Innopolysaccharide from inonotus obliquus and preparation method and application thereof

Abstract

The application discloses a dry powder of inonotus obliquus polysaccharide and a preparation method and application thereof, and belongs to the technical field of biotechnology. In the application, fruiting bodies of the inonotus obliquus are frozen and thawed to obtain broken-wall inonotus obliquus powder; the broken-wall inonotus obliquus powder is subjected to enzymolysis with water, papain and cellulase to obtain an enzymolysis liquid; a mixed bacteria liquid of the inonotus obliquus and lactobacillus plantarum is inoculated into the enzymolysis liquid to ferment to obtain a fermentation liquid, and fermentation supernatant is collected by centrifugation; polyethylene glycol 6000 and potassium dihydrogen phosphate are added, and after static stratification, the upper polysaccharide solution is collected, then decolorization, ultrafiltration and collection of the cut-off liquid are carried out; concentration and drying are carried out to obtain the dry powder of the inonotus obliquus polysaccharide. The method can obtain the dry powder of the inonotus obliquus polysaccharide with high purity, and the dry powder of the inonotus obliquus polysaccharide has the effects of significantly reducing blood sugar and regulating intestinal flora to treat constipation.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a dried powder of Inonotus obliquus polysaccharide, its preparation method and application. Background Technology

[0002] Inonotus obliquus is a medicinal fungus that grows on birch, poplar and other trees in cold regions. Its main active ingredients are polysaccharides, betulin, triterpenoids and other compounds. Among them, polysaccharides are one of the core active ingredients and have a variety of pharmacological activities such as enhancing immunity, anti-tumor and antioxidant effects.

[0003] Currently, the main methods for preparing Inonotus obliquus polysaccharides include hot water extraction, microwave extraction, ultrasonic extraction, and enzymatic hydrolysis. However, existing technologies have several shortcomings: First, the cell wall disruption effect is poor. The cell walls of Inonotus obliquus are hard, and traditional pulverization or single freeze-thaw disruption cannot fully release the polysaccharides within the cells, resulting in a low extraction rate. Second, active ingredients are easily destroyed during extraction. High temperatures and strong solvents can cause polysaccharide structure degradation, reducing its biological activity. Third, the purification process is complex. Traditional methods such as the Sevage method for protein removal and activated carbon decolorization are either cumbersome and inefficient, or they may introduce toxic and harmful substances, affecting product safety. Fourth, the drying methods are unreasonable. Single freeze-drying is costly and inefficient, while single spray drying easily leads to the loss of polysaccharide activity, and the resulting dry powder has poor solubility. Fifth, the existing preparation methods are diverse, resulting in polysaccharides with inconsistent activity. Therefore, developing a method for preparing Inonotus obliquus polysaccharide dry powder with high extraction rate, good activity retention, and significant effects on lowering blood sugar and regulating intestinal flora is key to overcoming the shortcomings of existing technologies and promoting the industrial application of Inonotus obliquus polysaccharides. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a dried powder of Inonotus obliquus polysaccharide, its preparation method and application, wherein the method prepares Inonotus obliquus polysaccharide with high purity and has significant effects on lowering blood sugar and regulating intestinal flora.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing Inonotus obliquus polysaccharide dry powder, comprising the following steps: freezing and thawing Inonotus obliquus fruiting bodies to obtain Inonotus obliquus powder with broken cell walls; enzymatically hydrolyzing the broken cell wall Inonotus obliquus powder with water, papain, and cellulase to obtain an enzymatic hydrolysate; inoculating the enzymatic hydrolysate with a mixed bacterial culture of Inonotus obliquus and Lactobacillus plantarum to obtain a fermentation broth, centrifuging and collecting the fermentation supernatant; adding polyethylene glycol 6000 and potassium dihydrogen phosphate, allowing it to stand and separate into layers, collecting the upper polysaccharide solution, then decolorizing and ultrafiltering, collecting the retentate; concentrating and drying to obtain Inonotus obliquus polysaccharide dry powder.

[0006] Preferably, the freezing temperature is -50~-70℃ and the time is 30-50 min, and the thawing temperature is 27-32℃ and the time is 0.5-1.5 h.

[0007] Preferably, the mass ratio of the cell-wall broken Inonotus obliquus powder to water is 1:15-22; after mixing the cell-wall broken Inonotus obliquus with water, the pH of the system is adjusted to 5.3-5.8.

[0008] Preferably, the amount of papain added is 0.1-0.3 w / w of the cellulase-infused birch bark powder; the amount of cellulase added is 0.2-0.4 w / w of the cellulase-infused birch bark powder; and the enzymatic hydrolysis temperature is 52-58℃, the rotation speed is 150-200 r / min, and the time is 1.5-3.5 h.

[0009] Preferably, the bacterial concentrations of both *Inonotus obliquus* and *Lactobacillus plantarum* are 0.5-1.5 × 10⁻⁶. 9 CFU / mL; The volume ratio of the Inonotus obliquus and Lactobacillus plantarum bacterial solutions in the mixed bacterial solution is (3-1):1.

[0010] Preferably, the inoculum size of the mixed culture of Inonotus obliquus and Lactobacillus plantarum is 1-5 w / w of the enzymatic hydrolysate; the fermentation conditions include: fermentation at 28-38℃ for 15-32 h, with stirring 1-3 times every 6-10 h during the fermentation process, each stirring lasting 5-15 min.

[0011] Preferably, the final mass fraction of the polyethylene glycol 6000 is 15-25%, and the final mass fraction of the potassium dihydrogen phosphate is 5-12%; the settling temperature is room temperature and the settling time is 5-12 minutes.

[0012] Preferably, the pore size of the ultrafiltration is 1500-2500 Da.

[0013] The present invention provides a dry powder of Inonotus obliquus polysaccharide obtained by the preparation method described above.

[0014] This invention provides the application of the dried powder of Inonotus obliquus polysaccharide obtained by the above preparation method in the preparation of drugs for lowering blood sugar and regulating intestinal flora.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention abandons the cumbersome steps of traditional methods such as cell wall disruption, defatting, and single water extraction, and introduces freezing cell wall disruption, enzymatic hydrolysis, and mixed bacterial fermentation technology to achieve a dual breakthrough in process simplification and efficacy enhancement. The co-fermentation of compound bacteria can directionally synthesize polysaccharide derivatives, which form a synergistic effect with the native polysaccharides of Inonotus obliquus, significantly enhancing the dual effects of lowering blood sugar and regulating intestinal flora, solving the problems of single efficacy and poor synergy in traditional products. The method of this invention can retain fermentation-derived active ingredients, which is significantly different from existing technologies, and the process and efficacy are both outstandingly innovative.

[0016] This invention, through animal model experiments on blood glucose reduction and gut microbiota regulation, demonstrates that the polysaccharide powder has significant dual effects of lowering blood glucose and regulating gut microbiota, and clarifies the synergistic mechanism of fermentation-enhanced dual effects, providing a scientific and reliable experimental basis for its application in the fields of blood glucose reduction and gut health. Detailed Implementation

[0017] This invention provides a method for preparing Inonotus obliquus polysaccharide dry powder, comprising the following steps: freezing and thawing Inonotus obliquus fruiting bodies to obtain broken-cell Inonotus obliquus powder; enzymatically hydrolyzing the broken-cell Inonotus obliquus powder with water, papain, and cellulase to obtain an enzymatic hydrolysate; inoculating the enzymatic hydrolysate with a mixed culture of Inonotus obliquus and Lactobacillus plantarum to obtain a fermentation broth, centrifuging and collecting the fermentation supernatant; adding polyethylene glycol 6000 and potassium dihydrogen phosphate, allowing it to stand and separate into layers, collecting the upper polysaccharide solution, then decolorizing and ultrafiltering, collecting the retentate; concentrating and drying to obtain Inonotus obliquus polysaccharide dry powder. Unless otherwise specified, the components and raw materials used in the above preparation method of this invention are obtained through commercially known channels in the art.

[0018] In this invention, the freezing temperature is -50 to -70°C for 30-50 minutes, and the thawing temperature is 27-32°C for 0.5-1.5 hours. Preferably, the freezing temperature is -55 to -65°C for 33-45 minutes, and the thawing temperature is 28-31°C for 0.8-1.3 hours. This invention achieves dual physical cell wall disruption through low-temperature ice crystal formation that ruptures the cell wall, followed by medium-temperature melting to allow cell swelling. This fully releases intracellular polysaccharides without damaging their activity. Simultaneously, it creates favorable conditions for subsequent enzymatic hydrolysis and fermentation, significantly improving polysaccharide extraction efficiency and the bioactivity of the final product.

[0019] In this invention, the mass ratio of the broken-cell wall Inonotus obliquus powder to water is 1:15-22, preferably 1:18-20; after mixing the broken-cell wall Inonotus obliquus with water, the pH of the system is adjusted to 5.3-5.8, preferably 5.4-5.7, and more preferably 5.5. The amount of papain added is 0.1-0.3 w / w% of the broken-cell wall Inonotus obliquus powder, preferably 0.2 w / w; the amount of cellulase added is 0.2-0.4 w / w% of the broken-cell wall Inonotus obliquus powder, preferably 0.3 w / w; the enzymatic hydrolysis temperature is 52-58℃, the rotation speed is 150-200 r / min, and the time is 1.5-3.5 h, preferably 53-57℃, the rotation speed is 160-190 r / min, and the time is 1.8-3.2 h. This invention utilizes a compound enzymatic hydrolysis method that synergistically degrades cell walls and removes protein impurities, allowing for the full dissolution of *Inonotus obliquus* polysaccharides and improving subsequent purification efficiency. The mild conditions of enzymatic hydrolysis preserve polysaccharide activity and optimize the system state, providing a high-quality substrate for subsequent compound bacterial fermentation. The papain and cellulase purchased for this invention have an activity specification of 50,000-150,000 U / g, preferably 80,000-120,000 U / g, and more preferably 100,000 U / g.

[0020] In this invention, the bacterial concentrations of both *Inonotus obliquus* and *Lactobacillus plantarum* are 0.5-1.5 × 10⁻⁶. 9 CFU / mL, preferably 0.8-1.3×10 9 CFU / mL, further preferably 1.0 × 10⁻⁶ 9 CFU / mL; the volume ratio of Inonotus obliquus to Lactobacillus plantarum in the mixed bacterial culture of Inonotus obliquus and Lactobacillus plantarum is (3-1):1, preferably (2.5-1.5):1, and more preferably 2:1. The inoculum amount of the mixed bacterial culture of Inonotus obliquus and Lactobacillus plantarum in this invention is 1-5 w / w% of the enzymatic hydrolysate, preferably 2-4 w / w%, and more preferably 3 w / w%; the fermentation conditions include: fermentation at 28-38℃ for 15-32 h, with stirring 1-3 times every 6-10 h during fermentation, each stirring for 5-15 min; preferably including: fermentation at 29-37℃ for 16-31 h, with stirring once every 7-9 h during fermentation, each stirring for 8-12 min. This invention utilizes the fermentation ability of the aforementioned compound bacteria to directionally synthesize polysaccharide derivatives, which synergistically enhance the dual effects of lowering blood sugar and regulating intestinal flora with native polysaccharides; it can also optimize the form of polysaccharides, improve their bioavailability, and make the system more suitable for subsequent purification, ensuring the purity and activity of the finished product.

[0021] In this invention, the final mass fraction of polyethylene glycol 6000 is 15-25%, and the final mass fraction of potassium dihydrogen phosphate is 5-12%, preferably 18-22% and 7-11%, more preferably 20% and 10%, respectively; the settling temperature is room temperature, and the settling time is 5-12 minutes. The pore size of the ultrafiltration in this invention is 1500-2500 Da, preferably 1800-2200 Da, and more preferably 2000 Da. This invention combines polyethylene glycol 6000 and potassium dihydrogen phosphate in an aqueous two-phase extraction technique for layered enrichment and ultrafiltration with appropriate pore size, achieving efficient separation and purification of polysaccharides, removing small molecule impurities and ineffective components, and significantly improving the purity of the finished polysaccharide. This invention utilizes a suitable amount of polyethylene glycol 6000 and potassium dihydrogen phosphate to extract the fermentation supernatant, resulting in a high content of polysaccharides extracted from the supernatant.

[0022] The present invention provides a dry powder of Inonotus obliquus polysaccharide obtained by the preparation method described above.

[0023] This invention provides the *Inonotus obliquus* polysaccharide powder obtained by the aforementioned preparation method, or the application of the *Inonotus obliquus* polysaccharide powder in the preparation of drugs for lowering blood sugar and regulating intestinal flora. This invention achieves a dual synergistic effect through compound bacterial fermentation: on the one hand, *Inonotus obliquus* polysaccharide directly exerts a hypoglycemic effect by inhibiting α-glucosidase activity, promoting glucose metabolism in hepatocytes, and improving insulin secretion; on the other hand, the polysaccharide derivatives and short-chain fatty acid precursors produced during fermentation can regulate the intestinal flora structure, increase the abundance of beneficial bacteria, reduce the abundance of harmful bacteria, promote the production of short-chain fatty acids, repair the intestinal barrier, reduce intestinal inflammation, and thus improve insulin sensitivity, indirectly assisting in lowering blood sugar. Simultaneously, the proliferation of beneficial bacteria can further promote the absorption and utilization of polysaccharides, effectively breaking the vicious cycle between diabetes and intestinal flora imbalance, achieving comprehensive conditioning for diabetic patients or patients with intestinal flora imbalance. Compared with *Inonotus obliquus* polysaccharide prepared by traditional methods, the conditioning effect is more comprehensive and longer-lasting.

[0024] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] Unless otherwise specified, the following embodiments are all conventional methods.

[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0027] In the following embodiments, the *Inonotus obliquus* was obtained from Ningbo Mingzhou Biotechnology Co., Ltd., product code BMZ339628. The *Lactobacillus plantarum* was obtained from Ningbo Mingzhou Biotechnology Co., Ltd., product code BMZ133561. The *Bifidobacterium longum* was obtained from Ningbo Mingzhou Biotechnology Co., Ltd., product code B12041. The *Lactobacillus fermentum* was obtained from Ningbo Mingzhou Biotechnology Co., Ltd., product code BMZ134603.

[0028] Example 1 1. Raw material pretreatment: Select 1 kg of Inonotus obliquus fruiting bodies that are free from mold and impurities, wash and dry them, and then grind them to 100 mesh using a pulverizer to obtain Inonotus obliquus coarse powder; freeze the coarse powder at -60℃ for 40 min, and then thaw it at 30℃ for 1 h. Repeat this process twice to obtain Inonotus obliquus cell wall broken powder.

[0029] 2. Enzymatic hydrolysis: Add 18 times the weight of purified water to the broken-cell wall Inonotus obliquus powder, adjust the pH of the system to 5.5, add 0.2 w / w% papain (100,000 U / g) and 0.3 w / w% cellulase (100,000 U / g) of the powder, and hydrolyze for 2.5 h at 55℃ and 180 r / min to inactivate the enzyme and obtain the enzymatic hydrolysate.

[0030] 3. Compound microbial fermentation treatment (1) Activation of fermentation strains: Inonotus obliquus and Lactobacillus plantarum were selected and inoculated into their respective activation media. They were cultured at 37℃ and 180 r / min for 12 h to obtain activated bacterial solutions. The concentration of the bacterial solutions was 1×10⁻⁶. 9 CFU / mL; Mix the bacterial suspensions of Inonotus obliquus and Lactobacillus plantarum at a volume ratio of 2:1 to obtain a compound bacterial suspension; (2) Fermentation: The total inoculation amount is 3w / w% of the enzyme hydrolysate mass. The compound bacterial solution is added to the enzyme hydrolysate and fermented at 30℃ for 18h. During the fermentation process, the mixture is stirred once every 8h for 10min each time. After the fermentation is completed, the mixture is centrifuged at 8000r / min for 15min, and the fermentation supernatant is collected and the precipitate is discarded.

[0031] 4. Purification: Add polyethylene glycol 6000 and potassium dihydrogen phosphate to the fermentation supernatant to adjust the mass fraction of polyethylene glycol 6000 to 20% and potassium dihydrogen phosphate to 7%. Let it stand at room temperature for 8 minutes. After separation, collect the upper polysaccharide-enriched phase. Dilute the polysaccharide-enriched phase with 1.2 times its volume of purified water. Decolorize using AB-8 macroporous adsorption resin. Pass the solution through the resin column at a flow rate of 1.2 BV / h. Then rinse the resin column with purified water at a flow rate of 2 BV / h (4 BV of purified water). Collect the effluent. Ultrafilter the effluent through an ultrafiltration membrane with a molecular weight cutoff of 2000 Da and collect the ultrafiltration retentate.

[0032] 5. Concentration and drying: The ultrafiltration retentate is concentrated using a nanofiltration membrane with a molecular weight cutoff of 400 Da to 1 / 6 of its original volume to obtain a polysaccharide concentrate; the concentrate is pre-frozen at -45℃ for 4 hours, and then freeze-dried under vacuum at -55℃ for 8 hours to obtain a polysaccharide powder, which is then passed through a 120-mesh sieve to obtain Inonotus obliquus polysaccharide powder.

[0033] 6. Sterilization and Packaging: The Chaga polysaccharide powder was sterilized using 60Co-γ rays (6 kGy). After sterilization, it was sealed in packaging and stored in a cool, dry place. Testing showed that the purity of the obtained Chaga polysaccharide powder was 96.3%.

[0034] Example 2 1. Raw material pretreatment: Select 1 kg of Inonotus obliquus fruiting bodies that are free from mold and impurities, wash and dry them, and then grind them to 80 mesh using a pulverizer to obtain Inonotus obliquus coarse powder; freeze the coarse powder at -50℃ for 30 min, and then thaw it at 25℃ for 1.8 h. Repeat this process twice to obtain Inonotus obliquus cell wall broken powder.

[0035] 2. Enzymatic hydrolysis: Add 15 times the weight of purified water to the broken-cell wall Inonotus obliquus powder, adjust the pH of the system to 5.3, add 0.1 w / w% papain and 0.2 w / w% cellulase by weight of the powder, and hydrolyze for 2 hours at 52℃ and 150 r / min to inactivate the enzymes and obtain the enzymatic hydrolysate.

[0036] 3. Compound microbial fermentation treatment (1) Activation of fermentation strains: The steps are the same as in Example 1; the compound bacterial solution is obtained by using the volume ratio of Inonotus obliquus bacterial solution to Lactobacillus plantarum bacterial solution of 1:1. (2) Fermentation: The total inoculation amount is 2w / w% of the enzyme hydrolysate mass. The compound bacterial solution is added to the enzyme hydrolysate and fermented at 32℃ for 15h. During the fermentation process, the mixture is stirred once every 6h for 15min each time. After the fermentation is completed, the mixture is centrifuged at 7000r / min for 10min, and the fermentation supernatant is collected and the precipitate is discarded.

[0037] 4. Purification: Add polyethylene glycol 6000 and potassium dihydrogen phosphate to the fermentation supernatant to adjust the mass fraction of polyethylene glycol 6000 to 15% and potassium dihydrogen phosphate to 6%. Let it stand at room temperature for 5 minutes, and collect the upper polysaccharide-enriched phase after separation. Dilute the polysaccharide-enriched phase with 0.9 times its volume of purified water, and decolorize it using AB-8 macroporous adsorption resin. Pass the solution through the resin column at a flow rate of 1.0 BV / h, and then rinse the resin column with purified water at a flow rate of 2 BV / h (purified water volume 3 BV). Collect the effluent. Ultrafilter the effluent through an ultrafiltration membrane with a molecular weight cutoff of 1500 Da and collect the ultrafiltration retentate.

[0038] 5. Concentration and drying: The ultrafiltration retentate is concentrated using a nanofiltration membrane with a molecular weight cutoff of 300 Da to 1 / 5 of its original volume to obtain a polysaccharide concentrate. The concentrate is pre-frozen at -40℃ for 5 hours, and then freeze-dried under vacuum at -50℃ for 9 hours to obtain polysaccharide powder. The powder is then passed through a 100-mesh sieve to obtain Inonotus obliquus polysaccharide powder.

[0039] 6. Sterilization and packaging: Same as in Example 1. The purity of the obtained Inonotus obliquus polysaccharide was tested to be 95.8%.

[0040] Example 3 1. Raw material pretreatment: Select 1 kg of Inonotus obliquus fruiting bodies that are free from mold and impurities, wash and dry them, and then grind them to 120 mesh using a pulverizer to obtain Inonotus obliquus coarse powder; freeze the coarse powder at -70℃ for 50 min, and then thaw it at 32℃ for 1.2 h, repeating the process twice to obtain Inonotus obliquus cell wall broken powder.

[0041] 2. Enzymatic hydrolysis: Add 22 times the weight of purified water to the broken-cell wall Inonotus obliquus powder, adjust the pH of the system to 5.8, add 0.3 w / w% papain and 0.3 w / w% cellulase of the powder, and hydrolyze for 3.0 h at 58℃ and 200 r / min to inactivate the enzyme and obtain the enzymatic hydrolysate.

[0042] 3. Compound microbial fermentation treatment (1) Activation of fermentation strains: The steps are the same as in Example 1; the compound bacterial solution is obtained by using the volume ratio of Inonotus obliquus bacterial solution to Lactobacillus plantarum bacterial solution of 3:1. (2) Fermentation: The total inoculation amount is 4w / w% of the enzyme hydrolysate mass. The compound bacterial solution is added to the enzyme hydrolysate and fermented at 38℃ for 22h. During the fermentation process, the mixture is stirred once every 10h for 15min each time. After the fermentation is completed, the mixture is centrifuged at 8500r / min for 20min, and the fermentation supernatant is collected and the precipitate is discarded.

[0043] 4. Purification: Add polyethylene glycol 6000 and potassium dihydrogen phosphate to the fermentation supernatant to adjust the mass fraction of polyethylene glycol 6000 to 25% and potassium dihydrogen phosphate to 10%. Let it stand at room temperature for 10 minutes. After separation, collect the upper polysaccharide-enriched phase. Dilute the polysaccharide-enriched phase with 2.0 times its volume of purified water. Decolorize using AB-8 macroporous adsorption resin. Pass the solution through the resin column at a flow rate of 2.1 BV / h. Then rinse the resin column with purified water at a flow rate of 4 BV / h (5 BV of purified water used). Collect the effluent. Ultrafilter the effluent through an ultrafiltration membrane with a molecular weight cutoff of 2500 Da and collect the ultrafiltration retentate.

[0044] 5. Concentration and drying: The ultrafiltration retentate is concentrated using a nanofiltration membrane with a molecular weight cutoff of 500 Da to 1 / 7 of its original volume to obtain a polysaccharide concentrate. The concentrate is pre-frozen at -50℃ for 5 hours, and then freeze-dried under vacuum at -60℃ for 10 hours to obtain polysaccharide powder. The powder is then passed through a 140-mesh sieve to obtain Inonotus obliquus polysaccharide powder.

[0045] 6. Sterilization and packaging: The steps are the same as in Example 1. The purity of the obtained Inonotus obliquus polysaccharide powder was tested to be 97.1%.

[0046] Comparative Example 1 1. Raw material pretreatment: Select 1 kg of Chaga mushroom fruiting bodies that are free from mold and impurities, wash and dry them, then grind them to 100 mesh using a pulverizer to obtain Chaga mushroom coarse powder. The process in steps 2-6 is the same as in Example 1.

[0047] Comparative Example 2 The enzymatic hydrolysis step in Example 1 was omitted. The enzymatic hydrolysate in step 3 of Example 1 was replaced with broken-cell wall Inonotus obliquus powder. All other steps were the same as in Example 1.

[0048] Comparative Example 3 Replace Inonotus obliquus and Lactobacillus plantarum in step 3 of Example 1 with Bifidobacterium longum and Lactobacillus fermentum. The remaining steps are the same as in Example 1.

[0049] Comparative Example 4 Remove step 3 from Example 1. In step 2, the enzymatic hydrolysate is centrifuged at 8000 rpm for 15 minutes, and the fermentation supernatant is collected and the precipitate is discarded. Then proceed to step 4 (same as in Example 1). The remaining steps are the same as in Example 1.

[0050] Experiment Example 1: Hypoglycemic Experiment in Diabetic Mice 1. Model establishment: Healthy Kunming mice (weight 20±2g) were selected and randomly divided into a normal control group and a model group after 7 days of acclimatization. Mice in the model group were injected intraperitoneally with streptozotocin (STZ, dose 150mg / kg), while mice in the normal control group were injected with an equal volume of physiological saline. After 7 days of normal feeding, blood was collected from the tail tip to measure fasting blood glucose levels. Mice with blood glucose levels between 10-25mmol / L were considered diabetic model mice.

[0051] 2. Grouping: The diabetic mice constructed above were randomly divided into model control group, Example 1-3 group, and Comparative Example 1-4 group, with 8 mice in each group; the normal control group (8 mice) and the model control group were administered an equal volume of physiological saline by gavage, while the other groups were administered gavage once a day at a dose of 300 mg / kg bw for 21 consecutive days.

[0052] 3. Index detection: Blood was collected from the tail tip of mice on days 0, 7, 14, and 21 after fasting for 12 hours following gavage to measure fasting blood glucose levels. After gavage, mouse body weight, serum insulin content, and liver glycogen content were measured.

[0053] 4. Experimental Results: The results are shown in Table 1. After 7, 14, and 21 days of gavage, the fasting blood glucose levels in Examples 1-3 showed a continuous decreasing trend and were significantly lower than those in the model control group (P<0.01). After 21 days of gavage, the blood glucose level was close to that of the normal control group. Although the blood glucose levels in the comparative groups decreased, the decrease was significantly less than that in Examples 1-3. Regarding body weight, after 21 days of gavage, the body weight in Examples 1-3 increased significantly (P<0.01), while the body weight in the comparative groups increased only slightly. Regarding serum insulin and liver glycogen levels, Examples 1-3 significantly increased serum insulin and liver glycogen levels (P<0.01), with Example 3 showing the best effect. The improvement effect in the comparative groups was significantly weaker than that in Examples 1-3. This indicates that specific cell wall disruption, enzymatic hydrolysis, and fermentation methods have a significant impact on the hypoglycemic effect of the prepared Inonotus obliquus polysaccharide dry powder. The technical methods described in this invention yielded Inonotus obliquus polysaccharide dry powder with a higher hypoglycemic effect.

[0054] Table 1. Results of glucose-lowering related indicators in mice of each group (n=8)

[0055] Note: Compared with the model control group. P<0.05, P<0.01 Experiment Example 2 1. Model Establishment Except for the normal control group, all other groups of mice were administered 200 μL of ampicillin (1 mg / mL) by gavage once a day for 7 consecutive days to establish a model of intestinal flora disorder.

[0056] 2. Group processing Mice with gut microbiota dysbiosis were randomly divided into a model control group, Example 1-3 groups, and Comparative Example 1-4 groups, with 8 mice in each group. The normal control group (8 mice) and the model control group were administered an equal volume of physiological saline by gavage, while the other groups were administered 200 mg / kg bw once daily by gavage for 14 consecutive days. During the administration period, the mice in all groups were kept under the same feeding conditions, and their diet, water intake, and general condition were recorded.

[0057] 3. Detection Indicators and Methods After administration, mice were fasted for 12 hours and deprived of water for 4 hours. Fresh fecal samples were collected for the following purposes: detection of intestinal flora abundance and short-chain fatty acid (SCFA) content.

[0058] Gut microbiota abundance detection: Bifidobacteria, lactobacilli (beneficial bacteria), and Escherichia coli (harmful bacteria) were isolated and cultured using selective culture media and counted. The results were expressed as 1g CFU / g feces. Detection of short-chain fatty acid (SCFA) content: Feces were treated using a HALO-F100 fecal treatment system. A 10% suspension was prepared, and 500 μL of the suspension was placed in a 1.5 mL centrifuge tube. 100 μL of crotonic acid metaphosphate solution was added, and the mixture was frozen at -30℃ for 24 h. After thawing, the mixture was centrifuged at 8000g for 3 min (4℃) to remove impurities such as proteins. The supernatant was filtered through a 0.22 μm aqueous filter membrane before being analyzed. The content of acetic acid, propionic acid, and butyric acid in the feces was detected. SCFAs are metabolites of beneficial intestinal bacteria and can reflect the metabolic function of the gut microbiota.

[0059] Table 2 shows that, compared with the normal control group, the abundance of Bifidobacterium and Lactobacillus in the feces of mice in the model control group was significantly decreased (P<0.01), while the abundance of Escherichia coli was significantly increased (P<0.01), indicating that the intestinal flora disorder model was successfully constructed. Compared with the model control group, the abundance of Bifidobacterium and Lactobacillus in Examples 1-3 was significantly increased, while the abundance of Escherichia coli was significantly decreased (P<0.01); the control group showed weaker effects and was not as effective as Examples 1-3 in improving the intestinal flora.

[0060] Table 2 Results of gut microbiota abundance detection

[0061] Note: Compared with the model control group, P<0.01.

[0062] As shown in Table 3, compared with the model control group, the improvement effect of the groups of Examples 1-3 in increasing the content of various SCFAs was significantly better than that of the comparative groups (P<0.05), indicating that the Inonotus obliquus polysaccharide prepared in the embodiments of the present invention can effectively promote the metabolism of beneficial bacteria and increase the production of SCFAs, further confirming its regulatory effect on intestinal flora.

[0063] Table 3 Comparison of short-chain fatty acid content in mouse feces of each group

[0064] Note: Compared with the model control group, P<0.01.

[0065] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a dried powder of Inonotus obliquus polysaccharide, characterized in that, The process includes the following steps: freezing and thawing the fruiting bodies of *Inonotus obliquus* to obtain *Inonotus obliquus* powder with broken cell walls; enzymatically hydrolyzing the *Inonotus obliquus* powder with water, papain, and cellulase to obtain an enzymatic hydrolysate; inoculating the enzymatic hydrolysate with a mixed bacterial culture of *Inonotus obliquus* and *Lactobacillus plantarum* to obtain a fermentation broth, centrifuging and collecting the fermentation supernatant; adding polyethylene glycol 6000 and potassium dihydrogen phosphate, allowing it to stand and separate into layers, collecting the upper polysaccharide solution, then decolorizing and ultrafiltration, collecting the retentate; concentrating and drying to obtain *Inonotus obliquus* polysaccharide dry powder.

2. The preparation method according to claim 1, characterized in that, The freezing temperature is -50~-70℃ and the time is 30-50 min, and the thawing temperature is 27-32℃ and the time is 0.5-1.5 h.

3. The preparation method according to claim 1, characterized in that, The mass ratio of the cell-wall broken Inonotus obliquus powder to water is 1:15-22; after mixing the cell-wall broken Inonotus obliquus with water, the pH of the system is adjusted to 5.3-5.

8.

4. The preparation method according to claim 1, characterized in that, The amount of papain added is 0.1-0.3 w / w of the cellulase in the broken-cell wall Inonotus obliquus powder; the amount of cellulase added is 0.2-0.4 w / w of the cellulase in the broken-cell wall Inonotus obliquus powder; the enzymatic hydrolysis temperature is 52-58℃, the rotation speed is 150-200 r / min, and the time is 1.5-3.5 h.

5. The preparation method according to claim 1, characterized in that, The bacterial concentrations of both *Inonotus obliquus* and *Lactobacillus plantarum* were 0.5-1.5 × 10⁻⁶. 9 CFU / mL; The volume ratio of the Inonotus obliquus and Lactobacillus plantarum bacterial solutions in the mixed bacterial solution is (3-1):

1.

6. The preparation method according to claim 1, characterized in that, The inoculum size of the mixed culture of Inonotus obliquus and Lactobacillus plantarum is 1-5 w / w of the enzymatic hydrolysate; the fermentation conditions include: fermentation at 28-38℃ for 15-32 h, with stirring 1-3 times every 6-10 h during the fermentation process, each stirring lasting 5-15 min.

7. The preparation method according to claim 1, characterized in that, The final mass fraction of the polyethylene glycol 6000 is 15-25%, and the final mass fraction of the potassium dihydrogen phosphate is 5-12%; the standing temperature is room temperature and the time is 5-12 minutes.

8. The preparation method according to claim 1, characterized in that, The ultrafiltration pore size is 1500-2500 Da.

9. The dried powder of Inonotus obliquus polysaccharide obtained by the preparation method according to any one of claims 1-8.

10. The use of the dried powder of Inonotus obliquus polysaccharide obtained by the preparation method according to any one of claims 1-8 in the preparation of drugs for lowering blood sugar and regulating intestinal flora.

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