan, is rich in active ingredients such as amino acids, proteins and polysaccharides in its mycelia. Previous studies have confirmed that its extracts have potential values such as antioxidant and anti-aging effects. However, current research on Thelephora ganbajun mostly focuses on the analysis of the nutritional components of the fruiting body under solid culture conditions, and there are few reports on the systematic research of its polysaccharide production by liquid fermentation, which limits its application in the development of high-value-added functional products. Therefore, it is necessary to further study the polysaccharides from Thelephora ganbajun, especially the intracellular polysaccharides of mycelia, from the perspective of improving brain cognitive function. Summary of the Invention

[0006] Aiming at the deficiencies of the existing technology, the present invention provides a polysaccharide from Thelephora ganbajun and its applications. Specifically, through experimental research, the present invention finds that the polysaccharides from the mycelia of Thelephora ganbajun prepared by a specific process, especially the zinc polysaccharides from the mycelia of Thelephora ganbajun, have many effects such as improving learning and memory abilities, regulating the intestinal flora and intervening in zinc deficiency-related aging indexes, thus providing a scientific basis for its application in the fields of functional foods, neuroprotective agents, etc. Based on the above research results, the present invention is completed.

[0007] To achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0008] In the first aspect of the present invention, there is provided the use of the polysaccharide of the Termitomyces albuminosus in the preparation of a product for improving learning and memory ability, regulating the intestinal flora, and / or protecting against degenerative changes caused by zinc deficiency and aging.

[0009] The product includes but is not limited to food, medicine, and feed.

[0010] The polysaccharide of the Termitomyces albuminosus in the present invention is specifically the polysaccharide of the mycelium of the Termitomyces albuminosus; the Termitomyces albuminosus can be obtained by ordinary fermentation culture or zinc-rich fermentation culture; the polysaccharide of the Termitomyces albuminosus can be the zinc-rich polysaccharide of the mycelium of the Termitomyces albuminosus.

[0011] Further, the polysaccharide of the Termitomyces albuminosus is a pyranose linked by β-glycosidic bonds; furthermore, the polysaccharide of the Termitomyces albuminosus mainly contains galactose, mannose, and glucose, and also contains a small amount of glucosamine, ribose, rhamnose, glucuronic acid, galactosamine, arabinose, and fucose.

[0012] In the second aspect of the present invention, there is provided a method for preparing the polysaccharide of the Termitomyces albuminosus, and the preparation method includes:

[0013] S1. Mix the mycelium of the Termitomyces albuminosus with water and then crush it, and take the supernatant;

[0014] S2. Add ethanol to the supernatant, mix and let it stand, and then collect the precipitate;

[0015] S3. The precipitate is decolorized, deproteinized, and separated by an anion exchange column to obtain the product.

[0016] In the third aspect of the present invention, there is provided a product having the functions of improving learning and memory ability, regulating the intestinal flora, and / or protecting against degenerative changes caused by zinc deficiency and aging. The product contains the polysaccharide of the Termitomyces albuminosus. Specifically, the polysaccharide of the Termitomyces albuminosus is prepared by the preparation method described in the second aspect above.

[0017] The beneficial technical effects of the above one or more technical solutions:

[0018] In the above technical solution, the mycelium of the Termitomyces albuminosus obtained by fermentation is used as the raw material. By optimizing the extraction and purification process conditions, the zinc polysaccharide TZIP and polysaccharide TIP of the mycelium of the Termitomyces albuminosus are finally obtained. At the same time, through experiments, it is verified that the polysaccharide of the mycelium of the Termitomyces albuminosus has multiple functions such as improving learning and memory ability, regulating the intestinal flora, and protecting against degenerative changes caused by zinc deficiency and aging. Among them, the zinc polysaccharide TZIP of the mycelium of the Termitomyces albuminosus shows better performance. Therefore, it can be widely applied to many fields such as food, medicine, and feed, thereby effectively improving the comprehensive utilization value of the Termitomyces albuminosus. Description of the Drawings

[0019] The accompanying drawings of the specification, which form a part of the present invention, are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0020] Figure 1 For Embodiment 1 of the present invention, it is the elution curve of the polysaccharide from Termitomyces albuminosus (the elution curves (A-I and A-II, DEAE-52 elution curves of TIP and TZIP; A-III and A-IV, Sephadex G-100 elution curves of TIP and TZIP)).

[0021] Figure 2 For Embodiment 2 of the present invention, it is the FT-IR spectrum of the polysaccharide from Termitomyces albuminosus.

[0022] Figure 3 For Embodiment 2 of the present invention, it is the FT-IR spectrum of the zinc polysaccharide from Termitomyces albuminosus.

[0023] Figure 4 For Embodiment 4 of the present invention, it is the HPGPC spectra of the zinc polysaccharide (TZIP) and the polysaccharide (TIP) from Termitomyces albuminosus. Figure 5 For Embodiment 5 of the present invention, it is the results of the T-maze behavioral test of zebrafish; A: The time for the first entry into the deep water area of the T-maze; B: The ratio of the swimming time in the deep water area to the total time; Note: Compared with the blank control group NC, ##P<0.01; compared with the model group MC, *P<0.05, **P<0.01. Note: NC, blank control group; MC, model group; Don, positive drug group; TIP-20, medium-concentration polysaccharide group from Termitomyces albuminosus; TZIP-10, TZIP-20, TZIP-40, low-, medium-, and high-concentration zinc polysaccharide groups from Termitomyces albuminosus.

[0024] Figure 6 For Embodiment 5 of the present invention, it is the results of the black and white box behavioral test of zebrafish; A: The proportion of the swimming distance in the black box of the black and white box; B: The proportion of the swimming time in the black box of the black and white box; Note: Compared with the blank control group, P<0.001; compared with the model group, *P<0.05, ***P<0.001. Note: NC, blank control group; MC, model group; Don, positive drug group; TIP-20, medium-concentration polysaccharide group from Termitomyces albuminosus; TZIP-10, TZIP-20, TZIP-40, low-, medium-, and high-concentration zinc polysaccharide groups from Termitomyces albuminosus. Figure 7 For Embodiment 5 of the present invention, it is the gene expression levels of zebrafish larvae creb1a (A), ache (B), chata (C), nrf2a (D), chrna1 (E), ngfβ (F); Note: The bar chart represents the fold change of the mRNA expression of creb1a (A), ache (B), chata (C), nrf2a (D), chrna1 (E), ngfβ (F); compared with NC, *P < 0.05, ** P < 0.01, *** P < 0.001; Compared with MC, # P < 0.05, ## P < 0.01, ### P < 0.001; Compared TIP-20 with TZIP-20, aa P < 0.01, aaa P < 0.001.

[0025] Figure 8 This is the category of the intestinal flora of zebrafish in Example 6 of the present invention; among them, A: Community composition at the family level. Note: Fusobacteriaceae; Shewanellaceae; Beijerinckiaceae; Aeromonadaceae; Rhodobacteraceae; Flavobacteriaceae; Pseudomonadaceae; Micrococcaceae; Burkholderiaceae_A; Microbacteriaceae; B: Community composition at the genus level; Note: Cetobacterium_A; Shewanella; Bosea; Aeromonas; Flavobacterium; Pseudomonas_E; Arthrobacter_E; Rubrivivax; Chitinibacter; Vibrio.

[0026] Figure 9 This is the content of lactic acid, IL-1β, CAT, GSH and SOD in the mouse brain in Example 7 of the present invention; Note: Compared with NC, ### P < 0.001; Compared with MC, ** P < 0.01, *** P < 0.001; Compared with AC, a P < 0.05; Compared with TIP-500, bbb P < 0.001. Detailed implementation

[0027] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0028] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should also be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. It should be understood that the protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for describing specific embodiments and not for limiting the protection scope of the present invention.

[0029] In a typical specific embodiment of the present invention, there is provided an application of the polysaccharide of the Termitomyces albuminosus in the preparation of a product for improving learning and memory ability, regulating intestinal flora, and / or protecting against degenerative changes caused by zinc deficiency and aging.

[0030] Specifically, through the zebrafish T-maze experiment and the black and white box experiment, the present invention proves that the polysaccharide of the Termitomyces albuminosus, especially the zinc polysaccharide of the Termitomyces albuminosus, has a good effect on improving learning and memory ability; at the same time, at the gene level, the present invention confirms that the polysaccharide of the Termitomyces albuminosus and the zinc polysaccharide have an obvious improvement effect on the abnormal gene expression related to learning and memory (such as brain nerve-related genes) in adult zebrafish. Therefore, the polysaccharide of the Termitomyces albuminosus and the zinc polysaccharide may improve abnormal learning and memory behaviors by regulating the abnormal gene expression related to the brain nerves of zebrafish with learning and memory impairment.

[0031] In addition, through experimental research, the present invention finds that compared with the blank control group, the diversity of the intestinal flora of the zebrafish in the model group is reduced. After treatment with the zinc polysaccharide of the Termitomyces albuminosus, the diversity of the intestinal flora increases, and to a certain extent, the abnormal changes in the flora structure are restored, indicating that the zinc polysaccharide of the Termitomyces albuminosus can regulate the community composition structure and restore its related material metabolism function.

[0032] Furthermore, the present invention also proves that the polysaccharide of the Termitomyces albuminosus, especially the zinc polysaccharide of the Termitomyces albuminosus, has a significant improvement effect on the brain lactic acid, inflammation, and oxidation indexes of zinc-deficient and aging mice, thus indicating that it has an excellent protective effect on the degenerative changes caused by zinc deficiency and aging. The above experiments fully prove that the polysaccharide of the Termitomyces albuminosus of the present invention has broad market value in the development of products for improving learning and memory ability, regulating intestinal flora, and / or protecting against degenerative changes caused by zinc deficiency and aging.

[0033] The products include but are not limited to foods, medicines, and feeds.

[0034] The polysaccharide of the Termitomyces albuminosus in the present invention is specifically the polysaccharide of the mycelium of the Termitomyces albuminosus; the Termitomyces albuminosus can be obtained by a common fermentation culture method or a zinc-rich fermentation culture method; therefore, in a specific embodiment of the present invention, the polysaccharide of the Termitomyces albuminosus is the zinc-rich polysaccharide of the mycelium of the Termitomyces albuminosus.

[0035] In another specific embodiment of the present invention, the Termitomyces albuminosus polysaccharide is a pyranose linked by β-glycosidic bonds; further, the Termitomyces albuminosus polysaccharide mainly contains galactose, mannose and glucose, and contains a small amount of glucosamine, ribose, rhamnose, glucuronic acid, galactosamine, arabinose and fucose.

[0036] In another specific embodiment of the present invention, a method for preparing Termitomyces albuminosus polysaccharide is provided, and the preparation method includes:

[0037] S1. Mix the Termitomyces albuminosus mycelium with water, break it, and heat to obtain the supernatant;

[0038] S2. Add ethanol to the supernatant, mix and let stand, and collect the precipitate;

[0039] S3. The precipitate is decolorized, deproteinized, and separated and purified by an anion exchange column and a Sephadex column to obtain the product.

[0040] Among them, in the step S1,

[0041] The mycelium can be obtained by ordinary fermentation culture or zinc-enriched fermentation culture; specifically, the ordinary fermentation culture method can be inoculating the Termitomyces albuminosus seed liquid into a fungal medium (such as potato dextrose liquid medium) for fermentation culture; the zinc-enriched fermentation culture method is the same as the ordinary fermentation culture method, the only difference being that the selected medium is a zinc-enriched fungal medium (such as potato dextrose liquid zinc-enriched medium), and the potato dextrose liquid zinc-enriched medium is based on the potato dextrose liquid medium and zinc element is added, and the zinc element is added in the form of zinc salt, specifically it can be zinc sulfate, and its concentration can be 100-800 mg / L, preferably 400 mg / L.

[0042] In another specific embodiment of the present invention, the mass-volume ratio of the Termitomyces albuminosus mycelium to water is 1:1-30, g / mL; preferably 1:20; the breaking can be carried out by ultrasonic breaking, and the specific breaking conditions are to treat for 1-30 min under the condition of 100-500 W, preferably 300 W for 10 min;

[0043] The heating can be carried out by water bath heating method, and the specific heating conditions are to treat for 1-5 h at 70-100 °C, preferably 90 °C for 2 h.

[0044] In the step S2, the supernatant can be concentrated to 1 / 2 to 1 / 3 of the original volume, and then ethanol with a volume 1 - 10 times (preferably 3 times) that of the concentrated supernatant is added. The ethanol is high-concentration ethanol, which can be ethanol with a concentration of 70% or more, such as 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% ethanol. In a specific embodiment of the present invention, the ethanol is 95% ethanol.

[0045] In the step S3, decolorization can be carried out by using activated carbon decolorization method;

[0046] Any method known in the art can be used for protein removal, such as the Sevag method, which is not specifically limited herein;

[0047] The anion exchange column is specifically a DEAE anion exchange column;

[0048] The Sephadex gel column is specifically a Sephadex G-100 Sephadex gel column.

[0049] After separation by the DEAE anion exchange column and the Sephadex G-100 Sephadex gel column, the polysaccharide of Termitomyces albuminosus is further purified, and the polysaccharide of Termitomyces albuminosus mycelium can be obtained, named TIP and TZIP respectively.

[0050] Specifically, the specific separation and purification processes of the TIP and TZIP are as follows:

[0051] The polysaccharide solution after protein removal is added to a DEAE-52 cellulose anion exchange column, and is eluted successively with deionized water and 0.05 and 0.1 mol / L NaCl solutions. The flow rate of the eluent is 0.5 - 5 mL / min (preferably 1 mL / min). 25 - 35 tubes (preferably 30 tubes) of eluent are collected for each elution concentration, and the volume collected for each tube is 0.5 - 5 mL (preferably 2 mL); Further, the elution fraction with 0.05 mol / L NaCl solution is collected, specifically tubes 40 - 52 are collected and freeze-dried; The first purified polysaccharide is obtained;

[0052] The first purified polysaccharide solution is further purified through a Sephadex G-100 dextran gel column, eluted with deionized water, the elution speed is 0.01 - 0.5 mL / min (preferably 0.1 mL / min), the volume collected per tube is 0.5 - 5 mL (preferably 2 mL), a total of 50 tubes are collected, and tubes 11 to 25 are taken and freeze-dried to obtain.

[0053] During the elution process, the phenol-sulfuric acid method is used to detect each tube one by one. With the tube number as the abscissa and the absorbance (A 490 nm ) as the ordinate, an elution curve is plotted.

[0054] In another specific embodiment of the present invention, a product with the ability to improve learning and memory ability, regulate the intestinal flora, and / or protect against degenerative changes caused by zinc deficiency and aging is provided. The product contains the polysaccharide of Termitomyces albuminosus. Specifically, the polysaccharide of Termitomyces albuminosus is prepared by the above preparation method.

[0055] The product includes but is not limited to foods, drugs, and feeds.

[0056] It should be noted that in the present invention, the term "food" should be understood in a broad sense, which can be understood as any form that can be eaten. For example, the foods in the present invention include ordinary foods and special foods. The special foods in the present invention include health foods and foods for special medical purposes; while ordinary foods are relative to special foods and are foods suitable for everyone.

[0057] The foods include but are not limited to solid foods and liquid foods; the solid foods include but are not limited to baked foods, candies, solid beverages, etc.; the liquid foods include but are not limited to liquid beverages, etc.

[0058] According to the present invention, when the product is a drug, the drug also includes at least one pharmaceutically inactive ingredient.

[0059] The pharmaceutically inactive ingredient can be a carrier, excipient, diluent, etc. commonly used in pharmacy. Moreover, according to the usual method, it can be made into dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc. for oral administration, external use, suppositories, and sterile injection solutions for use.

[0060] The carrier, excipient, diluent, etc. non-drug active ingredients that can be included are well-known in the field, and those of ordinary skill in the art can determine that they meet clinical standards.

[0061] In yet another specific embodiment of the present invention, the carriers, excipients and diluents include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate and mineral oil, etc.

[0062] In yet another specific embodiment of the present invention, the feed is the food for animals raised in agriculture or animal husbandry. The polysaccharide of Thelephora ganbajun can be added as a feed additive to any variety of feeds, and the feeds include, but are not limited to, complete formulated feeds, concentrated feeds and premixed feeds, and no specific limitation is made here.

[0063] The present invention will be further explained and illustrated by the following examples, but it does not constitute a limitation to the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. It should be noted that in the examples of the present invention, the Thelephora ganbajun numbered BNCC 230987 was purchased by the applicant from Beijing Beina Chuanglian Biotechnology Research Institute and has been disclosed in patents such as CN109880750A, and the public can obtain it through commercial sales and other means.

[0064] Example 1 Preparation of zinc polysaccharide from mycelium of Thelephora ganbajun (BNCC 230987) and polysaccharide of Thelephora ganbajun

[0065] Experimental method:

[0066] Transfer the slant culture of Thelephora ganbajun stored in a 4°C refrigerator to a PDA plate and incubate it in an incubator at 25°C for 7 days. Take 0.5 cm 2 The activated strain was inoculated into the seed medium and cultured on a shaker for 6 days (25°C, 130 r / min) to obtain the seed liquid. Take 0.5 mL of the seed liquid and transfer it into a potato dextrose liquid medium and a potato dextrose liquid zinc-rich medium (containing 400 mg / L of zinc element, calculated as zinc element, added in the form of zinc sulfate heptahydrate) respectively, and culture it on a shaker for 7 days (25°C, 130 r / min). The culture was filtered or centrifuged to separate the mycelium and the fermentation broth. The mycelium was dried at 55°C in an oven and pulverized.

[0067] The mycelium powder and zinc-enriched mycelium powder were separately mixed with deionized water in a beaker (1:20, g / mL), and ultrasonic fragmentation was carried out using an ultrasonic cell disruptor (300 W, 10 min). Then, it was placed in a water bath at 90 °C for 2 h, centrifuged (10000 r / min, 10 min, 4 °C), and the supernatant was taken. The above steps were repeated 3 times, and the supernatants were combined. The supernatant was concentrated to 1 / 2 to 1 / 3 of its volume using a rotary evaporator, 3 times the volume of 95% ethanol was added, and it was left to stand overnight. Centrifugation (10000 r / min, 10 min, 4 °C) was carried out to obtain a precipitate. The precipitate was dried at 55 °C, then dissolved in deionized water, centrifuged (10000 r / min, 10 min, 4 °C), and the supernatant was freeze-dried to obtain a crude intracellular polysaccharide freeze-dried product.

[0068] A certain amount of activated carbon was mixed with the crude polysaccharide solution (2 mg / mL), and the amount of activated carbon used was 2% of the polysaccharide solution. Stirring adsorption was carried out (60 °C and 1 h), and centrifugation (10000 r / min, 15 min) was carried out to obtain a decolorized polysaccharide solution. The Sevag method was used to remove proteins, and it was freeze-dried. The deproteinized polysaccharide was dissolved in deionized water, filtered through a 0.45 μm microporous filter membrane, 5 mL of the sample solution was added to a DEAE-52 cellulose anion exchange column (1.6 cm × 30 cm), and it was eluted with deionized water and 0.05 and 0.1 mol / L NaCl solutions. The flow rate of the eluent was 1 mL / min, 30 tubes were collected for each elution concentration, and the eluent was collected using an automatic fraction collector (2 mL / tube). The phenol-sulfuric acid method was used to detect each tube, and an elution curve was plotted with the tube number as the abscissa and the absorbance (A490 nm) as the ordinate.

[0069] The elution fractions with 0.05 mol / L NaCl solution were collected (polysaccharide tubes 40 to 52, zinc polysaccharide tubes 40 to 52), and they were freeze-dried. 0.1 g of the above polysaccharide fraction was dissolved in 4 mL of deionized water, and after dissolution, it was filtered through a 0.45 μm filter head and purified through a Sephadex G-100 dextran gel column (1.6 cm × 50 cm). It was eluted with deionized water, and the elution speed was 0.1 mL / min. 2 mL was collected in each tube, and a total of 50 tubes were collected. The phenol-sulfuric acid method was used to detect each tube, the absorbance was measured at 490 nm, and an elution curve was plotted.

[0070] The elution fractions with Sephadex G-100 were collected (polysaccharide tubes 11 to 25, zinc polysaccharide tubes 11 to 25), and they were freeze-dried to obtain the zinc polysaccharide (TZIP) and polysaccharide (TIP) of the mycelium of Termitomyces albuminosus (BNCC 230987). The polysaccharide contents of both were determined using the phenol-sulfuric acid method.

[0071] Experimental results

[0072] The polysaccharide contents in the mycelium zinc polysaccharide (TZIP) and the polysaccharide (TIP) of *Thelephora ganbajun* are 91 ± 3% and 92 ± 3% respectively.

[0073] Example 2 Structural Characterization of the Zinc Polysaccharide and the Polysaccharide of *Thelephora ganbajun*

[0074] Fourier transform infrared spectroscopy (FT-IR) analysis of the zinc polysaccharide (TZIP) and the polysaccharide (TIP) of *Thelephora ganbajun*. After drying the polysaccharides of *Thelephora ganbajun* and the zinc polysaccharide of *Thelephora ganbajun*, they were thoroughly ground with KBr in a mortar and pressed into circular samples using a tablet press. With the aid of FT-IR, scanning was carried out in the range of 4000 - 500 cm -1 .

[0075] As Figure 2 , Figure 3 shown, the infrared spectrogram results of the polysaccharide and the zinc polysaccharide of *Thelephora ganbajun* indicate that these two polysaccharide samples both have the general characteristic absorption peaks of polysaccharides. There is a strong and broad absorption peak at around 3300 cm -1 , and this strong band corresponds to the stretching vibration of OH - on the sugar ring. There is a narrow absorption peak at around 2900 cm -1 , which is attributed to the stretching vibration of the C-H bond. The absorption peak at around 1600 cm -1 is caused by the stretching vibration of the carbonyl group of the polysaccharide. The absorption peak at around 1400 cm -1 is caused by the bending vibration of the C-H bond. The absorption peak at around 1250 cm -1 is the sulfur-oxygen bond. The strong absorption peak at 1200 - 1000 cm -1 is caused by the superposition of the sugar ring vibration, the stretching vibration of the C-O-C glycosidic bond, and the stretching vibration of the C-OH side group. The characteristic absorption peak near 890 cm -1 proves that the polysaccharide and the zinc polysaccharide of *Thelephora ganbajun* are pyranoses linked by β-glycosidic bonds.

[0076] Example 3 Determination of Monosaccharide Composition

[0077] The monosaccharide compositions of the zinc polysaccharide and the polysaccharide of *Thelephora ganbajun* were analyzed by high performance liquid chromatography. Add 0.1 mg of the polysaccharide sample into an ampoule bottle, and add 5 mL of 2 mol / L trifluoroacetic acid solution (fill with nitrogen, seal, 120 °C, 2 h). Take out 1 mL of the hydrolyzate, add 1 mL of methanol, and dry it using nitrogen in a water bath at 70 °C. Repeat 3 times to remove the excess trifluoroacetic acid. Next, add 1 mL of 0.3 mol / L sodium hydroxide solution to the hydrolyzate to completely dissolve it, and prepare a polysaccharide hydrolysis solution to be measured.

[0078] Take 400 μL of the mixed monosaccharide standard solution or polysaccharide hydrolysis solution respectively and add them into 5-mL stoppered test tubes. Then, add 400 μL of PMP methanol solution to the test tubes and vortex mix. Place the test tubes in a water bath at 70 °C and react for 2 h, and then let it cool to room temperature. Add 400 μL of 0.3 mol / L hydrochloric acid to the test tubes for neutralization and adjust the pH to 7. Then add 1200 μL of water and an equal volume of chloroform, and vortex mix and shake. After standing, discard the chloroform layer, and repeat this extraction step twice. Finally, filter the aqueous phase through a 0.45-μm microporous membrane for HPLC injection analysis.

[0079] The HPLC used is equipped with an ultraviolet detector (Variable Wavelength Detector, VWD) and a C18 column chromatographic column (250 mm × 4.6 mm, 5 μm). The mobile phase is phase A (0.1 mol / L, PBS with a pH of 6.4) and phase B (acetonitrile; 0 - 44 min, 83%; 45 - 45 min, 55%; 51 - 57 min, 83%), the flow rate is 1 mL / min, and the injection volume is 20 μL. The column temperature is 30 °C. The standard monosaccharides are as follows, mannose (Man), glucosamine (Glc-N), ribose (Rib), rhamnose (Rha), glucuronic acid (Glc-UA), galacturonic acid (Gal-UA), galactosamine (Gal-N), glucose (Glc), galactose (Gal), xylose (Xyl), arabinose (Ara), and fucose (Fuc).

[0080] The experimental results show that the zinc polysaccharide (TIP) and the polysaccharide (TZIP) of Termitomyces albuminosus mycelium mainly contain galactose, mannose, and glucose, and may contain a small amount of glucosamine, ribose, rhamnose, glucuronic acid, galactosamine, arabinose, and fucose.

[0081] The specific data are as follows:

[0082] The proportions of mannose, glucosamine, ribose, rhamnose, glucuronic acid, galactosamine, glucose, galactose, arabinose, and fucose in the polysaccharide (TIP) are 27.97%, 1.49%, 0.21%, 0.01%, 0.21%, 0.17%, 17.99%, 51.44%, 0.35%, and 0.16% respectively.

[0083] The proportions of mannose, glucosamine, ribose, rhamnose, glucuronic acid, galactosamine, glucose, galactose, arabinose, and fucose in the zinc polysaccharide (TZIP) are 27.07%, 2.14%, 0.10%, 0.07%, 0.38%, 0.23%, 26.03%, 43.53%, 0.39%, and 0.06% respectively.

[0084] Determination of Molecular Weight in Example 4

[0085] Take 10 mg of the sample and dissolve it in 1 mL of 0.1 mol / L NaNO3 mobile phase. After filtration (0.45 μm), the molecular weight is measured. The measuring equipment is a Waters high-performance liquid chromatograph (e2695, Waters Company, USA) equipped with a gel filtration chromatography column (8 × 300 mm, TSKgel GMPWxl, Tosoh Company, Japan) and a Waters differential refractive index detector (RID) (2414, Waters Company, USA). The mobile phase is 0.1 mol / L NaNO3, and the flow rate is 0.6 mL / min. Pullulan polysaccharide standard is used as the standard. Chromatographic data collection and analysis are performed using Empower 3 chromatographic management software.

[0086] The weight-average molecular weights of Termitomyces albuminosus polysaccharide (TIP) and zinc polysaccharide of Termitomyces albuminosus (TZIP) are 21297 Da and 15094 Da, respectively.

[0087] Example 5 Efficacy of Zinc Polysaccharide of Termitomyces albuminosus and Termitomyces albuminosus Polysaccharide in Improving Learning and Memory Ability

[0088] (1) Experimental Grouping

[0089] The same batch of adult AB strain zebrafish is selected for the experiment and randomly divided into a blank control group (NC), a model group (MC, scopolamine 200 μmol / L), a positive drug group (Don, scopolamine 200 μmol / L + donepezil solution 10 mg / L), low-, medium-, and high-dose zinc polysaccharide of Termitomyces albuminosus groups (scopolamine 200 μmol / L + zinc polysaccharide of Termitomyces albuminosus 10, 20, 40 mg / L), and a medium-concentration Termitomyces albuminosus polysaccharide group (scopolamine 200 μmol / L + Termitomyces albuminosus polysaccharide 20 mg / L). Fresh medicinal solution is replaced daily until the day of the experiment.

[0090] (2) T-maze Experiment

[0091] Zebrafish in the blank control group were exposed to fish-raising water; zebrafish in the model group were exposed to hydrobromide scopolamine solution (200 μmol / L) for 1 h every day for 7 consecutive days; zebrafish in other experimental sample groups were first exposed to hydrobromide scopolamine solution (200 μmol / L) for 1 h every day, and then exposed to solutions of positive drug (donepezil: 10 mg / L), medium-concentration Termitomyces albuminosus polysaccharide (TIP: 20 mg / L), and different concentrations of Termitomyces albuminosus zinc polysaccharide (TZIP: 10, 20, 40 mg / L) for 7 consecutive days. From 3 to 6 days, the zebrafish were trained in a T-maze. On the 7th day, zebrafish in different groups were transferred to the T-maze one by one. After adapting to the environment for 5 min, behavioral tests were conducted, and the test duration was 1 min. Zeblab software was used for data processing to calculate the time (s) for zebrafish to first reach the deep water area and the proportion of the staying time in the deep water area, and Origin software was used for statistical analysis.

[0092] As Figure 5 It can be seen that the time for zebrafish in the model group (MC) to first enter the deep water area was significantly prolonged compared with that in the blank control group (NC) (P<0.01). Compared with the NC group, the ratio of the swimming time of zebrafish in the MC group to the total time in the deep water area was significantly reduced (P<0.01), and the learning and memory impairment model of zebrafish was successfully constructed. The intervention of Termitomyces albuminosus zinc polysaccharide (TZIP: 10, 20, 40 mg / L) restored the learning and memory behaviors of zebrafish in the T-maze to varying degrees, showing an obvious dose-effect relationship. The average time for zebrafish in the TZIP-40 group to first enter the deep water area was 13.63 s, which was 70.45% lower than that in the MC group (46.13 s). The ratio of the swimming time of zebrafish in the TZIP-40 group to the total time in the deep water area was 0.40, which was significantly increased compared with that in the MC group (0.14).

[0093] (3) Black and white box experiment

[0094] Zebrafish in the blank control group were raised in fish-raising water; zebrafish in the model group were exposed to hydrobromide scopolamine solution (200 μmol / L) for 1 h every day for 7 consecutive days; zebrafish in other experimental sample groups were first exposed to hydrobromide scopolamine solution (200 μmol / L) for 1 h every day, and then exposed to solutions of positive drug (donepezil: 10 mg / L), medium-concentration Termitomyces albuminosus polysaccharide (TIP: 20 mg / L), and different concentrations of Termitomyces albuminosus zinc polysaccharide (TZIP: 10, 20, 40 mg / L) for 7 consecutive days. On the 8th day, zebrafish in different groups were transferred to the black and white box one by one. After adapting to the environment for 10 min, behavioral tests were conducted, and the test duration was 1 min. Zeblab software was used for data processing to calculate the ratio of the swimming distance (or time) of zebrafish in the black area to the total swimming distance (or time) in the total area, and Origin software was used for statistical analysis.

[0095] As Figure 6 It can be seen that the proportion of the swimming distance and the proportion of the swimming time of zebrafish in the black box in the MC group were significantly lower than those in the NC group (P<0.001), indicating that the learning and memory impairment model of zebrafish was successfully established. The intervention of Termitomyces albuminosus zinc polysaccharide (TZIP: 10, 20, 40 mg / L) restored the learning and memory behaviors of zebrafish in the black and white box to varying degrees, showing an obvious dose-effect relationship. The proportion of the swimming distance and the proportion of the swimming time of zebrafish in the TZIP-40 group were 0.82 and 0.9 respectively, which were significantly higher than those in the MC group (0.26, 0.27).

[0096] Therefore, Termitomyces albuminosus polysaccharide and zinc polysaccharide have good effects on improving learning and memory ability.

[0097] (4) Effects on gene expression of adult zebrafish

[0098] After the behavioral experiment, the zebrafish were anesthetized and decapitated, and their brain tissues were quickly taken out on an ice box. The fish brains were placed in a 1.5 mL EP tube. After being washed repeatedly 3 times with PBS buffer, they were quickly frozen in a -80°C refrigerator. When extracting RNA, 350 μL of lysis buffer was added to each EP tube, and mechanical homogenization was carried out in a tissue grinder for 2 min. After centrifugation at 11000 r / min for 5 min in a high-speed centrifuge, the supernatant was aspirated and added to the purification column, and then centrifuged at 11000 r / min for 2 min. 350 μL of liquid was aspirated and added to an equal volume of 70% ethanol solution, quickly mixed evenly, and then all of it was transferred to the adsorption column and centrifuged again for 1 min. 700 μL of deproteinization solution was added, and it was allowed to stand at room temperature for 1 min. After centrifugation at high speed for 1 min, 500 μL of washing solution was added to wash twice. Finally, the adsorption column was placed in an empty centrifuge tube (RNase free), and 40 μL of pure water (RNase free water) was added. After standing at room temperature for 5 min, it was centrifuged at high speed for 1 min, and the extracted RNA was immediately placed on an ice box. The concentration of RNA was measured using an ultra-micro spectrophotometer, and the A260 / A280 was strictly controlled between 2.0 and 2.5.

[0099] Immediately reverse transcribe RNA into cDNA using the C1000 Touch thermal cycler, strictly following the instructions of the reverse transcription kit (gDNA Purge). Measure the cDNA concentration of each group using a ultra-micro spectrophotometer. Dilute the reverse-transcribed cDNA to a concentration of 100 ng / μL with double-distilled water, aliquot immediately, and store at -20 °C in a refrigerator. Use the reverse-transcribed cDNA of each group as a template, with rpl13a as the internal reference gene, and perform a sample addition experiment using 100 μL volume 8-strip EP tubes. After adding the primers to be tested, detect the changes in gene expression of each group by real-time fluorescence quantitative PCR on a Roche real-time fluorescence quantitative PCR instrument. After the amplification is completed, output the CT (Cycle Threshold) values of the target genes of each group and the internal reference gene rpl13a, and calculate the relative expression levels of genes in different groups based on the 2-ΔΔCT relative quantification method. The steps of total RNA extraction, concentration measurement, reverse transcription into cDNA, and amplification calculation are the same as those for gene detection in zebrafish larvae.

[0100] The primer sequence information required for this experiment is shown in Table 1.

[0101] Table 1 Primer sequences of zebrafish learning and memory-related genes

[0102]

[0103] Note:

[0104] creb1a: Involved in the development of the midbrain-hindbrain boundary and expressed in the nervous system.

[0105] ache: Localized to multiple cellular components, including cell junctions, extracellular regions, and synapses. The human ortholog of this gene is associated with Alzheimer's disease. Expressed in multiple structures, including the nervous system and neural tube. Homologous to human ACHE (acetylcholinesterase).

[0106] Chata: Choline O-acetyltransferase A; predicted to have choline O-acetyltransferase activity. Involved in motor behavior. Predicted to be localized to the cytoplasm and neuronal processes. Used to study congenital myasthenic syndrome. The human ortholog of this gene is associated with Alzheimer's disease and congenital myasthenic syndrome; expressed in the brain, neurons, spinal cord, and spinal neural tube. Homologous to human CHAT (choline O-acetyltransferase).

[0107] nrf2a: nfe2 like bZIP transcription factor 2a; involved in multiple processes, including the cellular response to oxidative stress, etc. The human ortholog of this gene is associated with Alzheimer's disease, cataract, liver cancer, and macular degeneration. Expressed in multiple structures, including the digestive system, gills, heart, pleural region, and sensory system. Homologous to human NFE2L2 (nuclear factor).

[0108] chrna1: Cholinergic receptor nicotinic alpha 1 (muscle); exhibits cation channel activity. Involved in multiple processes, including chemical synaptic transmission, chondrocyte intercalation involved in growth plate cartilage morphogenesis, and neuron generation. Localized to the acetylcholine-gated channel complex. Used in the study of congenital myasthenic syndromes. The human ortholog of this gene is associated with congenital myasthenic syndrome 1A and congenital myasthenic syndrome 1B. Expressed in the paraxial cell and muscle tissue systems. Homologous to human CHRNA1 (choline receptor nicotinic alpha 1 subunit).

[0109] ngfβ: Nerve growth factor B (beta polypeptide); predicted to have growth factor activity. Predicted to be involved in multiple processes, including cell surface receptor signaling pathways, negative regulation of the neuron apoptotic process, and nervous system development. Predicted to be localized to multiple cellular components, including axons, dendrites, and synaptic vesicles. The human ortholog of this gene is associated with multiple diseases, including IgA glomerulonephritis, end-stage renal failure, hereditary sensory and autonomic neuropathy type 5, interstitial cystitis, and neurogenic bladder. Expressed in multiple structures, including the nasal placode midbrain region, head mesenchyme, nervous system, periderm, and segmented plate. Homologous to human NGF (nerve growth factor).

[0110] As Figure 7 It can be seen that compared with the blank control group NC, the mRNA levels of learning and memory-related genes creb1a, ache, chrna1, and ngfβ in the model group (MC) zebrafish were significantly decreased, while the mRNA levels of chata and nrf2a were significantly increased. The construction of the zebrafish learning and memory model was successful. Compared with the model group, under the intervention of polyporus tuber-regium polysaccharide or zinc polysaccharide, the mRNA expression levels of creb1a, ache, chrna1, and ngfβ in zebrafish increased, while the mRNA expression levels of chata and nrf2a decreased. The differences were statistically significant. Therefore, polyporus tuber-regium polysaccharide and zinc polysaccharide have obvious improvement effects on the abnormal expression of genes related to learning and memory (such as brain nerve-related genes) in adult zebrafish. Therefore, polyporus tuber-regium polysaccharide and zinc polysaccharide may improve the abnormal learning and memory behavior by regulating the abnormal expression of brain nerve-related genes in learning and memory-impaired zebrafish.

[0111] Example 6 Improvement effect of polyporus tuber-regium zinc polysaccharide and polyporus tuber-regium polysaccharide on intestinal flora

[0112] After the behavioral experiments were completed, the zebrafish were anesthetized and decapitated. The abdomen of the zebrafish was cut open with small scissors, and its intestinal tissue was quickly removed on an ice box. The intestinal tissue of every two fish was placed in a 1.5 mL EP tube and quickly frozen in an -80 °C refrigerator. The microbial diversity in the intestine was measured.

[0113] As Figure 8It can be seen that based on the community composition analysis at the family level, the dominant bacterial families in the zebrafish intestinal flora of the blank control group (NC), the model group (MC), and the high-concentration Termitomyces albuminosus zinc polysaccharide group (TZIP-40) are Fusobacteriaceae, Shewanellaceae, Beijerinckiaceae, and Aeromonadaceae, respectively. Compared with the blank control group, the relative abundances of Fusobacteriaceae and Shewanellaceae in the model group increased, while the relative abundances of Beijerinckiaceae and Aeromonadaceae decreased. Compared with the model group, the relative abundances of Fusobacteriaceae and Shewanellaceae in the high-concentration Termitomyces albuminosus zinc polysaccharide group decreased, while the relative abundance of Beijerinckiaceae increased.

[0114] As Figure 8 It can be seen that based on the community composition analysis at the genus level, the dominant bacterial genera in the zebrafish intestinal flora of the blank control group (NC), the model group (MC), and the high-concentration Termitomyces albuminosus zinc polysaccharide group (TZIP-40) are Cetobacterium_A, Shewanella, Bosea, and Aeromonas, respectively. Compared with the blank control group, the relative abundances of Cetobacterium_A and Shewanella in the model group increased, while the relative abundances of Bosea, Aeromonas, and Flavobacterium decreased. Compared with the model group, the relative abundances of Cetobacterium_A and Shewanella in the high-concentration Termitomyces albuminosus zinc polysaccharide group decreased, while the relative abundance of Bosea increased.

[0115] The intestinal flora participates in the bidirectional communication between the intestine and the brain through neuroendocrine, neuroimmune signal transduction and other pathways, and long-term stress can cause disorders of the host intestinal flora. In this experiment, compared with the blank control group, the diversity of the zebrafish intestinal flora in the model group decreased. After treatment with Termitomyces albuminosus zinc polysaccharide, the diversity of the intestinal flora increased, and the abnormal changes in the flora structure were restored to a certain extent, indicating that Termitomyces albuminosus zinc polysaccharide can regulate the community composition structure and restore its related material metabolism function.

[0116] Example 7 Protective effects of Termitomyces albuminosus polysaccharide and zinc polysaccharide on degenerative changes caused by zinc deficiency and aging

[0117] Adult male ICR strain mice were adaptively fed for 7 days and randomly divided into 7 groups. (NC) Blank control group: fed normal diet, gavaged with deionized water, and subcutaneously injected with normal saline in the neck; (AC) Aging control group: fed normal diet, gavaged with deionized water, and subcutaneously injected with D-galactose (100 mg / kg / d) in the neck; (MC) Zinc-deficient aging model group: fed low-zinc diet, gavaged with deionized water, and subcutaneously injected with D-galactose (100 mg / kg / d) in the neck; TIP intervention control group (I) and TZIP intervention groups (II, III, IV), fed low-zinc diet, gavaged with TIP or TZIP samples, and subcutaneously injected with D-galactose (100 mg / kg / d) in the neck. The gavage dose of TIP in group I was 500 mg / kg / d, and the gavage doses of TZIP in groups II, III, and IV were 100, 300, and 500 mg / kg / d, respectively.

[0118] It can be seen from Figure 9 that the contents of lactic acid and IL-1β in the brains of mice in the MC group were significantly higher than those in the NC group, while the contents of CAT, GSH, and SOD were significantly lower than those in the NC group, indicating that the zinc-deficient aging mouse model was successfully established. Moreover, the lactic acid content in the MC group was significantly higher than that in the AC group, and the GSH content was significantly lower than that in the AC group. Therefore, zinc deficiency would exacerbate the changes in brain physiological and biochemical indexes caused by aging. Under the intervention of TZIP, the contents of lactic acid and IL-1β in the brains of mice were significantly decreased, while the contents of CAT, GSH, and SOD were significantly increased. The intervention effects of TZIP-500 on lactic acid, IL-1β, CAT, GSH, and SOD in the brain were significantly better than those of TIP-500 (P < 0.001). Compared with the MC group, the contents of lactic acid and IL-1β in the brains of mice under the intervention of TZIP-500 were decreased by 40.38% and 30% respectively, while the contents of CAT, GSH, and SOD were increased by 62.7%, 45.41%, and 56.34% respectively. Therefore, both TIP and TZIP had significant improvement effects on the brain lactic acid, inflammation, and oxidation indexes of zinc-deficient aging mice, and the improvement effect of TZIP was more ideal.

[0119] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the given examples, those of ordinary skill in the art can modify or equivalently replace the technical solutions of the present invention as needed without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. Application of the polysaccharide from the dried mushroom in the preparation of a product for improving learning and memory ability, regulating intestinal flora and / or protecting against degenerative changes caused by zinc deficiency and aging.

2. The application according to claim 1, wherein The product includes food, medicine and feed.

3. The application according to claim 1, characterized in that The polysaccharide from the dried mushroom is the polysaccharide from the mycelium of the dried mushroom; the dried mushroom is obtained by ordinary fermentation culture or zinc-enriched fermentation culture; further, the polysaccharide from the dried mushroom is the zinc-enriched polysaccharide from the mycelium of the dried mushroom.

4. A method for preparing the polysaccharide of the dried mushroom, characterized in that, The preparation method includes: S1. Mix the mycelium of the dried mushroom with water, break it, and heat to obtain the supernatant. S2. Add ethanol to the supernatant, mix and let stand, and collect the precipitate. S3. The precipitate is decolorized, deproteinized, and separated and purified by an anion exchange column and a Sephadex column to obtain the product.

5. The preparation method according to claim 4, characterized in that, In step S1, The mycelium is obtained by ordinary fermentation culture or zinc-enriched fermentation culture; specifically, the ordinary fermentation culture method is to inoculate the seed solution of the dried mushroom into a fungal medium for fermentation culture; the zinc-enriched fermentation culture method is the same as the ordinary fermentation culture method, except that the selected medium is a zinc-enriched fungal medium.

6. The preparation method according to claim 4, characterized in that, In step S1, The mass-volume ratio of the mycelium of the dried mushroom to water is 1:1 - 30, g / mL; preferably 1:20; the breaking can be carried out by ultrasonic breaking, and the specific breaking conditions are to treat for 1 - 30 min under the condition of 100 - 500 W, preferably 300 W for 10 min; The heating is carried out by water bath heating, and the specific heating conditions are to treat for 1 - 5 h at 70 - 100 °C, preferably 90 °C for 2 h.

7. The preparation method according to claim 4, characterized in that, In step S2, the supernatant is concentrated to 1 / 2 to 1 / 3 of the original volume, and then ethanol with a volume 1 - 10 times (preferably 3 times) that of the concentrated supernatant is added, and the ethanol is high-concentration ethanol.

8. The preparation method according to claim 4, wherein In step S3, decolorization is carried out by using activated carbon decolorization. The method for deproteinization is the Sevag method.

9. The preparation method according to claim 4, characterized in that In step S3, the anion exchange column is specifically a DEAE anion exchange column; The Sephadex column is specifically a Sephadex G-100 Sephadex column; After separation by the DEAE anion exchange column and the Sephadex G-100 Sephadex column, the polysaccharide from the dried mushroom is further purified to obtain the polysaccharide from the mycelium of the dried mushroom, named TIP and TZIP respectively; Specifically, the specific separation and purification process of TIP and TZIP is as follows: Add the deproteinized polysaccharide solution to a DEAE-52 cellulose anion exchange column, and elute successively with deionized water and 0.05 and 0.1 mol / L NaCl solutions. The flow rate of the eluent is 0.5 - 5 mL / min (preferably 1 mL / min), and 25 - 35 tubes (preferably 30 tubes) of eluent are collected for each elution concentration, and the volume collected for each tube is 0.5 - 5 mL (preferably 2 mL); further, collect the elution fraction with 0.05 mol / L NaCl solution, specifically collect 40 - 52 tubes and freeze-dry; Obtain the first purified polysaccharide; The first purified polysaccharide solution is further purified through a Sephadex G-100 dextran gel column, and eluted with deionized water at an elution rate of 0.01 - 0.5 mL / min (preferably 0.1 mL / min). The volume collected in each tube is 0.5 - 5 mL (preferably 2 mL), and a total of 50 tubes are collected. Tubes 11 to 25 are taken and obtained after freeze-drying.

10. A product having the ability to improve learning and memory, regulate the intestinal flora, and / or protect against degenerative changes caused by zinc deficiency and aging, characterized in that, The product contains the polysaccharide of the dried Tricholoma matsutake, specifically, the polysaccharide of the dried Tricholoma matsutake is prepared by the preparation method according to any one of claims 4 - 9; The product includes, but is not limited to, foods, drugs, and feeds.

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