Cordyceps militaris polysaccharide, and preparation method and application thereof

Abstract

The application belongs to the technical field of food and medicine, and particularly relates to a cordyceps militaris polysaccharide, a preparation method and application thereof. The application provides a method for improving the yield of cordyceps militaris polysaccharide by optimizing a liquid fermentation process, and proves that the cordyceps militaris polysaccharide has a good blood glucose lowering effect on T2DM mice. The application provides a liquid fermentation method capable of significantly improving the yield of cordyceps militaris polysaccharide, and the yield of the cordyceps militaris polysaccharide is improved to 82.7 mg / g mycelium, and the culture time is short. The cordyceps militaris polysaccharide obtained has a clear key structure characteristic range. The cordyceps militaris polysaccharide obtained has a good blood glucose lowering effect on T2DM mice, and by remodeling the intestinal flora, improving the SCFAs level, and improving a plurality of high blood glucose related indexes, the T2DM related high blood glucose symptoms are relieved.

Description

Technical Field

[0001] This invention belongs to the field of food and pharmaceutical technology, specifically relating to a Cordyceps militaris polysaccharide, its preparation method, and its application. Background Technology

[0002] Cordyceps militaris, a traditional and valuable edible and medicinal fungus belonging to the same genus as Cordyceps sinensis, possesses polysaccharides as its active ingredient, believed to have various physiological functions such as immunomodulation, anti-tumor activity, antioxidant activity, and hypoglycemia, making it highly valuable for development. However, wild Cordyceps militaris resources are scarce and limited by region and season, making it difficult to meet market demand; while artificially cultivated fruiting bodies also face bottlenecks such as long cycles (usually 2-3 months), susceptibility to pests and diseases, and unstable content of active ingredients, severely restricting its industrial development. Currently, liquid submerged fermentation technology has become a key alternative for the large-scale, industrialized production of Cordyceps militaris active substances. Numerous studies have shown that the types and contents of the main active ingredients (such as polysaccharides and cordycepin) in Cordyceps militaris mycelium obtained through liquid fermentation are similar to those in artificially cultivated fruiting bodies, and their core pharmacological activities are essentially the same. More importantly, this method has significant advantages such as a significantly shortened cultivation cycle (only a few days), completely controllable production environment, no influence from climate and region, and ease of achieving aseptic and automated large-scale production, providing a reliable technical platform for obtaining stable and uniform active products. Therefore, optimizing the liquid fermentation process to efficiently produce Cordyceps militaris polysaccharides with clearly defined activities has become a research hotspot.

[0003] Currently, while research on producing active substances from Cordyceps militaris using liquid fermentation technology is widespread, most existing studies focus on optimizing single intermediate indicators such as mycelial biomass, extracellular polysaccharide yield, or cordycepin yield, resulting in unstable activity of the obtained polysaccharide products. Although some studies have reported the hypoglycemic effect of Cordyceps militaris polysaccharides, these studies primarily focus on crude polysaccharides obtained through water extraction and alcohol precipitation, failing to conduct systematic and in-depth structural analysis of the obtained polysaccharides (such as molecular weight distribution, monosaccharide composition, and glycosidic bond linkage). The unclear product structure makes it impossible to establish a reliable "structure-characteristic activity" correlation. At the application level, existing research on the improvement of type 2 diabetes mellitus (T2DM) by Cordyceps militaris polysaccharides largely limits the exploration of its mechanism of action to the observation of direct phenotypic indicators such as blood glucose and insulin, or sporadic analyses of antioxidant and anti-inflammatory effects. These studies have failed to systematically reveal how it exerts comprehensive benefits by regulating gut microbiota homeostasis and influencing key pathways such as the liver lipidome and intestinal short-chain fatty acids from the integrated perspective of "multi-organ metabolic network" and "host-microbe interaction", thus limiting its in-depth development and application.

[0004] Therefore, it is of great significance to develop a new method for preparing Cordyceps militaris polysaccharide and to study its application. Summary of the Invention

[0005] The technical problem to be solved by this invention is to address the shortcomings of existing technologies by providing a method for increasing the yield of intracellular polysaccharides from Cordyceps militaris through optimized liquid fermentation processes. Furthermore, this invention also relates to structurally well-defined Cordyceps militaris intracellular polysaccharides obtained by this method, and their application in the preparation of drugs or health foods for the prevention or improvement of type 2 diabetes and the regulation of metabolic disorders.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] The first aspect of this invention provides a method for preparing Cordyceps militaris polysaccharide, comprising the following steps:

[0008] S1. The Cordyceps militaris spawn was inoculated into PDA solid medium for activation culture, and the Cordyceps militaris spores were washed off with distilled water to obtain Cordyceps militaris spore solution; the Cordyceps militaris spore solution was inoculated into seed medium and cultured to obtain Cordyceps militaris seed solution; the Cordyceps militaris seed solution was inoculated into fermentation medium, fermented and cultured, and the mycelium was collected by filtration and freeze-dried to obtain Cordyceps militaris mycelium;

[0009] S2. The mycelium of Cordyceps militaris was extracted with hot water, filtered, and compressed under reduced pressure to obtain a concentrated extract of Cordyceps militaris. The concentrated extract of Cordyceps militaris was mixed with an ethanol solution, allowed to stand, centrifuged to collect the precipitate, and then reconstituted with water to obtain a crude polysaccharide extract of Cordyceps militaris. The crude polysaccharide extract of Cordyceps militaris was decolorized and protein removed, and then freeze-dried to obtain crude polysaccharide of Cordyceps militaris.

[0010] S3. The crude polysaccharide of Cordyceps militaris was purified by DEAE Sepharose Fast Flow and Sephacryl S-300 chromatography. The purified liquid was collected, dialyzed, concentrated and freeze-dried to obtain the Cordyceps militaris polysaccharide.

[0011] In some embodiments of the present invention, in S1, the formula of the PDA solid culture medium is: 200g peeled potato, 20g glucose, 15g agar, and distilled water to a final volume of 1000mL.

[0012] In some embodiments of the present invention, in S1, the activation culture is carried out under the following conditions: cultured at 25°C for 3 days.

[0013] In S1, the concentration of the Cordyceps militaris spore solution is 1×10⁻⁶. 6 ~1×10 8 cfu / mL.

[0014] In some embodiments of the present invention, in S1, the concentration of the Cordyceps militaris spore liquid is 0.5 × 10⁻⁶. 7 cfu / mL.

[0015] In some embodiments of the present invention, the inoculation volume of the Cordyceps militaris spore liquid is 30 μL.

[0016] In some embodiments of the present invention, in S1, the seed culture medium is formulated as follows: 20g glucose, 10g peptone, 3g KH2PO4, 1.5g MgSO4, and distilled water to a final volume of 1000mL, with a volume of 50mL / bottle.

[0017] In some embodiments of the present invention, in S1, the seed culture is carried out under the following conditions: 25°C, 150 rpm, for 3 days.

[0018] In S1, the inoculation amount of the Cordyceps militaris seed liquid is 3-6% by volume; the fermentation culture conditions are: culture temperature 24-28℃, rotation speed 140-170rpm, and culture time 7-11 days.

[0019] In some embodiments of the present invention, in S1, the inoculation amount of the Cordyceps militaris seed liquid is 4% by volume.

[0020] In some embodiments of the present invention, in S1, the fermentation culture is carried out under the following conditions: culture temperature 28°C, rotation speed 160 rpm, and culture time 9 days.

[0021] In S1, the fermentation medium is formulated as follows: 40g glucose, 10g beef extract, 0.5g KH2PO4, 0.5g MgSO4, 0.5g CaCO3, and distilled water to a final volume of 1000mL, with a volume of 100mL per bottle.

[0022] In S2, the hot water extraction conditions are as follows: the temperature is 70~85℃, the solid-liquid ratio of Cordyceps militaris mycelium to pure water is 1:(20~40), the number of extractions is 1~3 times, and the extraction time for each extraction is 4h.

[0023] In some embodiments of the present invention, in S2, the hot water extraction is performed under the following conditions: the temperature is 80°C, the solid-liquid ratio of Cordyceps militaris mycelium to pure water is 1:30, the extraction is performed 3 times, and the extraction time for each extraction is 4 hours.

[0024] In some embodiments of the present invention, in S2, the reduced pressure compression is performed under the following conditions: the temperature is 65°C, and the volume is concentrated to 1 / 10 of the original volume.

[0025] In S2, the volume ratio of the Cordyceps militaris extract concentrate to the ethanol solution is 1:4; the volume concentration of the ethanol solution is 90%.

[0026] In S2, the protein removal is performed under the following conditions: Cordyceps militaris crude polysaccharide extract is mixed with Sevage reagent at a volume ratio of 1:3; the Sevage reagent is a mixture of chloroform and n-butanol at a volume ratio of 1:4.

[0027] In S3, the eluent for the DEAE Sepharose Fast Flow purification column is pure water; the eluent for the Sephacryl S-300 purification column is 0.1 mol / L NaCl.

[0028] A second aspect of the present invention provides a Cordyceps militaris polysaccharide.

[0029] In some embodiments of the present invention, Cordyceps militaris polysaccharide was successfully prepared by the preparation method provided in the first aspect of the present invention. Through analysis of its composition and microstructure, it was proved that Cordyceps militaris polysaccharide is a heteropolysaccharide and is characterized by discrete particles with uneven particle size and a loose porous distribution.

[0030] The third aspect of this invention provides the application of Cordyceps militaris polysaccharide in the preparation of hypoglycemic foods.

[0031] The fourth aspect of this invention provides the use of Cordyceps militaris polysaccharide in the preparation of drugs for treating and / or preventing diabetes.

[0032] The diabetes mentioned is type 2 diabetes (T2DM).

[0033] In some embodiments of the present invention, studies on the application of Cordyceps militaris polysaccharide in hyperglycemic symptoms in type 2 diabetes mellitus (T2DM) mice have shown that Cordyceps militaris polysaccharide alleviates T2DM-related hyperglycemic symptoms by reshaping the gut microbiota, increasing SCFA levels, and improving multiple hyperglycemia-related indicators. This demonstrates the application potential of Cordyceps militaris polysaccharide in the preparation of hypoglycemic foods and / or drugs.

[0034] Beneficial effects:

[0035] This invention provides a liquid fermentation method that can significantly increase the yield of intracellular polysaccharides from Cordyceps militaris, increasing the polysaccharide yield to 82.7 mg / g mycelium with a short culture time; and the obtained Cordyceps militaris intracellular polysaccharides have a clearly defined range of key structural features.

[0036] The intracellular polysaccharide of Cordyceps militaris obtained in this invention has a good hypoglycemic effect on T2DM mice. By reshaping the intestinal flora and increasing the level of SCFAs, it improves a number of hyperglycemia-related indicators, thereby alleviating T2DM-related hyperglycemia symptoms. Attached Figure Description

[0037] The present invention will be further described in detail below with reference to the accompanying drawings, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0038] Figure 1 The images show the high-performance liquid chromatogram, infrared spectrum, and scanning electron microscope image of the Cordyceps militaris polysaccharide prepared in Example 2 of this invention.

[0039] Figure 2 The values ​​represent the changes in body weight of mice in each group during the 4-week experiment in Example 3 of this invention; different letters indicate significant differences (p < 0.05).

[0040] Figure 3 The values ​​represent the changes in fasting blood glucose levels in each group of mice during the 4-week experiment in Example 3 of this invention; different letters indicate significant differences (p < 0.05).

[0041] Figure 4 The results of the oral glucose tolerance test (OGTT), area under the glucose tolerance curve (AUC of OGTT), and glycated serum protein (GSP) of each group of mice in Example 3 of this invention after 4 weeks of experimentation; different letters indicate significant differences (p < 0.05).

[0042] Figure 5 The effect of CMP on pancreatic islet function in T2DM mice in Example 3 of this invention; different letters indicate significant differences (p < 0.05).

[0043] Figure 6 The effect of CMP on serum and liver lipid metabolism in T2DM mice in Example 3 of this invention; different letters indicate significant differences (p < 0.05).

[0044] Figure 7 The values ​​represent the changes in the expression levels of genes at the mRNA level in liver tissue during the mouse experiments in Example 3 of this invention; different letters indicate significant differences (p < 0.05).

[0045] Figure 8 The changes in gut microbiota composition in each group of mice during the experiment in Example 4 of this invention are shown; different letters indicate significant differences (p < 0.05).

[0046] Figure 9 The values ​​represent the changes in the concentration of short-chain fatty acids, a metabolite of gut microbiota, in each group of mice during the experiments in Example 4 of this invention; different letters indicate significant differences (p < 0.05).

[0047] Figure 10 This is a heatmap showing the correlation between mouse gut microbiota and short-chain fatty acid concentrations and biochemical indicators in Example 4 of the present invention. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0049] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0050] The culture medium formulations used in the following examples are as follows:

[0051] PDA solid medium: 200g peeled potato, 20g glucose, 15g agar, distilled water to a final volume of 1000mL;

[0052] Seed culture medium: 20g glucose, 10g peptone, 3g KH2PO4, 1.5g MgSO4, distilled water to a final volume of 1000mL, 50mL / bottle.

[0053] Fermentation medium: 40g glucose, 10g beef extract, 0.5g KH2PO4, 0.5g MgSO4, 0.5g CaCO3, distilled water to a final volume of 1000mL, 100mL / bottle.

[0054] The Cordyceps militaris strain used in the following examples was purchased from the China Industrial Microbial Culture Collection Center, strain number CCIC 14013.

[0055] Example 1:

[0056] This embodiment provides a method for preparing Cordyceps militaris polysaccharide, which specifically includes the following steps:

[0057] S1. Inoculate 30 μL of Cordyceps militaris strain into PDA solid medium for activation culture. After culturing at 25°C for 3 days, wash off the Cordyceps militaris spores from the solid medium with 10 mL of distilled water to obtain Cordyceps militaris spore solution for later use.

[0058] 30 μL of Cordyceps militaris spore solution was inoculated into the seed culture medium, and the spore solution concentration was adjusted to 0.5 × 10⁻⁶ with pure water. 7 The concentration of cfu / mL was increased, and the culture conditions were 25℃ and 150rpm for 3 days to obtain Cordyceps militaris seed solution for later use.

[0059] Cordyceps militaris seed culture was inoculated into fermentation medium at an inoculation rate of 4% v / v and cultured at 28℃ and 150 rpm for 7 days. After fermentation, the mycelium was collected by filtration and washed three times with pure water, then freeze-dried to obtain Cordyceps militaris mycelium with a biomass of 15.66 g / L.

[0060] S2. Cordyceps militaris mycelium was mixed with pure water at a ratio of 1:30 (w / v, g / mL), and extracted at 80℃ for 4 hours. This extraction was repeated three times. The extract was collected by filtration and concentrated under reduced pressure at 65℃ to 1 / 10 of its original volume, yielding a concentrated Cordyceps militaris extract. The concentrated extract was then mixed with ethanol solution (90% by volume) at a ratio of 1:4 for alcohol precipitation. After standing for 12 hours, the precipitate was collected by centrifugation and redissolved in water to obtain a crude Cordyceps militaris polysaccharide extract. Macroporous resin was added to the crude Cordyceps militaris polysaccharide extract for decolorization, and the liquid was collected by filtration. The decolorized extract was thoroughly mixed with Sevage reagent (a mixture of chloroform and n-butanol at a ratio of 1:4 by volume) in a separatory funnel at a ratio of 1:3 to remove protein. The lower layer was collected to obtain a crude Cordyceps militaris polysaccharide solution. This solution was concentrated and freeze-dried to obtain crude Cordyceps militaris polysaccharide, with a yield of 82.7 mg / g mycelium.

[0061] S3. Cordyceps militaris crude polysaccharide was purified by DEAE Sepharose Fast Flow chromatography using pure water as the eluent. The eluent was collected, concentrated, and freeze-dried to obtain Cordyceps militaris polysaccharide semi-finished product. Then, using 0.1 mol / L NaCl as the eluent, it was purified by Sephacryl S-300 chromatography. The eluent was collected, concentrated, dialyzed, and freeze-dried to obtain Cordyceps militaris polysaccharide (CMP).

[0062] Example 2:

[0063] This embodiment provides a method for preparing Cordyceps militaris polysaccharide, which specifically includes the following steps:

[0064] S1. Inoculate 30 μL of Cordyceps militaris strain into PDA solid medium for activation culture. After culturing at 25°C for 3 days, wash off the Cordyceps militaris spores from the solid medium with 10 mL of distilled water to obtain Cordyceps militaris spore solution for later use.

[0065] 30 μL of Cordyceps militaris spore solution was inoculated into the seed culture medium, and the spore solution concentration was adjusted to 1 × 10⁻⁶ with pure water. 7 The concentration of cfu / mL was increased, and the culture conditions were 25℃ and 150rpm for 3 days to obtain Cordyceps militaris seed solution for later use.

[0066] Cordyceps militaris seed culture was inoculated into fermentation medium at an inoculation rate of 4% v / v and cultured at 25℃ and 160 rpm for 9 days. After fermentation, the mycelium was collected by filtration and washed three times with pure water, then freeze-dried to obtain Cordyceps militaris mycelium with a biomass of 14.35 g / L.

[0067] S2. Cordyceps militaris mycelium was mixed with pure water at a ratio of 1:30 (w / v, g / mL), and extracted at 80℃ for 4 hours. This extraction was repeated three times. The extract was collected by filtration and concentrated under reduced pressure at 65℃ to 1 / 10 of its original volume, yielding a concentrated Cordyceps militaris extract. The concentrated extract was then mixed with ethanol solution (90% by volume) at a ratio of 1:4 for alcohol precipitation. After standing for 12 hours, the precipitate was collected by centrifugation and redissolved in water to obtain a crude Cordyceps militaris polysaccharide extract. Macroporous resin was added to the crude Cordyceps militaris polysaccharide extract for decolorization, and the liquid was collected by filtration. The decolorized extract was then thoroughly mixed with Sevage reagent (a mixture of chloroform and n-butanol at a ratio of 1:4 by volume) in a separatory funnel at a ratio of 1:3 to remove protein. The lower layer was collected to obtain a crude Cordyceps militaris polysaccharide solution. This solution was concentrated and freeze-dried to obtain crude Cordyceps militaris polysaccharide, with a yield of 75.8 mg / g mycelium.

[0068] S3. Cordyceps militaris crude polysaccharide was purified by DEAE Sepharose Fast Flow chromatography using pure water as the eluent. The eluent was collected, concentrated, and freeze-dried to obtain Cordyceps militaris polysaccharide semi-finished product. Then, using 0.1 mol / L NaCl as the eluent, it was purified by Sephacryl S-300 chromatography. The eluent was collected, concentrated, dialyzed, and freeze-dried to obtain Cordyceps militaris polysaccharide (CMP).

[0069] The Cordyceps militaris polysaccharides prepared in this embodiment were analyzed. Figure 1 The images shown are the high-performance liquid chromatogram, infrared spectrum, and scanning electron microscope image of the Cordyceps militaris polysaccharide prepared in this embodiment. Figure 1 In the figure, A represents the high-performance liquid chromatogram of Cordyceps militaris polysaccharide, derived from... Figure 1 As shown in Figure A, high-performance liquid chromatography (HPLC) analysis of monosaccharide composition reveals that Cordyceps militaris polysaccharide is a heteropolysaccharide, mainly composed of mannose (49.267%) and galactose (42.131%), with a small amount of glucose (7.195%), and trace amounts of glucosamine hydrochloride (0.682%), glucuronic acid (0.303%), galactosamine hydrochloride (0.251%), and rhamnose (0.171%). The high proportion of mannose and galactose indicates that they constitute the structural framework of Cordyceps militaris polysaccharide.

[0070] Figure 1 In the image, B represents the infrared spectrum of Cordyceps militaris polysaccharide, derived from... Figure 1 As can be seen from B, 3397cm -1 and 2931cm -1 The strong absorption peaks at 1653 cm⁻¹ are the stretching vibration peaks of hydroxyl groups and C-hydrogen bonds, respectively, which are characteristic absorption peaks of polysaccharide structures; -1 and 1547cm -1 The absorption peak at 1420 cm⁻¹ is related to the carbonyl stretching vibration;-1 The absorption peak at 1042 cm⁻¹ indicates the presence of a carboxyl group, confirming that Cordyceps militaris polysaccharide contains uronic acid; -1 The absorption peak at 599 cm⁻ -1 The characteristic peak at 812 cm⁻¹ is the stretching vibration peak of the pyran ring in the sugar residue; -1 The characteristic absorption peak at the location confirms the presence of mannose units in Cordyceps militaris polysaccharides.

[0071] Figure 1 C and D in the image are scanning electron microscope images of Cordyceps militaris polysaccharide at magnifications of 1000× and 10000×, respectively. Figure 1 As can be seen from C and D, under 1000x magnification, Cordyceps militaris polysaccharide exhibits a heterogeneous structure with irregular flake-like and granular aggregates coexisting; under 10000x magnification, the polysaccharide appears as discrete particles of uneven particle size, distributed in a loose porous manner.

[0072] Example 3:

[0073] This embodiment further demonstrates the application of the Cordyceps militaris polysaccharide prepared in Example 2 in improving hyperglycemia symptoms in type 2 diabetic (T2DM) mice. The specific steps are as follows:

[0074] Thirty 8-week-old male db / db mice (SPF grade, provided by Yangzhou University Medical Center) were acclimatized for 7 days. Six mice were randomly selected as the normal control group (Normal group), while the other mice were induced to develop type 2 diabetes mellitus (T2DM) using a high-fat diet combined with streptozotocin. The T2DM mice were then randomly divided into four groups of six mice each: the model control group (Model group, saline), the low-dose Cordyceps militaris polysaccharide group (CMP-L group, 100 mg / kg), and the model control group (Model group, saline), and the low-dose Cordyceps militaris polysaccharide group (CMP-L group, 100 mg / kg). -1 d -1 Cordyceps militaris polysaccharide high-dose group (CMP-H group, 300 mg kg) -1 d -1 ) and the positive control metformin group (MET group, 100 mg kg) -1 d -1 ) Administer once daily by gavage for 4 consecutive weeks.

[0075] The weight of the experimental mice was tested every two weeks. Figure 2 The change in body weight of mice in each group during the 4-week experimental period was measured by... Figure 2 It can be seen that the body weight of T2DM mice was significantly lower than that of the normal group. Both high and low doses of CMP administration significantly alleviated this abnormal weight loss after 4 weeks, but MET administration did not significantly improve the abnormal weight loss.

[0076] Mice were tested for fasting blood glucose (FBG) levels every two weeks. Figure 3 The changes in FBG in each group of mice during the 4-week experimental period were calculated by... Figure 3 It can be seen that the FBG of T2DM mice was significantly higher than that of the normal group. Both CMP and MET administration significantly reduced the FBG level. Among them, the CMP-H group had the best hypoglycemic effect, and the FBG level was not significantly different from that of the normal group.

[0077] The oral glucose tolerance test (OGTT), area under the glucose tolerance curve (AUCof OGTT), and glycated serum protein (GSP) levels of mice in each group were measured after 4 weeks of experimentation. Figure 4 The results of OGTT, AUC of OGTT, and GSP for each group of mice after 4 weeks of experimentation are shown. Figure 4 In this context, A represents the OGTT result. Figure 4 In this context, B represents the AUC of the OGTT result. Figure 4 C in the figure represents the GSP result, derived from... Figure 4 It can be seen that CMP can significantly improve the hyperglycemia symptoms in T2DM mice, and the CMP-H group has a better effect.

[0078] To investigate the effects of CMP on pancreatic islet function in T2DM mice, pancreatic islet function indices were assessed using the Homeostasis Model Assessment (HOMA), including HOMA-insulin sensitivity (HOMA-IS), HOMA-insulin resistance (HOMA-IR), and HOMA-pancreatic β-cell function (HOMA-β). Figure 5 The effect of CMP on pancreatic islet function in T2DM mice, among which, Figure 5 In this context, A represents the HOMA-IS index, B represents the HOMA-β index, and C represents the HOMA-IR index, such as... Figure 5 As shown, the HOMA-IS and HOMA-β index levels in T2DM mice were significantly lower than those in the normal group, while the HOMA-IR index level was significantly higher. CMP and MET administration significantly reversed these phenomena, and the CMP-H group showed significantly better results than the CMP-L and MET groups, indicating that high-dose Cordyceps militaris polysaccharide had the best effect on improving pancreatic function in T2DM mice.

[0079] To investigate the effects of CMP on serum and liver lipid metabolism in T2DM mice, changes in serum and liver lipid profiles of mice in each group were tested. Figure 6 The effects of CMP on serum and liver lipid metabolism in T2DM mice, among which Figure 6In this table, A represents serum total cholesterol, B represents serum triglycerides, C represents serum low-density lipoprotein (LDL) cholesterol, D represents serum high-density lipoprotein (HDL) cholesterol, E represents the LDL / HDL ratio, F represents liver total cholesterol, G represents liver triglycerides, H represents liver LDL cholesterol, I represents liver HDL cholesterol, and J represents the liver LDL / HDL ratio. Figure 6 It can be seen that CMP and MET administration significantly reduced the levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-c), and low-density lipoprotein cholesterol / high-density lipoprotein cholesterol (LDL-c / HDL-c) in the serum and liver of T2DM mice, while increasing the level of high-density lipoprotein cholesterol (HDL-c), indicating that both CMP and MET have a significant restorative effect on lipid metabolism disorders in T2DM mice.

[0080] To investigate the effect of CMP on the molecular mechanism of glucose uptake in mice, the transcriptional levels of akt-1 and glut-2 genes were analyzed using real-time quantitative PCR. Figure 7 The changes in the expression levels of genes at the mRNA level in liver tissue during the experiment in each group of mice were shown. Figure 7 In this context, A represents akt-1 and B represents glut-2, such as... Figure 7 As shown, CMP administration significantly upregulated the transcriptional levels of akt-1 and glut-2 genes in T2DM mice, and the effect was better than that of MET. This indicates that CMP enhances the liver's ability to take up glucose from the systemic circulation by regulating the transcription of akt-1 and glut-2 genes, promotes intracellular energy metabolism, and thus improves the hyperglycemic symptoms in T2DM mice.

[0081] Example 4:

[0082] This embodiment further performed high-throughput 16S rRNA sequencing on the cecal contents of mice from each group in Example 3 to study the changes in the gut microbiota structure at the phylum level and the relative abundance of differentially expressed microbiota at the genus level in T2DM mice. The content of short-chain fatty acids was determined using gas chromatography. The relationship between biochemical indicators and gut microbiota was analyzed using correlation heatmaps. The specific experimental steps are as follows:

[0083] 1. Gut microbiota sequencing

[0084] Metagenomic DNA was extracted from cecal contents using the CTAB method. PCR amplification of the 16S rRNA gene (V3-V4 region) was performed using primers 341F (5'-CCTAYGGGRBGCASCAG-3') and 806R (5'-GGACTACNNGGTATC Taat-3'). The amplified PCR products were sequenced on an Illumina NovaSeq PE250 platform. The composition, abundance, and diversity of the microbial community were classified and analyzed based on the sequencing results. Principal component analysis (PCA) was used to compare differences in microbial community composition among the groups. The Kruskal-Wallis test was used to compare microbial abundance at the phylum and genus levels among the groups. Linear discriminant analysis (LEfSe) was used to identify bacteria with significant differences among the CMP-L, CMP-H, and model groups. Spearman correlations between gut microbiota and biochemical indicators were calculated using Wekemo Bioincloud.

[0085] 2. Determination of cecal short-chain fatty acids (SCFAs)

[0086] 50 mg of cecal contents were mixed with 0.5 mL of deionized water, homogenized, and then phosphoric acid was added. After vortexing and mixing, SCFAs were extracted twice with diethyl ether. After centrifugation, the supernatant was filtered through a 0.22 µm nylon filter and analyzed by gas chromatography. The Spearman correlation between gut microbiota and SCFAs was calculated using Wekemo Bioincloud.

[0087] Results analysis:

[0088] 1. Effects of CMP on the gut microbiota structure of T2DM mice

[0089] Figure 8 The changes in gut microbiota composition in each group of mice during the experiment are shown. Figure 8 In the diagram, A represents principal component analysis, B represents the gut microbiota composition at the phylum level, C represents the differentially expressed microbiota between the low-dose Cordyceps militaris polysaccharide group and the model group at the genus level, and D represents the differentially expressed microbiota between the low-dose Cordyceps militaris polysaccharide group and the model group at the genus level. Figure 8 It can be seen that the principal component analysis results ( Figure 8 A) The gut microbiota of each group of mice showed obvious clustering characteristics: the normal group samples were distributed in the second and third quadrants, showing obvious spatial separation from the model group samples; after high-dose intervention with Cordyceps militaris polysaccharide, the microbiota structure of T2DM mice shifted significantly towards the normal group.

[0090] At the phylum level, Firmicutes and Bacteroidetes account for 90% of the intestinal flora and are the dominant bacterial groups. For example... Figure 8As shown in B, compared with the model group, both low and high doses of Cordyceps militaris polysaccharide intervention significantly inhibited the excessive proliferation of Firmicutes and enriched the abundance of Bacteroidetes; while the abundance of these two phyla did not change significantly in the MET group compared with the model group.

[0091] Figure 8 C and Figure 8 D shows the differences at the genus level between the CMP-L and CMP-H groups and the model group. The results indicate that low-dose intervention with Cordyceps militaris polysaccharides can significantly increase [the levels of these polysaccharides].

[0092] Comparative analysis showed that low-dose intervention with Cordyceps militaris polysaccharides significantly increased the abundance of *Eubacterium ventriosum group* and *Delftia*, while decreasing the abundance of *Lachnoclostridium*, *Adlercreutzia*, and *Intestinimonas*. Meanwhile, high-dose intervention with Cordyceps militaris polysaccharides significantly increased the abundance of *Eubacterium oxidoreducens group* and *Paraprevotella*, and continuously decreased the abundance of harmful fungal genera such as *Adlercreutzia* and *Intestinimonas*.

[0093] 2. Effects of CMP on the content of short-chain fatty acids in the intestine of T2DM mice

[0094] Short-chain fatty acids (acetic acid, propionic acid, and butyric acid) are key metabolites produced by the anaerobic fermentation of non-digestible carbohydrates by gut microbiota. They play an important mediating role in the body's metabolic regulation by strengthening the intestinal mucosal barrier, regulating glycolipid homeostasis, and alleviating systemic inflammation. Figure 9 The changes in the concentration of short-chain fatty acid metabolites in the gut microbiota of each group of mice during the experiment are shown. Figure 9 In this context, A represents acetic acid, B represents propionic acid, and C represents butyric acid. For example... Figure 9 As shown, 4 weeks of CMP intervention significantly altered the short-chain fatty acid profile of T2DM mice: compared with the model group, the concentrations of acetic acid, propionic acid, and butyric acid in the cecum of mice in all treatment groups were significantly increased; and the CMP-L and CMP-H groups were significantly better than the MET group in enriching short-chain fatty acids (p<0.05), indicating that CMP has a stronger ability to achieve metabolic remodeling by regulating the gut microbiota.

[0095] 3. Correlation heatmap analysis

[0096] To further elucidate the interaction between gut microbiota structure and SCFA metabolites, a correlation heatmap was constructed at the genus level. Figure 10 A heatmap showing the correlation between mouse gut microbiota and short-chain fatty acid concentrations and biochemical indicators, in which... Figure 10In the diagram, A is a heatmap showing the correlation between gut microbiota and short-chain fatty acid concentration, and B is a heatmap showing the correlation between mouse gut microbiota and biochemical indicators.

[0097] like Figure 10 As shown in Figure A, the abundance of Intestinimonas and Lachnoclostridium was significantly negatively correlated with the concentrations of acetic acid, propionic acid, and butyric acid; while the genera Massiliomicrobiota, Blautia, Peptococcus, and Delftia were significantly positively correlated with the above-mentioned short-chain fatty acids, indicating that these genera are key contributors to the recovery of SCFA concentrations in the mouse intestine after Cordyceps militaris polysaccharide intervention.

[0098] To further explore the functional significance of CMP-induced changes in the gut microbiota, the association between representative differentially expressed bacterial genera and biochemical indicators was systematically analyzed, such as... Figure 10 As shown in Figure B, *Lachnoclostridium* showed a significant positive correlation with serum LDL-c / HDL-c, FBG, serum TG, and AUC of OGTT, while showing a significant negative correlation with acetic acid, propionic acid, butyric acid levels, and the expression of hepatic akt-1 and glut-2 genes. Meanwhile, *Adlercreutzia* showed a significant positive correlation with serum LDL-c, LDL-c / HDL-c, and hepatic TC levels; *Intestinimonas* showed a significant negative correlation with acetic acid, propionic acid, and butyric acid levels. Conversely, *Agathobaculum* showed a significant positive correlation with HOMA-β and HOMA-IS indices, while showing a significant negative correlation with serum TC, LDL-c, and hepatic LDL-c / HDL-c.

[0099] The above results indicate that 4 weeks of CMP intervention can effectively improve physiological and metabolic abnormalities in T2DM mice: CMP significantly restored mouse body weight, normalized blood glucose levels such as FBG, AUC of OGTT, and GSP, and enhanced pancreatic endocrine function. A clear dose-response relationship was observed in the study; in key indicators such as homeostasis model assessment (HOMA-IS, HOMA-β, HOMA-IR) and serum and liver lipid profiles, the CMP-H group showed significantly better results than the CMP-L group. Furthermore, CMP intervention restructured the gut microbiota, reducing the relative abundance of harmful bacteria such as *Adlercreutzia* and *Intestinimonas*, and increasing the relative abundance of beneficial bacteria such as *Eubacterium ventriosum* group and *Paraprevotella*. Correlation analysis further confirmed that CMP improves multiple hyperglycemia-related indicators by reshaping the gut microbiota and increasing SCFA levels, thereby alleviating T2DM-related hyperglycemia symptoms.

[0100] This invention provides a method for preparing Cordyceps militaris polysaccharide and its applications. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for preparing Cordyceps militaris polysaccharide, characterized in that, Includes the following steps: S1. The Cordyceps militaris strain was inoculated into PDA solid medium for activation culture, and the Cordyceps militaris spores were washed off with distilled water to obtain Cordyceps militaris spore solution; the Cordyceps militaris spore solution was inoculated into seed medium and cultured to obtain Cordyceps militaris seed solution; Cordyceps militaris seed liquid was inoculated into a fermentation medium, and after fermentation, the mycelium was collected by filtration and freeze-drying to obtain Cordyceps militaris mycelium. S2. The mycelium of Cordyceps militaris was extracted with hot water, filtered, and compressed under reduced pressure to obtain a concentrated extract of Cordyceps militaris. The concentrated extract of Cordyceps militaris was mixed with an ethanol solution, allowed to stand, centrifuged to collect the precipitate, and then reconstituted with water to obtain a crude polysaccharide extract of Cordyceps militaris. The crude polysaccharide extract of Cordyceps militaris was decolorized and protein removed, and then freeze-dried to obtain crude polysaccharide of Cordyceps militaris. S3. The crude polysaccharide of Cordyceps militaris was purified by DEAE Sepharose Fast Flow and Sephacryl S-300 chromatography. The purified liquid was collected, dialyzed, concentrated and freeze-dried to obtain the Cordyceps militaris polysaccharide.

2. The preparation method according to claim 1, characterized in that, In S1, the concentration of the Cordyceps militaris spore liquid is 1×10⁻⁶. 6 ~1×10 8 cfu / mL.

3. The preparation method according to claim 1, characterized in that, In S1, the inoculation amount of the Cordyceps militaris seed liquid is 3-6% by volume; the fermentation culture is carried out under the following conditions: culture temperature 24-28℃, rotation speed 140-170rpm, and culture time 7-11 days.

4. The preparation method according to claim 1, characterized in that, In S1, the fermentation medium formula is: 40g glucose, 10g beef extract, 0.5g KH2PO4, 0.5g MgSO4, 0.5g CaCO3, and distilled water to a final volume of 1000mL, with a volume of 100mL / bottle.

5. The preparation method according to claim 1, characterized in that, In S2, the hot water extraction is performed under the following conditions: the temperature is 70-85℃, the solid-liquid ratio of Cordyceps militaris mycelium to pure water is 1:(20-40), the number of extractions is 1-3, and the extraction time for each extraction is 4 hours.

6. The preparation method according to claim 1, characterized in that, In S2, the volume ratio of the Cordyceps militaris extract concentrate to the ethanol solution is 1:4; the volume concentration of the ethanol solution is 90%.

7. The preparation method according to claim 1, characterized in that, In S3, the eluent for the DEAE Sepharose FastFlow purification column is pure water; the eluent for the Sephacryl S-300 purification column is 0.1 mol / L NaCl.

8. Cordyceps militaris polysaccharide prepared by the preparation method according to any one of claims 1 to 7.

9. The application of the Cordyceps militaris polysaccharide according to claim 8 in the preparation of hypoglycemic foods.

10. The use of the Cordyceps militaris polysaccharide according to claim 8 in the preparation of a drug for treating and / or preventing diabetes.

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