CN114292343B - A method for preparing exopolysaccharide and intracellular polysaccharide of Perennial ash and its application in regulating intestinal microbial flora and lowering blood sugar - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly discloses a preparation method of perennial cerasus extracellular polysaccharide and intracellular polysaccharide, and application of the perennial cerasus extracellular polysaccharide and intracellular polysaccharide in regulating intestinal microbial flora and reducing blood sugar. The perennial cereus extracellular polysaccharide and intracellular polysaccharide can be used as prebiotics, can regulate the intestinal bacteria flora structure of diabetic mice, improve the relative abundance of flora such as intestinal lactobacillus, lachnospirillum, coprobacter, bacteroides and the like, reduce the relative abundance of bacillus producing, campylobacter and enterobacter, simultaneously regulate the fungus flora structure, reduce the number ratio of basidiomycota and ascomycota, reduce the relative abundance of inflammation-promoting fungi such as aspergillus, mortierella and the like, and improve the relative abundance of penicillium and candida albicans. In addition, the extracellular polysaccharide and the intracellular polysaccharide of the fraxinus chinensis can improve the insulin level, the glycosylated hemoglobin level and the function of islet beta cells of a mouse so as to improve the blood sugar of a diabetic mouse.

Description

A preparation method of exopolysaccharide and intracellular polysaccharide of perennial bacteria and its role in regulating intestinal Tract Microflora and Hypoglycemic Application

technical field

The invention belongs to the field of biological technology, and in particular relates to a method for preparing exopolysaccharides and intracellular polysaccharides of perennial bacteria and its application in regulating intestinal microbial flora and lowering blood sugar.

Background technique

Perenniporia fraxinea belongs to Basidiomycotina, Agaricomycetes, Polyporales, Polyporaceae, and Perenniporia. Medicinal fungi. White wax perennial bacteria contain a variety of physiologically active components, including polysaccharides, polyphenols, plasminase and so on. Studies have shown that perennial perennials has anti-tumor effect and antioxidant activity, but there are relatively few studies on the extraction method and medicinal value of polysaccharides from perennial perennials perennials.

Bacteria, archaea, and colonic fungi in the gut are collectively referred to as "gut microbes." The number of microorganisms in the gastrointestinal tract exceeds 10 to ¹⁴ species, which constitute the intestinal mucosal barrier and play an important regulatory role in the intake and metabolism of nutrients, the maturation of immune tissues, and the prevention of the reproduction of pathogenic microorganisms. Health comes with a lot of responsibility. Conversely, dysbiosis of the gut microbiota may be involved in the development of diseases such as: obesity, asthma, celiac disease, type 1 and type 2 diabetes, inflammatory bowel disease (Crohn's disease, ulcerative colitis), irritable bowel disease, Irritation syndrome, Clostridium difficile infection, HIV infection, colorectal cancer, kidney stones and periodontitis, to name a few.

With urbanization and development and changes in people's diet, type 2 diabetes mellitus (T2DM) and its complications have become one of the major diseases that endanger human health. Insulin injections and oral hypoglycemic agents are currently more effective treatments for controlling blood sugar. However, they have significant side effects, including hypoglycemia and gastrointestinal problems. Therefore, effective alternatives with reduced diabetes complications and lower side effects are urgently needed. In recent years, the search for alternative medicines against diabetes has received a great deal of attention. More and more studies have shown that edible and medicinal fungal polysaccharides have obvious effects, and have the effect of preventing and treating T2DM, and are natural hypoglycemic substances.

So far, no research has been found on the effect of polysaccharides from Perennial spp. on the intestinal microbiota to exert a hypoglycemic effect.

Contents of the invention

The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and provide a method for preparing exopolysaccharides and intracellular polysaccharides of perennial bacteria. The method is stable, reliable and practical.

Another object of the present invention is to provide the exopolysaccharide and intracellular polysaccharide of Perennialia cerevisiae prepared by the method.

Another object of the present invention is to provide the exopolysaccharides and intracellular polysaccharides of Perennial cerevisiae, which can promote the proliferation of intestinal probiotics, promote the production of intestinal short-chain fatty acids, regulate the structure of intestinal flora, and/or Or the application of hypoglycemic products.

The object of the present invention is achieved through the following technical solutions:

A method for preparing exopolysaccharides and intracellular polysaccharides of perennial bacteria, comprising the steps of:

(1) inoculating the perennial cereus strains in a liquid medium for fermentation and cultivation, separating the solid and liquid to obtain the perennial perennial cerevisiae fermented liquid and mycelia, and concentrating the perennial perennial perennial perennial ceruleum fermentation liquid for subsequent use;

(2) drying, pulverizing and sieving the mycelium of the perennial asteroids obtained in the step (1) to obtain mycelium powder; then adding petroleum ether for degreasing treatment, and drying to obtain dry mycelium powder of the perennial asteraceae;

(3) adding the dry powder of the perennial mycelia obtained in the step (2) into water, and carrying out hot water extraction to obtain the extract of the perennial perennial;

(4) After concentrating the fermented liquid of the perennial bacteria obtained in step (1) and the extract concentrate of the perennial bacteria obtained in (3), the trypsin combined with the Sevage method is used to remove the protein respectively, and the extracellular protein after the protein removal is obtained successively. Polysaccharide extract and intracellular crude polysaccharide extract;

(5) adding the extracellular crude polysaccharide extract and the intracellular crude polysaccharide extract obtained in step (4) into the ethanol solution respectively, standing still, centrifuging to collect the precipitate, and then adding water to redissolve to obtain the extracellular crude polysaccharide solution and Intracellular crude polysaccharide solution; followed by dialysis, concentration, and freeze-drying to obtain extracellular crude polysaccharide extract and intracellular crude polysaccharide extract of perennial bacteria;

(6) The extracellular crude polysaccharide extract and the intracellular crude polysaccharide extract of the perennial bacteria obtained in step (5) are chromatographically purified respectively, then dialyzed, and freeze-dried to obtain the extracellular polysaccharide (EPPF) and Intracellular polysaccharide (IPPF).

The formula of the liquid fermentation medium described in step (1) is: for every 1000mL liquid fermentation medium, glucose 30-40g, K ₂ HPO ₄ 5-8g, yeast extract powder 6-10g, add water to 1000mL, adjust pH =5~7. Preferably, the formula of the liquid fermentation medium described in step (1) is: for every 1000mL liquid fermentation medium, glucose 37.7g, K ₂ HPO ₄ 6.31g, yeast extract powder 8.3g, replenish water to 1000mL, adjust pH to 6.

The fermentation culture described in step (1) is preferably realized through the following steps:

① After activation and culture of the slant parent species of Perennial spp., transfer it to the solid medium of the slant, and cultivate it at 20-37°C for 8-15 days to obtain the slant strain of Bacillus perennial slant;

②Pick 4-6 pieces of bacteria from the slant of the white wax perennial bacteria obtained in step ①, inoculate them in 150-200mL liquid fermentation medium, let them stand at room temperature, and keep the wounds of the bacteria pieces healed, and culture them at a constant temperature and avoid light to obtain Fermentation broth and mycelia.

The formula of the slant solid medium described in step ① is: 200 g of peeled potatoes, 20 g of glucose, 1 g of peptone, (NH ₄ ) ₂ SO ₄ 2 g, 1 g of MgSO ₄ 7H ₂ O, 1 g of KH ₂ PO ₄ , 20 g of agar , add water to make up to 1000mL, and adjust the pH to 6.5.

The constant-temperature shaking culture in step ② is specifically 8-9 days at 25-32°C and 100-250 rpm in the dark for constant temperature shaking culture after the wound of the bacteria block has healed.

The solid-liquid separation described in step (1) is separated by means of filtration, centrifugation or suction filtration.

The concentrated fermented liquid of the perennial bacterium in the step (1) is 1/10 to 1/4 of the original volume; preferably 1/5 of the original volume.

The drying temperature described in step (2) is 50-70°C; preferably 60°C. The sieving described in the step (2) is through a 40-60 mesh sieve; preferably through a 40 mesh sieve.

The solid-to-liquid ratio of the mycelium powder and petroleum ether described in step (2) is 1:0.5-2 (g/mL); preferably 1:1 (g/mL).

The time for the degreasing treatment described in step (2) is 2 to 4 days; preferably 2 days. The frequency of the degreasing treatment described in step (2) is 1 to 3 times; preferably 2 times.

The solid-liquid ratio of the mycelia dry powder of Perennial aureus aureus described in step (3) to water is 1:20-40 (g/mL); preferably 1:25 (g/mL).

The temperature of the extraction described in step (3) is 50-90°C, preferably 80°C. The extraction time described in step (3) is 1.5-3.5 hours; preferably 3 hours. The number of extractions described in step (3) is 1 to 3 times; preferably 2 times.

The concentration described in the step (3) is to concentrate under reduced pressure. The concentration temperature in step (3) is 50-70°C; preferably 55-65°C; more preferably 60°C.

The concentration described in the step (3) is concentrated to 1/10-1/4 of the volume of the extract solution of the perennial bacteria; preferably concentrated to 1/5 of the volume of the extract solution of the perennial bacteria.

Adopting trypsin and Sevage method to remove protein described in step (4) is preferably realized through the following steps:

(1) adjust the pH to 8.0 respectively with the leaching concentrate obtained in the step (3) and the fermented concentrate obtained in the step (1), then add the trypsin solution respectively, fully shake and then water bath Inactivate the enzyme, cool to room temperature, and obtain the fermented liquid and the intracellular extract of the perennial bacteria aureus after enzymolysis;

(II) Add the mixed solution of chloroform and n-butanol to the fermented liquid and intracellular extract of the enzymolysis obtained in step (I), centrifuge after shaking, discard the protein layer and organic solution, and collect the supernatant solution, which is the intracellular crude polysaccharide extract.

The concentration of the trypsin solution described in the step (I) is 2% to 5% by mass to volume; preferably 2%.

The volume ratio of the trypsin solution described in the step (I) to the concentrated perennial bacteria fermentation broth and extract is 1:15-30; preferably 1:20.

The shaking in the step (I) is shaking at 37±2° C., 80-150 rpm for 30-60 minutes.

The conditions for inactivating the enzyme in the step (I) are: inactivating the enzyme at 100° C. for 5 to 15 minutes; preferably inactivating the enzyme at 100° C. for 10 minutes.

The volume ratio of chloroform and n-butanol in the chloroform-n-butanol mixture described in step (II) is 3-7:1, preferably 5:1.

The volume ratio of the chloroform-n-butanol mixture described in step (II) to the fermented liquid and intracellular extract of the perennial bacteria after enzymolysis is 1:0.1-0.25; preferably 1:0.2.

The oscillation time in step (II) is 30-40 minutes; the centrifugation condition is: centrifugation at 5000-10000 rpm for 5-15 minutes; preferably: centrifugation at 8000 rpm for 10 minutes.

Preferably, step (II) is repeated 3 to 5 times until no protein layer appears to obtain the intracellular crude polysaccharide extract after protein removal.

The volume ratio of the ethanol solution described in step (5) to the protein-removed fermented concentrated liquid and the extract of the perennial bacteria is 2-5:1; preferably 3-5:1; more preferably 4:1.

The ethanol solution described in step (5) is an ethanol solution with a mass fraction of 95%.

The standing condition described in step (5) is: standing at 0-4°C for 24-48h; preferably: standing at 4°C for 48h. The centrifugation conditions in step (5) are: centrifugation at 5000-10000 rpm for 5-15 minutes; preferably: centrifugation at 10000 rpm for 10 minutes.

The dialysis described in step (5) is to use a dialysis bag with a molecular weight cut-off of 6000-8000 Da for dialysis; preferably, a dialysis bag with a molecular weight cut-off of 6000-8000 Da is used for dialysis for 72 hours, and the deionized water is replaced every 8 hours.

The concentration described in step (5) is concentrated to 1/5～1/2 of the volume of the extract solution of exopolysaccharide and intracellular polysaccharide of perennial bacteria; 1/3 of the extract volume.

The chromatographic purification described in step (6) is preferably realized by the following steps:

S1. Column packing: add water to the chromatography column, open the liquid outlet at the lower end, and then pour DEAE-Sepharose Fast Flow filler into the chromatography column to allow it to settle naturally in the chromatography column;

S2. Balance: use a constant flow pump to pump the NaCl buffer solution into the chromatography column, open the lower end liquid outlet until the pH value of the effluent is the same as that of the NaCl buffer solution;

S3, sample loading and elution:

S31. Take the aqueous solution of extracellular crude polysaccharide and intracellular crude polysaccharide, centrifuge and take the supernatant, and slowly add it into the equilibrated chromatographic column by using a constant flow pump. First wash off the unadsorbed substances with buffer solution, then elute them with NaCl solution gradient respectively, set the collection time of the partial collector, collect, and measure the total sugar content, combine the single absorption peak samples, concentrate and dialyze, then The dialysate is concentrated and vacuum freeze-dried to obtain purified polysaccharides EPPF and IPPF.

The size of the chromatographic column described in step S1 is: 1.5 cm×20 cm. The volume of water added in step S1 is 1/3 of the column volume, and the volume of the filler is 2/3 of the column volume.

The pH value of the NaCl buffer solution in step S2 is 7.0-7.6; preferably 7.0. The flow rate of the effluent in step S2 is 0.5-2 mL/min, preferably 1 mL/min.

The concentrations of the aqueous solution of extracellular crude polysaccharide and the aqueous solution of intracellular crude polysaccharide in step S31 are both 8-12 mg/mL, preferably 10 mg/mL.

The centrifugation conditions in step S3 are: centrifugation at 5000-10000 rpm for 5-15 minutes, preferably 10 minutes at 8000 rpm. The concentration of NaCl solution used for elution in step S31 is 0-0.5 mol/L, and the flow rate of elution is 1 mL/min.

The collection described in step S31 is specifically to collect 20 tubes for each gradient, and each tube is collected for 10 minutes; the total sugar content of the sample collected in step S31 is determined by the phenol-sulfuric acid method.

The dialysis described in step (6) is performed by using a dialysis bag with a molecular weight cut-off of 6000-8000 Da; preferably, the dialysis is performed with a dialysis bag with a molecular weight cut-off of 6000-8000 Da for 48-72 hours.

An exopolysaccharide and an intracellular polysaccharide of Perennial cerevisiae, prepared by the above method.

The application of the exopolysaccharide and intracellular polysaccharide of perennial cereus in the preparation of probiotics in the intestinal tract, promoting the production of short-chain fatty acids in the intestinal tract, regulating the structure of intestinal flora, and/or reducing blood sugar. . The exopolysaccharide and intracellular polysaccharide of the perennial bacteria have good effects of improving intestinal microecology and lowering blood sugar.

The product is at least one of medicines, health products and functional foods.

The dosage of exopolysaccharides and intracellular polysaccharides of Perennialia cerevisiae is 50-200 mg/kg/d; preferably 100-200 mg/kg/d; more preferably 100 mg/kg/d.

The intestinal probiotics include Bacteroides and Lactobacillus.

The short-chain fatty acid is at least one of acetic acid, propionic acid, butyric acid and valeric acid; both the exopolysaccharide and the intracellular polysaccharide of Perennialia cerevisiae can induce and activate the expression of short-chain fatty acid receptors GPR41 and GPR43.

The intestinal flora is intestinal flora and/or intestinal fungi; preferably intestinal flora.

The intestinal bacteria include: Proteobacteria, Firmicutes, Bacteroidetes, Verrucomicrobia, Patescibacteria, Epsilonbacteraeota, Actinobacteria, Tenericutes, Acidobacteria, Deferribacteres, preferably Proteobacteria, Firmicutes, Bacteroidetes. In this study, high-fat diet combined with STZ induction can increase the relative abundance of probiotics such as Lactobacillus, Bacteroides, and Faecalibacterium in the intestinal flora, while increasing Enterobacter, Bacillus and Δproteobacteria and Relative abundance of harmful bacteria such as Campylobacter. After the intervention of exopolysaccharides and intracellular polysaccharides of perennial bacteria, the relative abundance of intestinal Lactobacillus, Lachnospira, Faecalibacterium, and Bacteroides increased, and the relative abundance of Bacillus, Campylobacter, and Enterobacteriaceae decreased. abundance.

Preferably, the intestinal fungi include: Ascomycota, Basidiomycota, Mortierellomycota, Chytridiomycota, Rozellomycota, Oiltridiomycetes Phylum (Olpidiomycota), Glomeromycota (Glomeromycota), Insectivomycota (Zoopagomycota) and Mucoromycota (Mucoromycota), the white wax perennial bacteria exopolysaccharide and intracellular polysaccharide of the present invention can reduce the difference between Basidiomycota and Ascomycota. Ratio, increased the relative abundance of Penicillium and Nosdida, decreased the relative abundance of Aspergillus and Mortierella.

Compared with the prior art, the present invention has the following advantages and effects:

(1) The liquid fermentation medium used in the present invention has clear ingredients, high repeatability, high fermentation efficiency, stable product, good mycelium growth, thick fermentation liquid, and easy separation and purification of polysaccharides.

(2) The present invention rapidly obtains the fermented liquid and mycelia of perennial bacteria through liquid submerged fermentation, and extracts extracellular polysaccharides and intracellular polysaccharides of perennial bacteria. Good property; the preparation process is simple, the conditions are easy to control, the cycle is short, the yield is high, and mass production can be realized.

(3) The present invention uses DEAE-Sepharose Fast Flow column chromatography to purify extracellular polysaccharides and intracellular polysaccharides of perennial bacteria, and through gradient elution, the polysaccharide components can be obtained quickly and efficiently. The polysaccharides are of good purity and the process is simple and easy to operate, avoiding multiple The loss of polysaccharide components caused by the secondary purification process.

(4) The extracellular polysaccharides and intracellular polysaccharides of the perennial bacteria of the present invention can be used as prebiotics, which can not only regulate the intestinal bacterial flora structure of diabetic mice, but also improve intestinal lactobacillus, Lachnospira, Faecalibacterium, Bacteroides, etc. The relative abundance of the flora reduces the relative abundance of Bacillus, Campylobacter, and Enterobacter, and at the same time adjusts the structure of the fungal flora, reduces the ratio of the number of Basidiomycota to Ascomycota, and reduces the number of Aspergillus and Mortierella The relative abundance of pro-inflammatory fungi such as the genus Penicillium and Candida albicans was increased. The polysaccharide of the perennial bacteria of the invention provides a new idea for intestinal regulation health food.

(5) Both the exopolysaccharide and the intracellular polysaccharide of perennialia cerevisiae of the present invention can improve the insulin level, the glycosylated hemoglobin level and the function of the islet β cells in mice so as to improve blood sugar in diabetic mice.

(6) The preparation process of the exopolysaccharide and intracellular polysaccharide of perennial bacteria of the present invention is simple, the product is stable, and can be produced in large quantities with high efficiency. The structure of intestinal flora and the field of hypoglycemic drugs or health food.

Description of drawings

Fig. 1. Histograms of the species distribution of the polysaccharides obtained in Example 4 on the intestinal bacteria of T2DM mice at different levels.

Fig. 2. Analysis of intestinal bacterial diversity of T2DM mice by polysaccharides obtained from Perennials perennials obtained in Example 4.

Fig. 3. The Veen diagram of the action of the polysaccharide from Perennialia cerevisiae obtained in Example 4 on the OTU of the fungus in T2DM mice.

Fig. 4. Species distribution diagram of the phylum level of the polysaccharide obtained from Perennials perennials acting on T2DM mice intestinal fungi obtained in Example 4.

Fig. 5. The effect of polysaccharides obtained from Perennials perennials obtained in Example 4 on specific fungal phyla in the intestinal tract of T2DM mice.

Fig. 6. The effect of the polysaccharide obtained from Perennial cerevisiae in Example 4 on short-chain fatty acids in feces of T2DM mice.

Fig. 7. Effects of polysaccharides obtained from Perennial perennials in Example 4 on hypoglycemic-related indicators in T2DM mice.

Fig. 8. Effects of polysaccharides from Perennialia perennials obtained in Example 4 on insulin function and β-cell function in T2DM mice.

Fig. 9. Analysis of expression of short-chain fatty acid receptors GPR41 and GPR43 regulated by the polysaccharide of Perennialia cerevisiae in Example 4.

Detailed ways

The present invention will be further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. The test method that does not indicate specific experimental conditions in the following examples is usually in accordance with conventional experimental conditions. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

The strain of Perenniporia fraxinea in the examples was provided by the School of Life Sciences, South China Normal University.

The medium formula involved in the embodiment:

Slope solid medium and plate solid medium formula: 200g potato (peeled), glucose 20g, K ₂ HPO ₄ 1g, MgSO ₄ 1g, peptone 1g, (NH ₄ ) ₂ SO ₄ 1g, vitamin B ₁ 0.01g, agar 20g was melted, added pure water to 1000mL, pH was 6.5.

Liquid submerged fermentation medium: glucose 37.7g, K ₂ HPO ₄ 6.31g, yeast extract powder 8.3g, add pure water to 1000mL, pH 6.

Embodiment 1: Preparation of intracellular polysaccharide of perennial bacteria

(1) Perennial perennial ash is activated by bacterial classification, cultured on a slanted surface and submerged in liquid fermentation to obtain mycelium of perennial perennial ash. The specific methods are as follows:

①Cultivation of slant strains: After activation and cultivation of the slant parent species of Perennial aureus, inoculate in sterilized slant solid medium, and cultivate at 28°C for 8 days for later use;

② Liquid submerged fermentation culture: pick 4 to 6 soybean-sized bacteria blocks from the slant of the slanted strain of the white wax perennial bacteria, inoculate in 150mL sterile liquid fermentation medium, and shake at 30°C and 180rpm at a constant temperature in the dark Cultivate for 9 days to obtain the liquid fermentation product of the perennial bacteria, and separate the mycelium by centrifugation and vacuum filtration, and dry it at 60°C;

(2) Degreasing: After the mycelium is dried to a constant weight, it is pulverized by a pulverizer to pass through a 40-mesh sieve, and the mycelium powder above 40 mesh is collected. Put the mycelium powder in the Erlenmeyer flask, mix the mycelium powder and petroleum ether according to the solid-to-liquid ratio of 1:1 (V:Vg/mL), shake on the shaker for 48 hours, centrifuge to save the precipitate, and repeat this step once . The obtained precipitate was dried at 60°C and placed in a drying oven for later use;

(3) Hot water leaching: extract the mycelia of the perennial mycelia after degreasing and drying in step (2) with hot water, and collect the extract; wherein: the extraction temperature is 80°C, the extraction times are 2 times, and the extraction Time 2h, the ratio of solid to liquid is 1:30(g:ml);

(4) The yield of total sugar in the extract was measured by the phenol-sulfuric acid method, and the yield of total sugar in the extract was 9.22%;

(5) Concentration: use a rotary evaporator to concentrate the extract collected in step (3) under reduced pressure in a constant temperature water bath at 60°C to 1/5 of the original volume to obtain a concentrated extract of the perennial bacteria;

(6) Trypsin and sevage method for protein removal and centrifugation: Weigh 2.0g of trypsin and dissolve it in 100mL of distilled water to obtain a trypsin solution; Add it to the extract concentrate of white wax perennial bacteria, and then put it in a 37°C water bath to preheat for 10 minutes, fully mix the enzyme solution and the two concentrates separately, and shake at 120rpm for 30 minutes, then put it in a 100°C water bath for 10 minutes to inactivate the enzyme , cooled to room temperature; 0.2 times volume of chloroform-n-butanol mixture (chloroform:n-butanol (V:V)=5:1) was added, and vigorously shaken for 30 min. Then centrifuge at 8000rpm for 10min, discard the protein layer and organic solution, reclaim the supernatant, repeat this process twice until the protein layer no longer appears, and obtain the crude polysaccharide extract after protein removal;

(7) Precipitation of polysaccharides with ethanol: add 4 times the volume of ethanol with a mass fraction of 95% to the extract solution of perennial perennial polysaccharides after protein removal, mix evenly, let stand at 4°C for 12 hours, and centrifuge to separate the perennial perennial polysaccharides collect sediment;

(8) Polysaccharide dialysis and drying:

①Dissolve the polysaccharide precipitate of perennial bacteria in pure water, put it into a dialysis bag with a molecular weight cut-off of 6000-8000Da and dialyze for 72 hours, and replace the deionized water every 8 hours;

② After dialysis, the polysaccharide extract of Perenia cerevisiae was concentrated by rotary evaporation to 1/3 of the volume of the extract of polysaccharide, and then vacuum freeze-dried to obtain the crude polysaccharide in the cells of Perennial cerevisiae;

Embodiment 2: Preparation of intracellular polysaccharide of perennial bacteria

(1) Perennial perennial ash is activated by bacterial classification, cultured on a slanted surface and submerged in liquid fermentation to obtain mycelium of perennial perennial ash. The specific methods are as follows:

①Cultivation of slant strains: After activation and cultivation of the slant parent species of Perennial spp., inoculate in sterilized slant solid medium, culture at 28°C for 8 days, and set aside;

② Liquid submerged fermentation culture: pick 4 to 6 soybean-sized bacteria blocks from the slant of the slanted strain of the white wax perennial bacteria, inoculate in 150mL sterile liquid fermentation medium, and shake at 30°C and 180rpm at a constant temperature in the dark Cultivate for 9 days to obtain the liquid fermentation product of Perennial aceriformis, which is separated by centrifugation and vacuum filtration, and dried at 60°C to obtain the mycelium of Perennial perennial asteraceae;

(2) Degreasing: After the mycelium is dried to a constant weight, it is pulverized by a pulverizer to pass through a 40-mesh sieve, and the mycelium powder above 40 mesh is collected. Put the mycelium powder in the Erlenmeyer flask, mix the mycelium powder and petroleum ether according to the solid-to-liquid ratio of 1:1 (V:Vg/mL), shake on the shaker for 48 hours, centrifuge to save the precipitate, and repeat this step once . The obtained precipitate was dried at 60°C and placed in a drying oven for later use.

(3) Hot water leaching: extract the mycelia of the perennial mycelia after degreasing and drying in step (2) with hot water, and collect the extract; wherein: the extraction temperature is 80°C, the extraction times are 2 times, and the extraction The time is 2h, the ratio of solid to liquid is 1:25(g:ml);

(4) The yield of total sugar in the extract was measured by the phenol-sulfuric acid method, and the yield of total sugar in the extract was 10.75%;

(5) Concentration: use a rotary evaporator to concentrate the extract collected in step (3) under reduced pressure in a constant temperature water bath at 60°C to 1/5 of the original volume to obtain a concentrated extract of the perennial bacteria;

(6) Trypsin and sevage method for protein removal and centrifugation: Weigh 2.0g of trypsin and dissolve it in 100mL of distilled water to obtain a trypsin solution; Add it to the extract concentrate of white wax perennial bacteria, and then put it in a 37°C water bath to preheat for 10 minutes, fully mix the enzyme solution and the two concentrates separately, and shake at 120rpm for 30 minutes, then put it in a 100°C water bath for 10 minutes to inactivate the enzyme , cooled to room temperature; 0.2 times volume of chloroform-n-butanol mixture (chloroform:n-butanol (V:V)=5:1) was added, and vigorously shaken for 30 min. Then centrifuge at 8000rpm for 10min, discard the protein layer and organic solution, reclaim the supernatant, repeat this process twice until the protein layer no longer appears, and obtain the crude polysaccharide extract after protein removal;

(7) Precipitation of polysaccharides with ethanol: add 4 times the volume of ethanol with a mass fraction of 95% to the extract solution of perennial perennial polysaccharides after protein removal, mix evenly, let stand at 4°C for 12 hours, and centrifuge to separate the perennial perennial polysaccharides collect sediment;

(8) Polysaccharide dialysis and drying:

①Dissolve the polysaccharide precipitate of perennial bacteria in pure water, put it into a dialysis bag with a molecular weight cut-off of 6000-8000Da and dialyze for 72 hours, and replace the deionized water every 8 hours;

② After the dialysis, the polysaccharide extract of Perennial cerevisiae was concentrated by rotary evaporation to 1/3 of the volume of the extract of polysaccharide, and then vacuum freeze-dried to obtain the intracellular crude polysaccharide of Perennial cerevisiae.

Embodiment 3: Preparation of intracellular polysaccharide of perennial bacteria

(1) Perennial perennial ash is activated by bacterial classification, cultured on a slanted surface and submerged in liquid fermentation to obtain mycelium of perennial perennial ash. The specific methods are as follows:

①Cultivation of slant strains: After activation and cultivation of the slant parent species of Perennial spp., inoculate in sterilized slant solid medium, culture at 28°C for 8 days, and set aside;

② Liquid submerged fermentation culture: pick 4 to 6 soybean-sized bacteria blocks from the slant of the slanted strain of the white wax perennial bacteria, inoculate in 150mL sterile liquid fermentation medium, and shake at 30°C and 180rpm at a constant temperature in the dark Cultivate for 9 days to obtain the liquid fermentation product of Perennial aceriformis, which is separated by centrifugation and vacuum filtration, and dried at 60°C to obtain the mycelium of Perennial perennial asteraceae;

(2) Degreasing: After the mycelium is dried to a constant weight, it is pulverized by a pulverizer to pass through a 40-mesh sieve, and the mycelium powder above 40 mesh is collected. Put the mycelium powder in the Erlenmeyer flask, mix the mycelium powder and petroleum ether according to the solid-to-liquid ratio of 1:1 (V:Vg/mL), shake on the shaker for 48 hours, centrifuge to save the precipitate, and repeat this step once . The obtained precipitate was dried at 60°C and placed in a drying oven for later use.

(3) Hot water leaching: extract the mycelia of the perennial mycelia after degreasing and drying in step (2) with hot water, and collect the extract; wherein: the extraction temperature is 80°C, the extraction times are 2 times, and the extraction Time 2h, the ratio of solid to liquid is 1:30(g:ml);

(4) The yield of total sugar in the extract was measured by the phenol sulfuric acid method, and the yield of total sugar in the extract was: 9.75%

(5) Concentration: use a rotary evaporator to concentrate the extract collected in step (3) under reduced pressure in a constant temperature water bath at 60°C to 1/5 of the original volume to obtain a concentrated extract of the perennial bacteria;

(6) Trypsin and sevage method for protein removal and centrifugation: Weigh 2.0g of trypsin and dissolve it in 100mL of distilled water to obtain a trypsin solution; Add it to the extract concentrate of white wax perennial bacteria, and then put it in a 37°C water bath to preheat for 10 minutes, fully mix the enzyme solution and the two concentrates separately, and shake at 120rpm for 30 minutes, then put it in a 100°C water bath for 10 minutes to inactivate the enzyme , cooled to room temperature; 0.2 times the volume of chloroform-butanol mixture (chloroform: n-butanol (V:V) = 5:1) was added, and vigorously shaken for 30 min. Then centrifuge at 8000rpm for 10min, discard the protein layer and organic solution, reclaim the supernatant, repeat this process twice until the protein layer no longer appears, and obtain the crude polysaccharide extract after protein removal;

(7) Precipitation of polysaccharides with ethanol: add 4 times the volume of ethanol with a mass fraction of 95% to the extract solution of perennial perennial polysaccharides after protein removal, mix evenly, let stand at 4°C for 12 hours, and centrifuge to separate the perennial perennial polysaccharides collect sediment;

(8) Polysaccharide dialysis and drying:

① Dissolve the polysaccharide precipitate of perennial bacteria in pure water, put it into a dialysis bag with a molecular weight cut-off of 6000-8000Da and dialyze for 72 hours, and replace the deionized water every 8 hours;

② After dialysis, the polysaccharide extract of Perenia cerevisiae was concentrated by rotary evaporation to 1/3 of the volume of the extract of polysaccharide, and then vacuum freeze-dried to obtain the crude polysaccharide in the cells of Perennial cerevisiae;

(9) Determination of crude polysaccharide content of perennial bacteria

The total sugar yield of the extract was determined by the phenol sulfuric acid method, and the intracellular crude polysaccharide content was 52.97%.

Embodiment 4: Preparation of exopolysaccharide and intracellular polysaccharide of perennial bacteria

(1) Perennial cerevisiae is activated by strains, cultured on a slant and submerged in liquid to obtain fermented liquid and mycelia of perennial perennials. The specific methods are as follows:

①Cultivation of slant strains: After activation and cultivation of the slant parent species of Perennial spp., inoculate in sterilized slant solid medium, culture at 28°C for 8 days, and set aside;

② Liquid submerged fermentation culture: pick 4 to 6 soybean-sized bacteria blocks from the slant of the slanted strain of the white wax perennial bacteria, inoculate in 150mL sterile liquid fermentation medium, and shake at 30°C and 180rpm at a constant temperature in the dark Cultivate for 9 days to obtain the liquid fermentation product of perennial bacteria, which is separated by centrifugation and vacuum filtration to obtain the fermentation broth and mycelium, the fermentation broth is concentrated, and the mycelium is dried at 60°C;

(2) Degreasing: After the mycelium is dried to a constant weight, it is pulverized by a pulverizer to pass through a 40-mesh sieve, and the mycelium powder above 40 mesh is collected. Put the mycelium powder in the Erlenmeyer flask, mix the mycelium powder and petroleum ether according to the solid-to-liquid ratio of 1:1 (V:Vg/mL), shake on the shaker for 48 hours, centrifuge to save the precipitate, and repeat this step once . The obtained precipitate was dried at 60°C and placed in a drying oven for later use.

(3) Hot water leaching: extract the mycelia of the perennial mycelia after degreasing and drying in step (2) with hot water, and collect the extract; wherein: the extraction temperature is 80°C, the extraction times are 2 times, and the extraction Time 2h, the ratio of solid to liquid is 1:30(g:ml);

(4) Concentration: use a rotary evaporator to concentrate the extract collected in step (3) under reduced pressure in a constant temperature water bath at 60°C to 1/5 of the original volume to obtain a concentrated extract of the perennial bacteria;

(5) Trypsin and sevage method for protein removal and centrifugation: Weigh 2.0g of trypsin and dissolve it in 100mL of distilled water to obtain a trypsin solution; then adjust the pH of 1000mL of perennial bacteria fermentation concentrate and extraction concentrate to 8.0 , add the trypsin solution to the fermented concentrated liquid of perennial bacteria and the concentrated liquid extracted, and then put it in a 37°C water bath to preheat for 10 minutes, then fully mix the enzyme liquid and the two concentrated liquids, shake at 120rpm for 30 minutes, and then release Inactivate the enzyme in a 100°C water bath for 10 minutes, cool to room temperature; add 0.2 times the volume of chloroform-butanol mixture (chloroform:n-butanol (V:V)=5:1), and shake vigorously for 30 minutes. Then centrifuge at 8000rpm for 10min, discard the protein layer and organic solution, and reclaim the supernatant. This process is repeated twice until the protein layer no longer appears, and the extracellular crude polysaccharide extract and intracellular crude polysaccharide extract after protein removal are obtained;

(6) Precipitation of polysaccharides with ethanol: add 4 times the volume of ethanol with a mass fraction of 95% to the extracted extracellular crude polysaccharides and intracellular crude polysaccharides of Perennialia cerevisiae after protein removal, mix well, and let stand at 4° C. for 12 hours. After the polysaccharide is precipitated, the precipitate is collected by centrifugation;

(7) Polysaccharide dialysis and drying:

① Dissolve the extracellular crude polysaccharide and intracellular crude polysaccharide precipitates of perennial bacteria in pure water, put them into a dialysis bag with a molecular weight cut-off of 6000-8000Da and dialyze for 72 hours, and replace the deionized water every 8 hours;

② After dialysis, the extracted solution of extracellular crude polysaccharide and intracellular crude polysaccharide of Perenia perennialis was concentrated by rotary evaporation to 1/3 of the volume of the polysaccharide extract, and then vacuum freeze-dried to obtain crude extracellular polysaccharide and crude intracellular polysaccharide of Perennium perennialis. polysaccharide;

③ The mass fractions of the total sugar content of the extracellular crude polysaccharide and intracellular crude polysaccharide of Perennial cerevisiae were 64.38% and 52.97%, respectively, as determined by the phenol sulfuric acid method;

(8) The DEAE-Sepharose method for purifying the polysaccharide of Perennial cerevisiae, the method is as follows:

①Pretreatment of DEAE-Sepharose

Soak 10g of DEAE-Sepharose in distilled water for 24 hours, remove the superfine part by flotation, and collect the precipitated part. Add 15 mL of 0.5 mol/L NaOH per gram and soak in NaOH solution for 30 min, stir intermittently several times, put into a chromatography column (1.5 cm × 20 cm), wash with distilled water until neutral, and pour it out. Then soak in 0.5mol/L HCL solution for 30min, stir intermittently several times, put into a chromatography column, wash with distilled water until neutral, and pour it out again. Next, continue to soak in 0.5mol/L NaOH solution for 30 minutes, pack the column, and finally wash it with distilled water until the pH is neutral, and set it aside.

② Polysaccharide purification

1) Column packing: After the chromatography column is cleaned, fix it vertically on the iron stand, add about 1/3 column volume of deionized water to the chromatography column, open the lower liquid outlet, and then slowly deionize the DEAE-Sepharose Pour it into the chromatographic column and allow it to settle naturally in the chromatographic column. The volume of the filler is about 2/3 of the column volume, and stop packing the column. The liquid inlet at the upper end of the chromatography column is connected with a constant flow pump.

2) Equilibrium: Use a constant flow pump to pump distilled water into the chromatography column, open the liquid outlet at the lower end of the chromatography column, and keep the flow rate of the equilibrium buffer at about 1mL/min. When the pH values of the equilibration buffers are the same, it proves that the column has reached equilibrium.

3) Loading and elution: Dissolve 0.1g of extracellular crude polysaccharide and intracellular crude polysaccharide of perennial bacteria in 10mL of distilled water, centrifuge at 5000rpm for 10min, take the supernatant, and slowly add it to the balanced chromatography column with a constant flow pump middle. First wash off the unadsorbed substances with buffer solution, and then elute them with a gradient of 0-0.5mol/L NaCl solution. Set the collection time of the partial collector to collect, collect 20 tubes for each gradient, and collect 10 tubes for each tube. minute. The total sugar content was determined by the phenol-sulfuric acid method, and the samples with a single absorption peak were combined and concentrated, then dialyzed with distilled water for 72 hours, and the distilled water was replaced every 8 hours. After dialysis, the dialysate is concentrated and vacuum freeze-dried to obtain purified polysaccharides EPPF and IPPF.

The obtained EPPF and IPPF are easily soluble in pure water, but insoluble in organic solvents such as absolute ethanol, dimethyl sulfoxide, anhydrous ether and ethyl acetate. The mass fractions of total sugar in the exopolysaccharide and intracellular polysaccharide of perennial sp. were determined by phenol-sulfuric acid method to be 92.30% and 87.24%, respectively.

Example 5: Evaluation of the effect of the polysaccharide of Perennial cerevisiae (prepared in Example 4) on regulating the intestinal flora

(1) Kunming mice: SPF level, male, body weight 16-20 g, age 28-30 days, provided by Guangdong Medical Experimental Animal Center. License number: SCXK (Guangdong) 2018-0002. Feed: common control feed (D12450B), high-fat feed (D12451), provided by Guangdong Medical Experimental Animal Center. License number: SCXK (Guangdong) 2018-0002.

(2) Adaptive feeding and grouping: After the mice were purchased, they were divided into cages (9 groups, 10 in each group) in the animal room, at room temperature 20-22 ° C, relative humidity 50-60% RH, free to eat and drink ( Sterilization of drinking water, sterilization of basal feed by ultraviolet irradiation) to adapt to feeding for 6 days. Afterwards, the mice were randomly divided into groups, including normal control group (Normal control, NC), T2DM model group (Diabetes model, DM), positive drug group (Positive control, PC, positive drug is metformin), treated with polysaccharide Diabetic group (divided into low, middle and high dose groups of exopolysaccharide and intracellular polysaccharide). The mice in the normal control group were fed the standard control diet D12450B (total energy 3.85kacl/g, fat energy supply ratio 10%); the mice in the other experimental groups were fed high-fat diet D12451 total energy 4.73kacl/g, fat energy supply ratio 45% %).

(3) Construction of the T2DM mouse model: the mice in the experimental group were fed with a high-fat diet for 3 weeks, and then intraperitoneally injected streptozotocin (STZ, 50 mg/kg dissolved in 0.1 mol of pH 4.5) on the first and third days, respectively. /L citric acid buffer solution), and the normal control group was injected with an equal dose of 0.1mol/L citric acid buffer solution at the same time. Fasting blood glucose was measured on the 7th day after injection, and mice with blood glucose levels>11.1mmol/L were defined as T2DM.

(4) Polysaccharide administration test: After successful modeling, the experimental group was given the test substance by oral gavage every day, and the volume of gavage was 1% (mL/g) BW/d. The amount of gavage was adjusted according to the weekly weight gain and loss, and the experimental period was 28 days. White wax perennial exopolysaccharide low-dose treatment group (50 mg/kg/d), white wax perennial exopolysaccharide medium dose treatment group (100 mg/kg/d) and white wax perennial high-dose exopolysaccharide treatment group (dose is 200mg/kg/d), white wax perennial bacteria intracellular polysaccharide low-dose treatment group (dose is 50mg/kg/d), white wax perennial bacteria intracellular polysaccharide middle dose treatment group (dose is 100mg/kg/d) and The high-dose treatment group (dose of 200 mg/kg/d) of intracellular polysaccharides of perennial bacteria, the positive drug group (30 mg/kg/d of metformin), and the normal control group were given equal volumes of sterile water. Record the food intake of the mice every day, and weigh the experimental mice in each group before gavage and on the 7th, 14th, 21st, and 28th day of the experiment, measure the fasting blood sugar, and record their body weight changes, blood sugar changes and death before and after the experiment Rate.

(5) After gavage on the last day, the mice were fasted for 12 hours without food and water, and the fresh feces samples of the mice were aseptically collected by the forced method, put in a sterilized 1.5mL centrifuge tube, and quickly frozen with liquid nitrogen, and the bacterial 16S rRNA High-throughput sequencing analysis of sequences, high-throughput sequencing analysis of fungal ITS sequences, and detection of short-chain fatty acids in mouse feces by gas chromatography-mass spectrometry.

(6) Experimental results

1) Effects of polysaccharides from Perennial cerevisiae on intestinal bacterial flora

Fig. 1 is the histogram of the species distribution of the polysaccharides of perennial bacteria on the intestinal tract of T2DM mice at different levels (NC: normal control group, the mice were fed the modeling control feed D12450B, and 1% (mL/g) BW was administered orally every day Sterile water; DM: Diabetes model group, mice were fed high-fat diet, fed with 1% (mL/g) BW sterile water every day; EPPF: middle-dose exopolysaccharide group of perennials perennials, mice were fed high-fat diet , 100mg/kg EPPF was administered intragastrically every day; IPPF: middle dose group of intracellular polysaccharide of white wax perennial bacteria, the mice were fed high-fat diet, and 100mg/kg IPPF was administered intragastrically every day. Compared with the normal control group, #: P<0.05, # #: P<0.01; compared with T2DM model group, *: P<0.05, **: P<0.01.)

As shown in Figure 1-A, the intestinal microbial flora of mice is at the phylum level, and the top ten phyla at the abundance level are: Proteobacteria, Firmicutes, and Bacteroidetes , Verrucomicrobia, Patescibacteria, Epsilonbacteraeota, Actinobacteria, Tenericutes, Acidobacteria, Deferribacteres. Compared with the normal control group, Proteobacteria, Epsilonbacteraeota and Molluscum significantly increased by 831.28%, 520.25% and 29.28% respectively; The phyla, acidobacteria and deferrobacteria decreased by 49.47%, 56.48%, 99.27%, 17.96%, 32.89%, 20.70% and 65.76%, respectively. Among them, the ratio of Firmicutes/Bacteroidetes was higher than that of normal group. After 28 days of exopolysaccharide and intracellular polysaccharide administration, compared with the DM model group, the intestinal proteobacteria decreased by 10.98% and 23.87%, respectively. The Bacteroides phylum in the intracellular polysaccharide group increased by 36.11%. Epsilonbacteraeota was first identified as a pathogenic bacterium known for its pathogenic genera Campylobacter, Helicobacter and, to a lesser extent, Vibrio. Both exopolysaccharides and intracellular polysaccharides of Perennialia cerevisiae could reverse the increase of relative abundance of Epsilonbacteraeota. In addition, a large number of studies have confirmed that the intestinal Proteobacteria can reflect dysbiosis or unstable intestinal microbial community structure, which is often due to the continuous increase in the abundance of Proteobacteria. Selecting the relative abundance of Proteobacteria, Firmicutes, Bacteroidetes and Epsilonbacteraeota for mapping can more intuitively see the changes of these bacterial phyla between different treatment groups. From Figure 1-B, it was found that the abundance of Proteobacteria in the normal control group was low, but it was significantly increased in the model group, and the abnormal increase was a manifestation of intestinal microecological imbalance. Both EPPF and IPPF can reduce the abnormal increase of Proteobacteria, but there are still significant differences compared with the normal control group.

As shown in Figure 1-C, the top ten of the mouse intestinal flora at the genus level are: Escherichia coli-Shigella (Escherichia-Shigella), Thyme (Muribaculaceae), Dubosiella, Faecalibacterium (Faecalibaculum), Lactobacillus, Bacteroides, Desulfovibrionaceae, Lachnospiraceae_NK4A136_group, Alloprevotella and Aerococcus . Select the genus Pseudoprevotella, Aerobacillus, Dubosiella and Lactobacillus to draw Figure 1-D separately, so that you can see the changes of these bacteria more intuitively. Compared with the normal control group, the genera of Escherichia coli and Aerobacter in the model group were significantly increased by 2888.99% and 1495.25%, while the genera of Dubosiella and Lactobacillus were significantly reduced by 73.72% and 87.10%. After IPPF administration, the relative abundances of Escherichia coli and Aerobacillus decreased by 32.06% and 75.35%, respectively, and the relative abundances of Dubosiella and Lactobacillus increased by 200.67% and 367.87%, respectively. In addition, the relative abundance of Thyme, Bacteroides, Desulfovibrio and Lachnospira genera was increased. After EPPF administration, the relative abundance of Dubosiella and Lactobacillus increased by 305.00% and 187.27%, respectively.

High-fat diet combined with low-dose STZ induced intestinal homeostasis in T2DM mice, marked by abnormally elevated Proteobacteria. However, EPPF and IPPF intervention can effectively reduce the relative abundance of harmful bacteria such as Proteobacteria and Bacillus-forming bacteria in the intestinal flora of mice at the level of phylum, class, order, family, genus and species, and increase the beneficial bacteria such as Lactobacillus and Bacteroides. The relative abundance of bacteria tends to the level of the normal control group and maintains the stability of the intestinal microbiota.

Alpha diversity (Alpha diversity) reflects the species abundance and diversity of a single sample through multiple metrics. The Simpson index is used to measure species diversity, which is affected by species abundance and species evenness in the sample community. The smaller the Simpson index value, the higher the species diversity of the species. It can be seen from Figure 2-A that compared with the normal control group, the Simpson index of the DM model group increased, while the Simpson index of both EPPF and IPPF intervention decreased, but it was still not statistically significant. The β-diversity analysis can reflect the differences in species diversity among samples, and the closer the samples on the coordinate map, the greater the similarity. Principal Component Analysis (PCA) can be seen in Figure 2-B. There is a certain distance between the intestinal flora of the model group and the flora of the normal control group, while the information on the flora of mice intervened by IPPF is mostly overlapped with that of the normal control group , which is relatively close to the model group. The EPPF-intervened mouse flora has a certain distance from the normal control group, but tends to approach the normal control group. It shows that the colony composition of the IPPF group is more similar to that of the normal group.

2) Effects of polysaccharides from Perennialia cerevisiae on intestinal fungal flora

The OTUs in the intestinal tract of the mice were counted and compared, and the Veen diagram of the sample OTUs is shown in Figure 3. The number of OTUs shared by the intestinal fungi of the four treatment groups was 675, and the number of OTUs shared by the exopolysaccharide group, the normal control group and the model group The number of OTUs was 277, while the number of OTUs shared by the intracellular polysaccharide group, the normal control group and the model group was only 28. It is worth noting that the normal control group contained a specific OTU, the exopolysaccharide intervention group contained 4 specific OTUs, and the intracellular polysaccharide group contained up to 70 specific OTUs. It shows that the intervention of perennial bacteria can change the OTU types of intestinal fungal flora.

According to the ITS sequence, the species distribution histogram of each sample at the taxonomic level was obtained. To explore the effect of polysaccharides from Perennial cerevisiae on the distribution of fungal species in the intestinal tract of T2DM mice, the results are shown in Figure 4. Fig. 4 is the species distribution diagram of the polysaccharides of perennial bacteria acting on the intestinal fungus of T2DM mice at the phylum level (note: NC: normal control group, mice were fed with modeling control feed D12450B, and 1% (mL/g )BW sterile water; DM: diabetic model group, mice were fed high-fat diet, fed with 1% (mL/g) BW sterile water every day; Fatty feed, 100mg/kg EPPF was intragastrically administered every day; IPPF: in the middle dose group of intracellular polysaccharide of perennial bacteria, the mice were fed high-fat diet, and 100mg/kg IPPF was intragastrically administered every day.). The top nine phylum relative abundances of the intestinal fungal flora in mice are: Ascomycota, Basidiomycota, Mortierellomycota, Chytridiomycota, Roche Rozellomycota, Olpidiomycota, Glomeromycota, Zoopagomycota and Mucoromycota.

Figure 5 shows the effect of polysaccharides from perennialia cerevisiae on specific fungal phyla in the intestinal tract of T2DM mice (Note: A is the relative abundance of Ascomycota, Basidiomycota and Mortierella in the intestinal microbiota of mice in each treatment group; B Be each treatment group mouse intestinal microorganism Basidiomycota and Ascomycota number ratio.Normal control group: mice are fed modeling contrast feed D12450B, and irritate 1% (mL/g) BW sterile water every day; Diabetes model group Mice are fed with high-fat diet, fed with 1% (mL/g) BW sterile water every day; Positive drug group (positive drug is metformin): mice are fed with high-fat diet, fed with 30mg/kg metformin every day; white wax for many years Bacterial extracellular polysaccharide medium dose group: mice were fed high-fat feed, and 100mg/kg EPPF was intragastrically administered every day; white wax perennial bacteria intracellular polysaccharide medium-dose group: mice were fed high-fat feed, and 100mg/kg IPPF was intragastrically administered every day. Compared with the control group, #: P<0.05, ##: P<0.01; compared with the T2DM model group, *: P<0.05, **: P<0.01.). It can be seen from Figure 5 that, compared with the normal control group, the relative abundances of Basidiomycota and Mortierella in the model group were significantly increased by 203.16% and 261.38% respectively (P<0.01), while Ascomycota were relatively abundant. The degree was extremely significantly reduced by 30.57% (P<0.01). After 28 days of intracellular polysaccharide administration, the relative abundance of Basidiomycota and Mortierella decreased by 14.65% and 61.2% respectively (P<0.05), and the relative abundance of Ascomycota increased by 17.92% (P<0.05 ). After exopolysaccharide administration, the relative abundance of Basidiomycota decreased by 15.69% (P>0.05), but the relative abundance of Mortierella could not be reduced, and there was no significant effect on the relative abundance of Ascomycota. Furthermore, it has been shown that one of the hallmarks of intestinal fungal dysbiosis is an elevated ratio of Basidiomycota to Ascomycota. Compared with the normal control group, the ratio of the relative abundance of Basidiomycota and Ascomycota in the model group was significantly increased (P<0.01), and after the intervention of exopolysaccharides and intracellular polysaccharides, the ratio decreased, and intracellular polysaccharides Group effect was significant (P<0.05).

3) Effects of polysaccharides from Perennialia cerevisiae on short-chain fatty acids in feces of T2DM mice

Short-chain fatty acids (SCFAs) refer to fatty acids with a carbon number of 6 or less, including three major SCFAs: acetate, propionate, butyrate, and the two less abundant valeric and caproic acids. Among them, acetate was the most abundant SCFA in the colon, accounting for more than half of the total SCFA detected in feces. Mainly produced by bacterial fermentation in the gut, SCFAs can actively participate in host energy regulation and play key regulatory roles in brain, muscle, airway, white adipose tissue, brown adipose tissue, and vascular physiology. A large amount of evidence shows that increasing the production of SCFA is beneficial to the host to exert anti-obesity and anti-diabetes effects.

According to the GC-MS results, the content of short-chain fatty acids in each group is shown in Figure 6. Figure 6 shows the effect of polysaccharides from perennial bacteria on short-chain fatty acids in feces of T2DM mice (Note: A is acetic acid; B is propionic acid; C is butyric acid; D is isobutyric acid; E is valeric acid; F is isopentyl acid; normal control group: the mice were fed with modeling contrast feed D12450B, and 1% (mL/g) BW sterile water was fed by stomach every day; the diabetes model group: the mice were fed with high-fat diet, and 1% (mL/g) was fed by stomach every day ) BW sterile water; Positive drug group (positive drug is metformin): the mice are fed with high-fat feed, and 30mg/kg metformin is fed by stomach every day; Stomach 100mg/kg EPPF; white wax perennial intracellular polysaccharide medium dose group: mice were fed high-fat diet, and 100mg/kg IPPF was intragastrically administered every day. Compared with the normal control group, #: P<0.05, ##: P<0.01 ; Compared with T2DM model group, *: P<0.05, **: P<0.01.). It can be seen from Figure 6 that compared with the normal control group, the content of acetic acid, propionic acid and butyric acid in the model group decreased significantly (P<0.01), and the content of isobutyric acid and valeric acid also decreased significantly (P<0.05). Compared with the model group, IPPF can significantly increase the content of acetic acid, valeric acid and isovaleric acid by 48.19%, 295.57% and 170.67% respectively (P<0.01). The content of butyric acid increased by 199.11% and 87.63% respectively (P<0.05). While EPPF can also increase the content of acetic acid and butyric acid, but the effect is not significant. It shows that polysaccharides from Perennialia cerevisiae can increase the content of some short-chain fatty acids in T2DM mice, and the effect of intracellular polysaccharides is obviously better than that of exopolysaccharides.

Example 6: The polysaccharide of Perennial cerevisiae (prepared in Example 4) is used for hypoglycemic research

(1) Kunming mice: SPF level, male, body weight 16-20 g, age 28-30 days, provided by Guangdong Medical Experimental Animal Center. License number: SCXK (Guangdong) 2018-0002. Feed: common control feed (D12450B), high-fat feed (D12451), provided by Guangdong Medical Experimental Animal Center. License number: SCXK (Guangdong) 2018-0002.

(2) Adaptive feeding and grouping: After the mice were purchased, they were divided into cages (9 groups, 10 in each group) in the animal room, at room temperature 20-22 ° C, relative humidity 50-60% RH, free to eat and drink ( Sterilization of drinking water, sterilization of basal feed by ultraviolet irradiation) to adapt to feeding for 6 days. Afterwards, the mice were randomly divided into groups, including normal control group (Normal control, NC), T2DM model group (Diabetes model, DM), positive drug group (Positive control, PC, positive drug is metformin), treated with polysaccharide Diabetic group (divided into low, middle and high dose groups of exopolysaccharide and intracellular polysaccharide). The mice in the normal control group were fed the standard control diet D12450B (total energy 3.85kacl/g, fat energy supply ratio 10%); the mice in the other experimental groups were fed high-fat diet D12451 total energy 4.73kacl/g, fat energy supply ratio 45% %).

(3) Construction of the T2DM mouse model: the mice in the experimental group were fed with a high-fat diet for 3 weeks, and then intraperitoneally injected streptozotocin (STZ, 50 mg/kg dissolved in 0.1 mol of pH 4.5) on the first and third days, respectively. /L citric acid buffer solution), and the normal control group was injected with an equal dose of 0.1mol/L citric acid buffer solution at the same time. Fasting blood glucose was measured on the 7th day after injection, and mice with blood glucose levels>11.1mmol/L were defined as T2DM.

(4) Polysaccharide administration test: After successful modeling, the experimental group was given the test substance by oral gavage every day, and the volume of gavage was 1% (mL/g) BW/d. The amount of gavage was adjusted according to the weekly weight gain and loss, and the experimental period was 28 days. White wax perennial exopolysaccharide low-dose treatment group (50 mg/kg/d), white wax perennial exopolysaccharide medium dose treatment group (100 mg/kg/d) and white wax perennial high-dose exopolysaccharide treatment group (dose is 200mg/kg/d), white wax perennial bacteria intracellular polysaccharide low-dose treatment group (dose is 50mg/kg/d), white wax perennial bacteria intracellular polysaccharide middle dose treatment group (dose is 100mg/kg/d) and The high-dose treatment group (dose of 200 mg/kg/d) of intracellular polysaccharides of perennial bacteria, the positive drug group (30 mg/kg/d of metformin), and the normal control group were given equal volumes of sterile water. Record the food intake of the mice every day, and weigh the experimental mice in each group before gavage and on the 7th, 14th, 21st, and 28th day of the experiment, measure the fasting blood sugar, and record their body weight changes, blood sugar changes and death before and after the experiment Rate.

(5) Fasting blood glucose test in mice

After the mice were fasted for 12 hours, the mice were fixed in the mouse holder, the tail of the mice was disinfected with alcohol cotton balls, and the tail veins of the mice were pricked with a disposable blood collection needle, and measured with a blood glucose meter after bleeding fasting blood sugar level.

(6) Oral glucose tolerance test

On the 28th day, the fasting venous blood of the mice was taken to measure the fasting blood glucose, and 2 g/(kg·bw) of glucose was taken orally, and then the blood glucose values of 30 min, 60 min, and 120 min were measured respectively. Calculate the area under the curve (Area Under the Curve, AUC) according to the formula.

AUC＝0.5×(G0 h+G0.5 h)×0.5+0.5×(G2 h+G0.5 h)×1.5

(7) After intragastric administration on the last day, the mice were fasted without water for 12 hours. After the mice were anesthetized with 4% chloral hydrate, the blood was collected from the eyeballs, and the serum was separated to detect the expression levels of blood glucose-related indicators, including mouse insulin. (INS), glycated hemoglobin (HbA1c). The homeostatic model assessment index HOMA-IR and HOMA-β were used to evaluate insulin resistance (IR) and β-cell function, and the homeostasis assessment index was calculated using the following formula:

HOMA-IR＝[fasting insulin (mU/L)×fasting blood glucose (mmol/L)]/22.5

HOMA-β(%)=[20×fasting insulin (mU/L)]/[fasting blood glucose (mmol/L)-3.5]×100%

After the blood was collected, the mice were dissected quickly, and their livers were collected, and the expressions of short-chain fatty acid receptors GPR41 and GPR43 were determined by ELISA.

(8) Experimental results

1) Analysis of the hypoglycemic effect of polysaccharides from Perennial cerevisiae

High-fat diet combined with low-dose STZ induced hyperglycemia mice, and then gavaged with different concentrations of exopolysaccharides and intracellular polysaccharides of perennial bacteria for 28 days. The fasting blood glucose of mice was measured, and the results showed that the polysaccharide of perennial bacteria had a significant effect on the fasting blood glucose of mice. Figure 7 shows the effect of polysaccharides from perennial bacteria on the hypoglycemic indicators of T2DM mice (Note: A is the fasting blood glucose value of mice in each treatment group on days 0, 7, 14, 21, and 28; B is the mice in each treatment group C is the blood glucose AUC value; D is the glycosylated hemoglobin level of mice in each treatment group. From left to right at each time point, the groups are: NC: normal control group, mice fed with modeling control feed D12450B , gavage 1% (mL/g) BW sterile water every day; DM: diabetes model group, mice were fed high-fat diet, and gavage 1% (mL/g) BW sterile water every day; PC: positive drug group ( Positive drug is metformin), the mice were fed with high-fat feed, and 30mg/kg metformin was administered by intragastric administration every day; EPPF: low, medium and high dose groups of perennial sp. , 100mg/kg, 200mg/kg of EPPF; IPPF: low-, medium-, and high-dose groups of intracellular polysaccharides of Perennial cerevisiae, mice were fed high-fat feed, and 50mg/kg, 100mg/kg, and 200mg/kg of IPPF were administered intragastrically every day. Compared with the normal control group, #: P<0.05, ##: P<0.01; compared with the T2DM model group, *: P<0.05, **: P<0.01.) It can be seen from Figure 7-A , the low, medium and high doses of exopolysaccharides of Perennial cerevisiae and the middle dose of intracellular polysaccharides can significantly reduce the fasting blood glucose of T2DM mice. Blood sugar decreased by 19.59%, 15.76%, 30.94% and 24.93% respectively. Among them, the high-dose treatment group with exopolysaccharide of perennial cerevisiae had the best effect. The glucose tolerance response of the mice after 4 weeks of treatment was studied by glucose tolerance experiment. The results are shown in Figure 7-B. The results showed that after oral administration of the glucose solution, the blood glucose of the mice increased significantly at 30 minutes, slightly decreased at 90 minutes, and basically tended to normal at 120 minutes. Among them, the blood glucose values of mice in the model group were significantly higher than those in the blank control group before oral administration of glucose and 30 minutes, 90 minutes and 120 minutes after oral administration. And after oral glucose 120min, the blood glucose value of the mice with low, middle and high doses of exopolysaccharide groups and middle doses of intracellular polysaccharides decreased by 36.05%, 30.38%, 47.95% and 30.59% respectively compared with the model group ( P<0.01), and they were all close to the normal control group, indicating that the mice in the above polysaccharide treatment group had better glucose tolerance. In addition, combined with the AUC of the area under the blood glucose line (Figure 7-C), it can be seen that after 2 hours of oral administration of glucose, the blood glucose AUC of the mice in the low-, medium-, and high-dose groups of exopolysaccharide and the middle-dose group of intracellular polysaccharide were all significantly Compared with the model group (P<0.05), blood glucose AUC decreased by 26.02% (P<0.01), 22.79% (P<0.05), 35.19% (P<0.01) and 27.55% (P<0.01 ). And compared with the positive drug group, there was no significant difference, which indicated that the effects of the three doses of exopolysaccharide and the medium dose of intracellular polysaccharide of perennial bacteria were close to those of metformin. From the level of glycosylated hemoglobin (Figure 7-D), it can be seen that the treatment groups with low and medium doses of exopolysaccharide and low dose of intracellular polysaccharide can significantly reduce the level of HbA1c in mouse serum, compared with the model group Respectively decreased by 10.90% and 8.48%. However, there was no significant difference in the level of HbA1c between the model group, the normal control group and the positive drug group.

2) Analysis of insulin function and islet β-cell function in the serum of T2DM mice by the polysaccharide of perennial bacteria

Figure 8 is the effect of polysaccharides from perennial bacteria on T2DM mice insulin function and islet β cell function (Note: A is the insulin level in the serum of mice in each treatment group; B is the HOMA-IR value of mice in each treatment group; C Be the HOMA-β value of each treatment group mouse.Groups: NC: normal control group, mice are fed modeling contrast feedstuff D12450B, irritate 1% (mL/g) BW sterile water every day; DM: diabetes model group , the mice were fed with a high-fat feed, and 1% (mL/g) BW sterile water was administrated every day; PC: the positive drug group (positive drug was metformin), the mice were fed with a high-fat feed, and 30mg/kg metformin was administrated every day; EPPF: low, medium, and high dose groups of perennial perennial bacteria exopolysaccharide, the mice were fed high-fat diet, and 50mg/kg, 100mg/kg, 200mg/kg of EPPF were gavaged every day; IPPF: low, In the middle and high dose groups, the mice were fed with high-fat feed, and 50mg/kg, 100mg/kg, and 200mg/kg IPPF were intragastrically administered every day. Compared with the normal control group, #: P<0.05, ##: P<0.01; Compared with the T2DM model group, *: P<0.05, **: P<0.01.) As can be seen from Figure 8-A, compared with the normal control group, the insulin level of the model group was significantly increased by 32.03% (P <0.01), indicating low insulin sensitivity and poor utilization in the model group. Compared with the model group, the serum insulin content of the mice in the metformin treatment group was reduced by 13.27% (P<0.05), the exopolysaccharide medium-dose group was reduced by 14.90% (P<0.05), and the exopolysaccharide high-dose group was reduced 13.84% (P<0.05), L-IPPF decreased by 19.91% (P<0.05). The low-dose exopolysaccharide group can also reduce the insulin content, but there is no significant difference with the model group. It can be seen from Fig. 8-B that compared with the normal control group, the HOMA-IR value of the model group was significantly increased by 101.23% (P<0.01). Compared with the model control group, the positive drug group, the low, medium and high dosage groups of exopolysaccharide and the middle dosage group of intracellular polysaccharide could significantly reduce the value of HOMA-IR by 37.75% respectively (P<0.01 ), 40.05% (P<0.01), 40.74% (P<0.01), 40.05% (P<0.01) and 34.46% (P<0.01), and there was no significant difference with the normal control group. It shows that these treatment groups can significantly reduce the insulin resistance of T2DM mice, which is consistent with the results of hypoglycemia. In terms of improving islet β-cell function, as shown in Figure 8-C, compared with the model group, the low-dose and high-dose groups of exopolysaccharides and the middle-dose group of intracellular polysaccharides could significantly increase the value of HOMA-β, respectively The increase was 97.7% (P<0.01), 62.63% (P<0.05) and 90.35% (P<0.01), and there was no significant difference with the normal control group. In addition, the positive drug group and the medium dose group of exopolysaccharide can also increase the value of HOMA-β, but not significantly. It shows that polysaccharides from perennialia can improve blood sugar level by reducing insulin content, improving insulin resistance and improving islet β-cell function.

3) Analysis of the effect of polysaccharides from Perennialia cerevisiae on the expression of short-chain fatty acid receptors GPR41 and GPR43

Short-chain fatty acid receptors GPR41 and GPR43 are expressed in the intestine, adipose tissue, liver, pancreas and many immune cell subtypes, and both can play important physiological roles in the body by mediating short-chain fatty acids. GPR41 is mainly activated by short-chain fatty acids such as butyrate and pentanoate, while GPR43 has a higher affinity for acetate and propionate. In the cecum and large intestine, 95% of the short-chain fatty acids produced are rapidly absorbed by colonic cells, and the absorbed SCFAs stimulate the corresponding receptors through enterohepatic circulation to express in intestinal cells, adipose tissue, liver and other cells.

The analysis of the effect of polysaccharides on the regulation of the expression of short-chain fatty acid receptors GPR41 and GPR43 by the polysaccharide of Perennial cerevisiae is shown in Figure 9. It can be seen from Figure 9-A that compared with the DM model group, the positive drug metformin significantly increased the expression of GPR43 by 8.91% (P <0.05). The medium dose and high dose of exopolysaccharide significantly increased the expression of GPR43 by 29.21% (P<0.01) and 38.26% (P<0.01). The low, medium and high doses of intracellular polysaccharides significantly increased the expression of GPR43 by 31.72% (P<0.01), 34.19% (P<0.01) and 34.97% (P<0.01), respectively. It can be seen from Fig. 9-B that, compared with the normal control group, the expression level of GPR41 in the model group was significantly decreased by 12.28% (P<0.01). Compared with the model group, the positive drug metformin significantly increased the expression of GPR41 in the liver by 10.58% (P<0.01). Also compared with the model group, the low-, medium-, and high-dose groups of exopolysaccharides significantly increased the expression of GPR41 by 10.19% (P<0.01), 24.02% (P<0.01) and 23.36% (P<0.01 ). Corresponding doses of intracellular polysaccharides significantly up-regulated the expression of GPR41 by 20.17% (P<0.01), 21.38% (P<0.01) and 22.26% (P<0.01), and there was a dose-effect relationship.

Combining the results of Examples 5 and 6, it can be seen that the polysaccharides of Perennial cerevisiae can be used as a prebiotic to act on the intestinal flora to increase the proportion of beneficial flora; in turn, the beneficial flora regulates the metabolism of the body with the help of their metabolites SCFAs. Combined with the exploration of the hypoglycemic effect of perennial polysaccharides, we found that polysaccharides from perennial bacteria mediated the expression of proteins related to carbohydrate metabolism by regulating the composition and diversity of intestinal microbial flora and the content of short-chain fatty acids in intestinal derivatives, which together played a role to hypoglycemic effect. This study verified the hypoglycemic activity of perennial polysaccharides, and explored its relationship with hypoglycemia as a prebiotic from the perspective of regulating intestinal flora. For the first time, the present invention verifies the hypoglycemic activity of perennial polysaccharides from the perspective of intestinal flora, provides new ideas for the research on the hypoglycemic mechanism of perennial polysaccharides, and also regulates intestinal disorders for the preparation of polysaccharides from perennial perennials and other medicinal fungi Drugs and clinical treatment of diabetes open up new areas.

Claims (10)

1. A preparation method of extracellular polysaccharide and intracellular polysaccharide of Fraxinus chinensis planch is characterized by comprising the following steps:

(1) Inoculating the wax perennial strain into a liquid culture medium for fermentation culture, performing solid-liquid separation to obtain wax perennial strain fermentation liquor and mycelium, and concentrating the wax perennial strain fermentation liquor for later use;

(2) Drying, crushing and sieving the perennial wax mycelia obtained in the step (1) to obtain mycelium powder; then adding petroleum ether for degreasing treatment and drying to obtain cervus chinensis perennial mycelium dry powder;

(3) Adding the mycelium dry powder of the cerumen et cacumen Myrianthi obtained in the step (2) into water, and leaching with hot water to obtain a cerumen et cacumen Myrianthi leaching solution; then concentrating the extract to obtain a concentrated solution of the perennial white wax fungus extract;

(4) Removing protein of the perennial white wax fungus fermentation liquor obtained in the step (1) and the perennial white wax fungus leaching concentrated liquor obtained in the step (3) by adopting trypsin combined Sevage method respectively to obtain an extracellular polysaccharide extracting solution and an intracellular crude polysaccharide extracting solution after protein removal in sequence;

(5) Respectively adding the extracellular crude polysaccharide extracting solution and the intracellular crude polysaccharide extracting solution obtained in the step (4) into an ethanol solution, standing, centrifuging, collecting precipitates, and respectively adding water for redissolving to obtain an extracellular crude polysaccharide solution and an intracellular crude polysaccharide solution; dialyzing, concentrating, and freeze drying to obtain crude extracellular polysaccharide extract and crude intracellular polysaccharide extract of Cera chinensis for many years;

(6) Respectively carrying out chromatography purification on the crude extracellular polysaccharide extract and the crude intracellular polysaccharide extract of the perennial Fraxinus obtained in the step (5), then dialyzing, and freeze-drying to obtain extracellular polysaccharide and intracellular polysaccharide of the perennial Fraxinus;

the dialysis in the step (5) is carried out by adopting a dialysis bag with the molecular weight cutoff of 6000 to 8000 Da;

the dialysis in the step (6) is carried out by adopting a dialysis bag with the molecular weight cutoff of 6000 to 8000Da

The chromatographic purification in the step (6) is realized by the following steps:

s1, column assembling: adding water into the chromatographic column, opening a liquid outlet at the lower end, and pouring DEAE-Sepharose Fast Flow filler into the chromatographic column to naturally settle in the chromatographic column;

s2, balancing: pumping the NaCl buffer solution into the chromatographic column by using a constant flow pump, and opening a liquid outlet at the lower end until the pH value of the effluent liquid is the same as that of the NaCl buffer solution;

s3, loading and eluting:

s31, taking extracellular crude polysaccharide and intracellular crude polysaccharide water solution, centrifuging, taking supernatant, and slowly adding the supernatant into a balanced chromatographic column by using a constant flow pump; washing off unadsorbed substances by using buffer solution, performing gradient elution by using NaCl solution respectively, setting the collection time of a part of collectors, collecting, measuring the total sugar content, combining single absorption peak samples, concentrating, dialyzing, and performing concentration and vacuum freeze drying treatment on dialysate to obtain purified polysaccharides EPPF and IPPF.

2. The method according to claim 1, wherein the protein removal by trypsin and Sevage in step (4) is performed by:

(I) Respectively adjusting the pH of the perennial white cereus leaching concentrated solution obtained in the step (3) and the perennial white cereus fermentation concentrated solution obtained in the step (1) to 8.0, then respectively adding trypsin solution, fully oscillating, then carrying out water bath enzyme deactivation, and cooling to room temperature to obtain the zymolytic perennial white cereus fermentation liquor and intracellular leaching liquor;

(II) adding the zymolytic white wax perennial bacterium fermentation liquor and the intracellular leaching liquor obtained in the step (I) into a mixed liquor of chloroform and n-butyl alcohol, violently oscillating for 30-40 min, then centrifuging, removing a protein layer and an organic solution, and collecting supernatant, namely the intracellular crude polysaccharide extracting solution.

3. The method of claim 2, wherein: the concentration of the trypsin solution in the step (I) is 2-5% of the mass-volume ratio; the volume ratio of the trypsin solution to the concentrated fermentation liquor and leaching liquor of the cercospora alba in the step (I) is 1:15 to 30; the volume ratio of chloroform to n-butanol in the chloroform-n-butanol mixed solution in the step (II) is 3 to 7; the volume ratio of the chloroform-n-butanol mixed liquor in the step (II) to the zymolyzed white wax perennial fungus fermentation liquor and the intracellular leaching liquor is 1:0.1 to 0.25.

4. The method of claim 1, wherein: the volume ratio of the ethanol solution to the protein-removed yew paraffin fungus fermentation concentrated solution and the leaching liquor in the step (5) is 2 to 5:1; the standing condition in the step (5) is as follows: standing for 24-48 h at 0-4 ℃; the centrifugation conditions in the step (5) are as follows: centrifuging at 5000 to 10000rpm for 5-15 min; the concentration in the step (5) is 1/5 to 1/2 of the volume of the extracellular polysaccharide extracting solution and the intracellular polysaccharide extracting solution of the perennial Fraxinus.

5. The method of claim 1, wherein: 1/10 to 1/4 of the original volume of the concentrated cerinus chinensis fermentation liquor in the step (1); the drying temperature in the step (2) is 50-70 ℃; sieving in the step (2) is to pass through a sieve of 40 to 60 meshes; the solid-to-liquid ratio of the mycelium powder to the petroleum ether in the step (2) is 0.5 to 2 (g/mL); the time of degreasing in the step (2) is 2 to 4 days, and the times of degreasing are 1 to 3;

the feed-liquid ratio of the cerinus chinensis perennial fungus mycelium dry powder to water in the step (3) is 1:20 to 40 (g/mL); the extraction temperature in the step (3) is 50-90 ℃, and the extraction time is 1.5-3.5 h; the extraction times are 1 to 3;

the concentration in the step (3) is performed by adopting a reduced pressure concentration mode; the concentration temperature is 50 to 70 ℃;

the concentration in the step (3) is 1/10 to 1/4 of the volume of the cerifera chinensis leaching liquor.

6. The method of claim 1, wherein: the fermentation culture in the step (1) is realized by the following steps:

(1) activating and culturing the perennial cerealose slant mother strain, transferring the strain into a slant solid culture medium, and culturing at 20-37 ℃ for 8-15d to obtain the perennial cerealose slant strain;

(2) selecting 4-6 bacterial blocks from the perennial Ceratophyllum brevicornum slant strains obtained in the step (1), inoculating the bacterial blocks into 150-200 mL of liquid fermentation medium, standing at room temperature, and carrying out light-shielding constant-temperature oscillation culture after the wounds of the bacterial blocks are healed to obtain fermentation liquor and mycelia.

7. A polysaccharide prepared by the method of any one of claims 1 to 6.

8. Use of extracellular and intracellular polysaccharides of Cera chinensis as claimed in claim 7 for the preparation of a product effective in promoting proliferation of intestinal probiotic bacteria, promoting production of short chain fatty acids in intestinal tract, regulating intestinal flora structure, and/or lowering blood glucose.

9. Use according to claim 8, characterized in that: the dosage of the extracellular polysaccharide and the intracellular polysaccharide of the perennial Fraxinus chinensis is 50 to 200mg/kg/d;

the intestinal probiotics comprise bacteroides and lactobacillus; the short-chain fatty acid is at least one of acetic acid, propionic acid, butyric acid and valeric acid; the intestinal flora is intestinal flora and/or intestinal flora.

10. Use according to claim 9, characterized in that the intestinal bacteria comprise: proteobacteria (Proteobacteria), firmicutes (Firmicutes), bacteroidetes (Bacteroidetes), verrucomicrobia (Verrucomicrobia), proteobacteria, episilonobacter, actinomyces (Actinobacteria), mollicutes (Tenericutes), acidobacteria (Acidobacteria), desferribacteroidetes (deferobacteres);

the intestinal fungi include: ascomycota (Ascomycota), basidiomycota (Basidiomycota), mortierella (Mortierella), chytridiomycota (Chytridiomycota), rozelomycota (Rozelomycota), olpidimycota (Olpidimycota), gliocladiomycota (Glomeromycota), thielavia (Zoopagomycota) and Mucor (Mucor).

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