Lentinan, its preparation and use

Abstract

The present application particularly relates to lentinula edodes stem oligosaccharide (hereinafter referred to as E-LEBG) with hypoglycemic activity and a preparation method and application thereof. The E-LEBG is prepared from lentinula edodes stem, a processing by-product of lentinula edodes, by means of composite enzyme-assisted hot water extraction β - glucan, and then is prepared by enzyme directional enzymolysis; the obtained E-LEBG has uniform molecular weight, strong water solubility and high purity, has a significant inhibitory effect on α - amylase and α - glucosidase in vitro, and can significantly reduce fasting blood glucose of type 2 diabetes mice, improve oral glucose tolerance and relieve weight loss in vivo. The present application firstly proves that the E-LEBG has hypoglycemic activity, and provides a new technical path for high-value utilization of lentinula edodes stem resources.

Description

Background Technology

[0001] Shiitake mushrooms are one of the most produced and widely used edible and medicinal fungi in my country, with an annual output exceeding ten million tons. During the fresh sale, drying, canning, and further processing of shiitake mushrooms, a large amount of stem byproducts are generated, accounting for approximately 30% to 40% of the fresh weight of the mushroom. For example, when extracting lentinan or even oligosaccharides from the fruiting body of shiitake mushrooms, many people typically remove the stem completely. Currently, shiitake mushroom stems are mostly discarded, incinerated, or used only as low-value animal feed, causing not only serious resource waste but also environmental pressure.

[0002] Similar to other fruiting body parts, the stem of shiitake mushrooms also contains β-glucan, but the extraction strategies differ. Currently, extracting polysaccharides solely from the stem is not common, and the use of a complex enzyme combination of cellulase, papain, and pectinase is even rarer. Literature using this complex enzyme has suggested that prolonged enzymatic hydrolysis can damage the glucan structure.

[0003] Currently, research on lentinan (shiitake mushroom polysaccharides) mainly focuses on high-molecular-weight polysaccharides. While studies have confirmed that lentinan possesses various biological activities, such as lowering blood sugar, many publications emphasize the need to preserve the skeletal structure and avoid degradation during extraction. The oligosaccharides derived from enzymatic hydrolysis are typically low-degree polymers formed by 2-20 monosaccharides linked by glycosidic bonds, and no studies have disclosed their ability to lower blood sugar. Furthermore, the enzymatic extraction process for preparing lentinan oligosaccharides tends to involve a brief enzymatic hydrolysis with glucanase followed by further enzymatic hydrolysis with cellulase or similar enzymes.

[0004] There are no reliable reports on the targeted preparation of hypoglycemic polysaccharides from shiitake mushroom stems; there are no reports on the hypoglycemic effects of their oligosaccharides; and there are no reports on technologies and products that combine in vitro enzyme inhibition with in vivo animal model system verification.

[0005] Based on this, the present invention addresses the shortcomings of existing technologies by providing a stable, green, efficient, and highly active method for preparing E-LEBG oligosaccharides from shiitake mushroom stems, and clarifies its hypoglycemic activity, thereby realizing the high-value utilization of shiitake mushroom processing by-products. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the defects of existing lentinan, such as large molecular weight, poor solubility, low hypoglycemic activity and low utilization rate, and to provide E-LEBG with low molecular weight, strong water solubility, high purity and excellent hypoglycemic activity, as well as its preparation method and application.

[0007] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0008] On one hand, the present invention provides a method for preparing lentinan E-LEBG from shiitake stems, comprising the following steps:

[0009] I. LEBG Extraction of Polysaccharides from Shiitake Mushroom Stems:

[0010] Shiitake mushroom stem powder is extracted with hot water to obtain supernatant 1 and residue 2. The residue is enzymatically hydrolyzed (pH 3-8, such as 5-6) using a complex enzyme containing cellulase, papain, and pectinase (mass ratio 2:1:1 to 1:2:2, such as 1:1:1) to obtain supernatant 2. The hydrolysis temperature can be 35-65℃ (such as 50-60℃), and the hydrolysis time can be 0.5-5h (such as 1-3h). Supernatant 1 and supernatant 2 are combined and precipitated with an alcohol solvent (such as ethanol) to obtain a polysaccharide precipitate. The polysaccharide precipitate is then dialyzed or ultrafiltered (with a molecular weight cutoff of 7-10kD for the semipermeable membrane) to remove small molecule impurities, yielding LEBG crude sugar.

[0011] 2. Targeted enzymatic hydrolysis (specific enzymes, such as endonucleases):

[0012] The LEBG sample obtained in the previous step was enzymatically hydrolyzed with β-glucanase (endo-β-1,3-glucanase, which can be derived from Trichoderma and has a content of 100,000-200,000 U / g) to obtain E-LEBG oligosaccharides.

[0013] The material-to-liquid ratio is defined in this paper as the conventional definition in this field, that is, the mass-to-volume ratio of raw material to extract, in g / mL.

[0014] Optionally, the pH of the directional enzymatic hydrolysis is 5-6, such as pH 5.5.

[0015] Alternatively, the enzymatic hydrolysis temperature of dextranase is 50-60℃.

[0016] Optionally, the amount of β-glucanase used is 2-4% of the dry weight of the LEBG sample, such as 2.5%, 3%, or 3.5%. Alternatively, the enzymatic hydrolysis time of the glucanase is 60-180 min, preferably 90-180 min (such as 90, 120, or 150 min), and more preferably 120-180 min.

[0017] Optionally, the ratio of the dry weight of the LEBG sample to be enzymatically hydrolyzed to the water used for enzymatic hydrolysis is 1:60-100, such as 1:70, 1:80, or 1:90.

[0018] Optionally, the enzymatic hydrolysis conditions in step two are: pH 5.5, temperature 55℃, material-to-liquid ratio 1:80, β-glucanase addition 4.0%, and hydrolysis for 180 min. Optionally, the supernatant 2 is obtained by enzymatic hydrolysis of the compound enzyme product without hot water extraction.

[0019] Optionally, the cellulase activity content is 50,000 to 500,000 U / g.

[0020] Optionally, the amount of papain can be 100,000-600,000 U / g, and the amount of pectinase can be 50,000-100,000 U / g.

[0021] Optionally, the complex enzyme is composed of cellulase, papain and pectinase.

[0022] Optionally, the enzymatic hydrolysis time of the compound enzyme is 1.5-2.5 hours, such as 2 hours.

[0023] Optionally, the LEBG crude sugar is pre-deproteinized with Sevage reagent before being subjected to targeted enzymatic hydrolysis. Sevage reagent can be a mixture of chloroform and n-butanol in a ratio of chloroform:n-butanol = 4:1.

[0024] Optionally, the material-to-liquid ratio used for extracting the supernatant 1 or for enzymatic hydrolysis by the compound enzyme is 1:15-20. Alternatively, the extraction temperature is 90-100℃.

[0025] Optionally, the shiitake mushroom stem powder described in step one is pre-soaked and washed with an aqueous solution of an alcohol solvent (such as ethanol) to remove impurities and convert it into residue 1. Then, residue 1 is used for extraction.

[0026] Optionally, the washing uses a 75% ethanol solution (in this art, % refers to volume percentage), which can be added at a material-to-liquid ratio of 1:10 (g / mL). The washing can be achieved by heating and reflux, with a washing temperature of 75°C and a washing time of 2 hours.

[0027] Optionally, the particle size of the shiitake mushroom stem powder used is less than 100 mesh.

[0028] Optionally, before the alcohol precipitation, supernatant 1 and supernatant 2 are first concentrated (e.g., by vacuum concentration) to one-third of their original volume. Alternatively, anhydrous ethanol is used for alcohol precipitation, and the volume of ethanol can be 4-6 times that of the purified supernatant.

[0029] Optionally, the number-average molecular weight of the obtained E-LEBG is 5.30 × 10⁻⁶. 4 Da, dispersion coefficient PD=1.96; E-LEBG monosaccharide composition is mainly glucose, with a molar percentage of up to 80.07%, and may also contain 9.12% galactose, 5.01% mannose and trace amounts of glucuronic acid; E-LEBG glycosidic bonds are mainly β-type, and the infrared spectrum is at 890 cm⁻¹. ⁻¹ It has a characteristic absorption peak.

[0030] A specific example of the preparation method of the above-mentioned E-LEBG with hypoglycemic activity provided by the present invention includes the following steps:

[0031] S1: Raw material pretreatment: Shiitake mushroom stems are dried at 40℃, pulverized and passed through a 100-mesh sieve to obtain shiitake mushroom stem powder; 75% ethanol is added at a material-to-liquid ratio of 1:10, refluxed at 75℃ for 2 h, centrifuged and the supernatant is discarded to remove pigments, small molecule sugars and fat-soluble impurities, and to obtain the impurity-removed residue.

[0032] S2: Extraction of crude polysaccharide (LEBG) from shiitake stems: The residue was added to distilled water at a material-to-liquid ratio of 1:15 and extracted in a 95℃ water bath for 4 hours. The supernatant 1 was obtained by centrifugation. A compound enzyme (cellulase:papain:pectinase = 1:1:1) was added to the residue at a concentration of 2% (w / w). After enzymatic hydrolysis, the enzyme was inactivated at 100℃ for 10 minutes and centrifuged to obtain supernatant 2. The supernatants were combined and concentrated under reduced pressure to 1 / 3 volume. Four times the volume of anhydrous ethanol was added, and the mixture was allowed to stand overnight at 4℃. The precipitate was collected by centrifugation. Small molecule impurities were removed by dialysis using a 7000 Da dialysis bag, and LEBG was obtained by freeze-drying.

[0033] S3: Sevage method for deproteinization: LEBG was prepared into a 10 mg / mL aqueous solution, and 20% volume of Sevage reagent (chloroform: n-butanol = 4:1) was added. The mixture was shaken vigorously and centrifuged at 8000 r / min for 10 min. The lower protein phase was discarded. The process was repeated until no white precipitate was observed. The deproteinized polysaccharide was then lyophilized.

[0034] S4: Targeted enzymatic hydrolysis with β-glucanase: Dissolve deproteinized polysaccharide in water, adjust pH to 5.5 and temperature to 55℃; material-to-liquid ratio 1:80, β-glucanase addition 4.0%, enzymatic hydrolysis for 180 min; boil at 100℃ for 10 min to inactivate enzyme, cool, centrifuge at 4000 r / min for 20 min, and collect the supernatant;

[0035] S5: Freeze-drying: The enzymatic hydrolysis supernatant is concentrated under reduced pressure and freeze-dried to obtain E-LEBG with hypoglycemic activity.

[0036] Furthermore, the present invention provides lentinan E-LEBG from shiitake stems prepared by the above method.

[0037] This invention also provides the application of the above-mentioned E-LEBG in the preparation of hypoglycemic drugs and health products that help maintain healthy blood glucose levels, especially suitable for type II diabetes. E-LEBG can reduce fasting blood glucose, improve oral glucose tolerance, and alleviate weight loss in diabetic patients.

[0038] The present invention also provides the application of the above-mentioned E-LEBG in the preparation of α-amylase and α-glucosidase inhibitors.

[0039] Accordingly, the present invention also provides health products and medicines containing E-LEBG.

[0040] Compared with the prior art, the advantages of the present invention are as follows:

[0041] By extracting from shiitake mushroom stems and employing directional enzymatic hydrolysis coupling technology, highly efficient hypoglycemic oligosaccharides were directly obtained. This process converts high-molecular-weight polysaccharides into oligosaccharides with controllable molecular weight, uniform distribution, and significantly improved water solubility. The in vitro hypoglycemic activity is significantly superior to that of the raw polysaccharide. The inventors also completed dual verification in vitro and in vivo animal studies, demonstrating a clear hypoglycemic effect, high safety, and no obvious toxic side effects. Furthermore, the process is stable, simple to operate, operates under mild conditions, and is easily scaled up industrially, making it suitable for direct application in the development of functional foods, health products, and adjunctive hypoglycemic drugs. Attached Figure Description

[0042] The following figures are used to demonstrate the characteristics and activity of lentinan β-glucan E-LEBG from shiitake stems:

[0043] Figure 1 This describes the effect of the feed-to-liquid ratio on the inhibition rate of α-amylase / α-glucosidase.

[0044] Figure 2 This describes the effect of enzyme addition amount on the inhibition rate of α-amylase / α-glucosidase.

[0045] Figure 3 This describes the effect of enzymatic hydrolysis time on the inhibition rate of α-amylase / α-glucosidase.

[0046] Figure 4 This is the ion chromatogram of LEBG.

[0047] Figure 5 This is an ion chromatogram of E-LEBG.

[0048] Figure 6 These are the FT-IR infrared spectra of LEBG and E-LEBG.

[0049] Figure 7 The effect of E-LEBG on fasting blood glucose in T2DM mice. Detailed Implementation

[0050] The principles and features of the present invention are described below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. For example, the determination of α-amylase inhibition rate and α-glucosidase inhibition rate are conventional methods, and acarbose is used as a positive control. Unless otherwise specified, the materials and reagents used are commercially available. The key enzyme activities used are: cellulase, papain, and pectinase, with activities of 500,000 U / g, 100,000 U / g, and 50,000 U / g, respectively; and endo-β-1,3-glucanase from Trichoderma, with an activity of 100,000 U / g.

[0051] Example 1: Preparation of E-LEBG

[0052] S1: Raw material pretreatment: Shiitake mushroom stems are dried at 40℃, pulverized and passed through a 100-mesh sieve to obtain shiitake mushroom stem powder; 75% ethanol is added at a material-to-liquid ratio of 1:10, refluxed at 75℃ for 2 h, centrifuged at 4000 r / min for 20 min, and the supernatant is discarded to obtain impurity-removed residue 1.

[0053] S2: Crude polysaccharide extraction: Residue 1 was added to distilled water at a material-to-liquid ratio of 1:15 and extracted in a 95℃ water bath for 4 h. After centrifugation, supernatant 1 and residue 2 were obtained. Residue 2 was added to a compound enzyme (cellulase:papain:pectinase mass ratio = 1:1:1) at a dosage of 2%. After enzymatic hydrolysis, the enzyme was inactivated at 100℃ for 10 min and centrifuged to obtain supernatant 2. The supernatants were combined, concentrated under reduced pressure to 1 / 3 volume, and 4 times the volume of anhydrous ethanol was added. The mixture was precipitated overnight at 4℃. The precipitate was collected by centrifugation, dialyzed in a 7000 Da dialysis bag for 48 h, and freeze-dried to obtain LEBG with a yield of 9.15±0.03%.

[0054] S3: Deproteinization: Prepare a 10 mg / mL aqueous solution of LEBG, add 20% Sevage reagent, shake and centrifuge to remove protein, repeat until no white precipitate is found, and freeze dry.

[0055] S4: LEBG component determination:

[0056] ① Determination of total sugar content: The total sugar content in LEBG was determined by the phenol-sulfuric acid method.

[0057] ② Protein content determination: The protein content in LEBG was determined using the Coomassie Brilliant Blue method.

[0058] ③ β-glucan content determination: The β-glucan content in LEBG was determined by the difference calculation method. The specific steps are as follows: (1) Determination of total glucan content (P); (2) Determination of α-glucan content (P1); (3) Standard curve and calculation: Using anhydrous glucose as standard, the total glucan content minus the α-glucan content is the β-glucan content (P2).

[0059] The LEBG was found to contain 89.58±0.99% total sugar, 1.53±0.03% protein, and 71.49±1.33% β-glucan.

[0060] S5: Targeted enzymatic hydrolysis with β-glucanase: The LEBG product obtained in step S3 is dissolved in water and enzymatically hydrolyzed with β-glucanase derived from Trichoderma at pH 5.5 and temperature 55℃. Then, the enzyme is inactivated by boiling at 100℃ for 10 min, cooled, and centrifuged at 4000 r / min for 20 min. The supernatant obtained is the E-LEBG oligosaccharide product.

[0061] Example 2: Optimization of the extraction method of lentinan

[0062] Different E-LEBG oligosaccharide products were obtained by conducting single-factor or orthogonal experiments according to the method of Example 1, and their enzyme inhibition rate and activity were compared: The LEBG product obtained in step S3 of the previous example was dissolved in water, and enzymatically hydrolyzed with β-glucanase at pH 5.5 and temperature 55°C (other conditions are described in the optimization experiment below). Then, the enzyme was inactivated by boiling water bath, cooled, and centrifuged to obtain the enzymatic supernatant, which is the E-LEBG oligosaccharide product.

[0063] S1: The inhibition rates of α-amylase and α-glucosidase in the oligosaccharides extracted from each group in the single-factor experiment were evaluated.

[0064] Optimization of the extraction solid-liquid ratio: With a fixed extraction pH of 5.5, enzyme dosage of 2%, and hydrolysis time of 120 min, different extraction solid-liquid ratios were set at 1:60, 1:70, 1:80, 1:90, and 1:100. The results are as follows: Figure 1 As shown, when the material-to-liquid ratio reaches 1:80, the inhibition rates of α-amylase and α-glucosidase are 58.36±1.19% and 43.97±1.22%, respectively. Therefore, 1:80 is selected as the optimal material-to-liquid ratio for extraction.

[0065] Optimization of extraction enzyme dosage: With the extraction pH fixed at 5.5, hydrolysis time at 120 min, and solid-liquid ratio at 1:80, different extraction enzyme dosages were set at 2%, 2.5%, 3%, 3.5%, and 4%. Results are as follows: Figure 2 As shown.

[0066] Extraction time optimization: With a fixed extraction pH of 5.5, a solid-liquid ratio of 1:80, and an enzyme dosage of 3.5%, different extraction times were set at 60, 90, 120, 150, and 180 min. Results are as follows... Figure 3 As shown.

[0067] S2: Orthogonal experimental design

[0068] Based on the single-factor experiments, the extraction material-liquid ratio A (1:70, 1:80, 1:90) (these three ratios are recorded as 1, 2, and 3 in Table 3 from left to right, and the BC factors are recorded in the same way), enzyme addition amount B (3.0%, 3.5%, 4.0%), and enzymatic hydrolysis time C (120 min, 150 min, 180 min) were selected as the factors to be investigated. The extraction process was optimized by orthogonal experiment. The orthogonal experimental design table and results are shown in Table 1.

[0069] Table 1. Results of the orthogonal experiment

[0070]

[0071] In the orthogonal experiment, a comprehensive scoring method was used to analyze the experimental results. The results showed that the order of factors was B>C>A: enzyme dosage, enzymatic hydrolysis time, and solid-liquid ratio. The optimal combination was A2B3C3, under which the α-glucosidase inhibition rate was 57.25±0.96%. Therefore, the optimal extraction process for polysaccharides was determined to be: an extraction solid-liquid ratio of 1:80 g / mL, an extraction enzyme dosage of 4%, and an extraction time of 180 min. Under this optimal process, the yield of E-LEBG was 68.61±1.82%.

[0072] S3: Freeze-drying: The enzymatic hydrolysate of the above-mentioned dextranase is concentrated under reduced pressure, precipitated with alcohol, and then freeze-dried to obtain the E-LEBG product. The product corresponding to the process of the above-mentioned optimal combination A2B3C3 is called E-LEBG1.

[0073] Example 3: Structural Analysis of LEBG and E-LEBG

[0074] A comparative analysis was performed on the deproteinized LEBG obtained in Example 1 and the E-LEBG1 obtained in Example 2:

[0075] S1: Molecular weight analysis by gel permeation chromatography (GPC): 5 mg was accurately weighed and dissolved completely in 1 mL of 0.1 mol / L NaNO3 solution as the mobile phase. A differential refractive index detector (RID) was used with Ultrahydrogel™ 500 Column and Ultrahydrogel™ 250 Column gel chromatography columns in series. The number-average molecular weights (Mn) of LEBG and E-LEBG were determined to be 2.18 × 10⁻⁶. 5 Da, 5.30×10 4 Meanwhile, the dispersion factor (PD) of E-LEBG decreased from 4.15 for LEBG to 1.96, indicating that the enzymatic hydrolysate E-LEBG has a lower molecular weight and a more uniform molecular weight distribution.

[0076] S2: Monosaccharide composition analysis:

[0077] The monosaccharide composition of LEBG and E-LEBG was determined by ion chromatography. Twelve monosaccharides—fucose (Fuc), rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), ribose (Rib), xylose (Xyl), mannose (Man), galacturonic acid (Gal-UA), glucuronic acid (Glc-UA), mannuronic acid (Man-UA), and guluronic acid (Glu-UA)—were used as standards, and a standard curve was established using the five-point external standard method. 2 mg of sample was accurately weighed into a hydrolysis flask, and 3 mL of 2 mol / L trifluoroacetic acid (TFA) aqueous solution was added. The flask was sealed and hydrolyzed in an oil bath at 121 °C for 3 h. After cooling to room temperature, the sample was completely dried using a nitrogen evaporator. After drying, 3 mL of methanol was added to each flask to reconstitute the solution, and the flask was dried again. This process was repeated three times to remove all TFA. Subsequently, 1 mL of ultrapure water was added and the mixture was repeatedly shaken to fully dissolve the dry matter in the bottle. The dissolved material was then transferred to a 2 mL centrifuge tube, thawed, and diluted to an appropriate concentration before analysis. An electrochemical detector ion exchange chromatography system (Thermo ICS-5000+) equipped with a Dionex CarboPac PA20 analytical column (3 × 150 mm, 10 μm) was used, employing a monosaccharide PA20 program for gradient elution to detect monosaccharides in the hydrolyzed sample. Results are as follows: Figure 4 , 5 As shown, the monosaccharide composition of both LEBG and E-LEBG is mainly glucose, and also contains a certain proportion of Gal, Man, etc. In E-LEBG, the main sugars are Glc (80.07 mol%), Gal (9.12 mol%), and Man (5.01 mol%). Compared with LEBG, the proportion of glucose in E-LEBG increases by 4.94 mol.

[0078] S3: Fourier transform infrared spectroscopy analysis:

[0079] Grind 2 mg of sample with 200 mg of KBr in a mortar, then place the powder into a tablet press, tighten it, apply a pressure of 15 MPa and maintain it for about 1 min. Slowly release the pressure to zero and remove the tablet. After turning on the machine, first acquire background data (using 200 mg KBr as a blank), then insert the sample for scanning. The scanning range is 4000–450 cm⁻¹. -1 FT-IR analysis was performed on the region at a resolution of 4 cm. -1 The number of scans was 32. The main peaks (intensity and wavenumber) of the test sample were identified and analyzed using the instrument software EZOMNIC 6.0 (Thermo Electron Corporation, MA, USA). The results are as follows: Figure 6As shown, the broad, strong absorption peaks around 3290 cm⁻¹ in both LEBG and E-LEBG correspond to the OH stretching vibration of sugars, the absorption near 2900 cm⁻¹ originates from the CH stretching vibration, the band at 1640 cm⁻¹ reflects the C=O stretching vibration of uronic acid, and 1370 cm⁻¹ is a characteristic peak of the CH bending vibration. In the fingerprint region, the strong absorption near 1020 cm⁻¹ is attributed to the COC and CO stretching vibrations of the pyranose ring, while the absorption at approximately 890 cm⁻¹ is a characteristic peak of the β-glycosidic bond configuration, indicating the presence of β-glycosidic bonds in both LEBG and E-LEBG.

[0080] Example 4: Comparison of in vitro hypoglycemic activity between LEBG and E-LEBG

[0081] A comparative analysis was performed on the deproteinized LEBG obtained in Example 1 and the E-LEBG1 obtained in Example 2, using acarbose as a positive control. The α-amylase inhibition rate and α-glucosidase inhibition rate of the two sugars were measured and calculated according to the formula. The results showed that:

[0082] Within the concentration range of 0.5–16.0 mg / mL, E-LEBG exhibited higher inhibition rates against both α-amylase and α-glucosidase than LEBG. At a concentration of 16 mg / mL, the inhibition rates of α-amylase and α-glucosidase by E-LEBG were 68.61 ± 0.89% and 61.06 ± 1.10%, respectively, significantly superior to LEBG and 2.21 times and 1.84 times that of LEBG, respectively.

[0083] Example 5: Verification of E-LEBG's in vivo hypoglycemic activity

[0084] Taking E-LEBG1 obtained in Example 2 as an example, the activity of oligosaccharides in fasting blood glucose and glucose tolerance indicators was verified:

[0085] S1: Animal Experiment Design: Purchase 80 SPF-grade male C57 BL / 6J mice (6 weeks old, 20-22g). All mice were housed in an environment with a temperature of 22±0.5℃, humidity of 60±5%, and a 12-hour light-dark cycle. They had free access to food and water.

[0086] After one week of acclimatization, 10 mice were randomly selected as the normal control group (NC) and fed a normal diet. The remaining mice were used as the model group and fed a high-fat diet (HFD) to induce obesity and insulin resistance. After three weeks of feeding, all mice were fasted overnight but allowed water for 12 hours. Mice in the model group were intraperitoneally injected for three consecutive days with 55 mg / kg BW 1% freshly prepared low-temperature streptozotocin (STZ) solution (dissolved in 0.1 mol / L citrate buffer, pH=4.5). Mice in the NC group were intraperitoneally injected with the same dose of citrate buffer. 72 hours after the intraperitoneal injection, all mice were again fasted overnight but allowed water for 12 hours. Fasting blood glucose levels were rapidly measured in each group using an ACCU-CHEK blood glucose meter via tail clipping. A fasting blood glucose level greater than or equal to 11.1 mmol / L was considered a successful establishment of the T2DM mouse model. Successfully constructed type 2 diabetes mellitus (T2DM) mice were randomly divided into five groups of 10 mice each: metformin group (MET group), diabetes model group (DM group), low-dose E-LEBG intervention group (LD group, 200 mg / kg BW), medium-dose E-LEBG intervention group (MD group, 400 mg / kg BW), and high-dose E-LEBG intervention group (HD group, 800 mg / kg BW). Mice in the LD, MD, and HD groups were administered different doses of E-LEBG solution dissolved in 0.9% sterile saline via gavage once daily. Mice in the MET group were administered 200 mg / kg BW of metformin hydrochloride solution dissolved in 0.9% sterile saline via gavage. Mice in the NC and DM groups were administered the same dose of 0.9% sterile saline via gavage. The intervention period was 4 weeks, and the body weight and fasting blood glucose (FBG) of each group were recorded weekly. After the experiment, all mice were fasted overnight, euthanized by cervical dislocation under isoflurane anesthesia, and biological samples were collected for further analysis.

[0087] S2: Measurement of fasting blood glucose and glucose tolerance: All mice were fasted overnight for 12 hours with free access to water on a fixed day each week. Fasting blood glucose was measured in each group of mice the following day by tail clipping. Oral glucose tolerance test was performed in the last week of E-LEBG intervention; and the area under the curve (AUC) of the time-blood glucose curve was calculated.

[0088] S3: Experimental Results: Effects of E-LEBG on Body Weight and Fasting Blood Glucose.

[0089] During the experiment, the body weight of mice in the DM group decreased continuously as their diabetes worsened, while the body weight of mice in the NC group steadily increased. Initially, all groups showed a significant increase in body weight compared to the NC group, exhibiting characteristics of obesity. After 4 weeks of intervention, the body weight of mice in the NC group consistently increased, while the body weight of mice in the DM group gradually decreased, consistent with the symptoms of weight loss in diabetic mice. Compared to the DM group, the symptoms of weight loss were alleviated in all intervention groups. At the end of the experiment, the average body weight of mice in the DM group decreased by 9.13%, while the body weight of mice in the MET group did not decrease significantly after the intervention. The body weight loss in the E-LEBG intervention groups was smaller, indicating that E-LEBG and metformin hydrochloride have a certain alleviating effect on weight loss in diabetic mice.

[0090] The effect of E-LEBG on hyperglycemia in type 2 diabetes mellitus (T2DM) mice was evaluated by measuring fasting blood glucose (FBG) weekly during the experiment. Figure 7 As shown, at the start of the gavage experiment (week 0), compared with the NC group, the FBG of mice in other groups was significantly increased (P<0.05), and all were much higher than 11.1 mmol / L, indicating that the T2DM mouse model was successfully established. After 4 weeks of intervention, the blood glucose levels of mice in the NC and DM groups did not change significantly and remained within a certain range; while the FBG levels of all E-LEBG intervention groups and the MET group were significantly lower than those in the DM group (P<0.05). Compared with the DM group, the blood glucose levels of mice in the low, medium, and high dose E-LEBG intervention groups decreased by 30.07%, 35.40%, and 39.61%, respectively, while the blood glucose levels of mice in the MET group decreased by 46.65%. The experimental results indicate that both E-LEBG and metformin hydrochloride have good hypoglycemic effects.

[0091] S4: Experimental Results: Effect of E-LEBG on glucose tolerance.

[0092] In all experimental groups, blood glucose levels rose rapidly after oral administration of glucose solution, peaking at 30 min, and then gradually decreased over the next 90 min. Compared with the other five groups, the NC group mice were more sensitive to changes in glucose, with blood glucose returning to normal within 120 min. However, blood glucose levels in the DM group mice remained high after 120 min, indicating severe deterioration of glucose tolerance in untreated T2DM mice. E-LEBG intervention significantly reduced the peak blood glucose level in T2DM mice. At the end of the oral glucose tolerance test (OGTT), blood glucose levels in the high, medium, and low dose E-LEBG intervention groups were significantly lower than those in the DM group (P<0.05). Further quantification of glucose tolerance by area under the curve (AUC) showed that the AUC values ​​in all diabetic groups were significantly higher than those in the NC group (P<0.05). Furthermore, the AUC values ​​in diabetic mice decreased sequentially from the DM, LD, MD, HD, and MET groups, indicating that all doses of E-LEBG significantly improved glucose tolerance in diabetic mice.

Claims

1. A method for preparing lentinan E-LEBG from shiitake mushroom stems, comprising the following steps:

1. Extraction of LEBG polysaccharides from shiitake mushroom stems: Shiitake mushroom stem powder is extracted with hot water to obtain supernatant 1 and residue. The residue is enzymatically hydrolyzed (pH 3-8, such as 5-6) using a complex enzyme containing cellulase, papain, and pectinase (mass ratio 2:1:1 to 1:2:2, such as 1:1:1) to obtain supernatant 2. The hydrolysis temperature can be 35-65℃ (such as 50-60℃), and the hydrolysis time can be 0.5-5h (such as 1-3h). Supernatant 1 and supernatant 2 are combined and precipitated with an alcohol solvent (such as ethanol) to obtain polysaccharide precipitate. The polysaccharide precipitate is then dialyzed or ultrafiltered (with a molecular weight cutoff of 7-10kD for the semipermeable membrane) to remove small molecule impurities, yielding LEBG crude sugar.

2. Targeted enzymatic hydrolysis: use β The LEBG sample obtained in the previous step was enzymatically digested with glucanase (endo-β-1,3-glucanase, which can be derived from Trichoderma, with an enzyme activity of 100,000-200,000 U / g) to obtain E-LEBG oligosaccharides.

2. The method as described in claim 1, characterized in that, The pH of the directed enzymatic hydrolysis is 5-6, such as pH 5.5; optionally, the enzymatic hydrolysis temperature is 50-60℃.

3. The method as described in any of the prior claims, characterized in that, The β The amount of dextranase used is 2-4% of the dry weight of the LEBG sample, such as 2.5%, 3%, or 3.5%. Optionally, the enzymatic hydrolysis time is 60-180 min, such as 90, 120, or 150 min. Optionally, the ratio of the dry weight of the LEBG sample to the water used for enzymatic hydrolysis is 1:60-100, such as 1:70, 1:80, or 1:

90.

4. The method as described in claim 1, characterized in that, The supernatant 2 is obtained by enzymatic hydrolysis of the compound enzyme product without hot water extraction.

5. The method as described in claim 1, characterized in that, The cellulase activity is 50,000-500,000 U / g, the papain activity is 100,000-600,000 U / g, and the pectinase activity is 50,000-100,000 U / g.

6. The method as described in claim 1, characterized in that, Before being subjected to targeted enzymatic hydrolysis, the LEBG crude sugar was deproteinized with Sevage reagent.

7. The method as described in claim 1, characterized in that, The material-to-liquid ratio used for extracting the supernatant 1 or for enzymatic hydrolysis of the compound enzyme is 1:15-20. Optionally, the extraction temperature is 90-100℃.

8. The method as claimed in any of the prior claims, characterized in that, Before soaking, the shiitake mushroom stem powder is pre-washed with an aqueous solution of an alcohol solvent (such as ethanol) to remove impurities and convert it into residue, which is then used for extraction.

9. E-LEBG prepared by the method of any of the prior claims.

10. The use of the E-LEBG as described in the preceding claim in the preparation of hypoglycemic products.

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