A polypeptide hydrogel based on oxidized Ganoderma lucidum polysaccharide and its application

Abstract

This invention discloses a polypeptide hydrogel based on oxidized Ganoderma lucidum polysaccharide and its applications. The preparation method of the polypeptide hydrogel of this invention is simple, the gelation is rapid, and it has good stability, antioxidant activity, and inhibition of tyrosinase activity. The polypeptide hydrogel of this invention provides a physical barrier for bioactive peptides, enabling them to overcome the influence of the gastrointestinal environment, allowing 71% of the peptides to be released in the intestine, improving the bioavailability of peptides, and effectively solving the problems of limitations in peptide use conditions.

Description

(I) Technical Field

[0002] This invention relates to a polypeptide hydrogel based on oxidized Ganoderma lucidum polysaccharide and its applications. (II) Background Technology

[0004] Pearl peptides are peptides extracted from pearls that possess various biological activities (antibacterial, antioxidant, anti-tyrosinase, etc.) (Jingying Pei, et al. Extraction, purification, bioactivities and application of matrix proteins from pearl powder and nacre powder: A review. Frontiers in Bioengineering and Biotechnology, 2021). However, proteins and peptides are highly sensitive to the environment; changes in pH and environmental ion concentration can denature and inactivate them (Jun Ye, Targeted release of soybean peptide from CMC / PVA hydrogels in simulateddintestinal fluid and their pharmacokinetics, Carbohydrate Polymers, 2023). Furthermore, the drastic pH changes and high ionic strength in the gastrointestinal tract result in extremely low bioavailability of peptides absorbed orally.

[0005] Hydrogels are soft materials with a three-dimensional network structure. Polysaccharide-based hydrogels possess excellent biocompatibility, biodegradability, and water retention. Furthermore, polysaccharides are readily available and have low toxicity, making them ideal carriers for orally administered small-molecule bioactive peptides (Arshied Manzoor, Recent insights into polysaccharide-based hydrogels and their potential applications in the food sector: A review, International Journal of Biological Macromolecules, 2022). By encapsulating bioactive peptides in hydrogels, the effects of gastric acid and enzymes on the peptides during gastrointestinal digestion can be avoided.

[0006] Reishi mushroom is an edible fungus used both as food and medicine, possessing anti-cancer, anti-inflammatory, and antioxidant activities. Reishi polysaccharides are one of its main active components (LiFang Zhu, Engineering of Ganoderma lucidum polysaccharide loaded polyvinyl alcohol nanofibers for biopharmaceutical delivery, Journal of Drug Delivery Science and Technology, 2019). However, current research on reishi polysaccharides mainly focuses on extraction, purification, and activity verification, while research on using reishi polysaccharides to prepare hydrogels and applying them to the transport of nutrients is extremely limited.

[0007] The patent "A Preparation Method of Ganoderma lucidum Polysaccharide Hydrogel and Its Application in Diabetic Wound Repair" (CN117752851 A) by Sui Xiaoyu et al. shows that the Ganoderma lucidum polysaccharide hydrogel prepared using Ganoderma lucidum polysaccharide and carboxymethyl chitosan as raw materials, with the addition of sodium alginate and a cross-linking agent, has a good effect on repairing wound damage. Animal experiments showed that the wound healing rate of the Ganoderma lucidum polysaccharide hydrogel group reached 98.94±0.77% on day 21, which was higher than that of the CMC / SA group and the saline group. However, the hydrogel prepared by this method contains a cross-linking agent, which has high toxicity.

[0008] The patent "A Galactosylated Sodium Alginate Derivative Gel Microsphere and Its Preparation Method and Application" (CN202310710208) by Zhang Liming et al. shows that galactosylated sodium alginate derivative gel microspheres can be used as a carrier material to encapsulate bovine serum albumin. When 5 mg / ml bovine serum albumin solution is added, the encapsulation rate reaches 64.24%. In deionized water, the maximum cumulative release is reached on the 12th day, almost reaching 100%. However, the gelation of this gel requires 5-15 minutes, which is relatively long. (III) Summary of the Invention

[0010] The purpose of this invention is to provide a polypeptide hydrogel based on oxidized Ganoderma lucidum polysaccharide and its application. The polypeptide hydrogel prepared by the method of this invention is simple to prepare and forms a gel rapidly. The prepared hydrogel has certain antioxidant and anti-tyrosinase activities and shows good inhibitory effects on Escherichia coli and Staphylococcus aureus. At the same time, the hydrogel is released in the intestine in a targeted manner, which improves the bioavailability of orally administered peptides and effectively solves the problem that peptides are easily inactivated by the gastrointestinal environment during oral administration of bioactive peptides.

[0011] The technical solution adopted in this invention is:

[0012] This invention provides a polypeptide hydrogel based on oxidized Ganoderma lucidum polysaccharide, wherein the polypeptide hydrogel is prepared by the following method:

[0013] (1) Preparation of oxidized Ganoderma lucidum polysaccharide

[0014] Ganoderma lucidum polysaccharide was added to distilled water and stirred until completely dissolved. Sodium periodate was added under light-protected conditions and the reaction was carried out at room temperature in the dark for 5-10 hours (preferably 6 hours). Ethylene glycol was added and stirred to terminate the reaction. The reaction mixture was placed in a dialysis bag and dialyzed in pure water for 1-3 days (preferably 2 days). The retentate was then freeze-dried (preferably at -80℃ for 48 hours) to obtain oxidized Ganoderma lucidum polysaccharide.

[0015] (2) Preparation of polypeptide hydrogels

[0016] Add a polypeptide to an aqueous solution of 2-10% carboxymethyl chitosan (CMCS) to achieve a polypeptide concentration of 0.05-1%, and obtain a polypeptide solution; mix an aqueous solution of 1-5% oxidized Ganoderma lucidum polysaccharide with the polypeptide solution at a volume ratio of 1:0.5-2, and allow to stand at room temperature to form a gel, thus obtaining a polypeptide hydrogel.

[0017] Furthermore, in step (1), Ganoderma lucidum polysaccharide is obtained by removing protein from Ganoderma lucidum extract, and the polysaccharide content is 55-65%.

[0018] Further, in step (1), Ganoderma lucidum polysaccharide is prepared as follows: Sevage reagent is added to the aqueous solution of Ganoderma lucidum extract, and after mixing and shaking, the lower water layer and denatured proteins at the interface are removed. This process is repeated several times until no denatured proteins precipitate, and the clear polysaccharide solution is collected. Then, anhydrous ethanol with a final volume concentration of 80% is added, and the mixture is left to stand overnight at room temperature. After centrifugation, the precipitate is collected. The precipitate is reconstituted with water, and the organic solvent is removed by rotary evaporation. The mixture is then freeze-dried at -80℃ for 48 hours to obtain Ganoderma lucidum polysaccharide. The Sevage reagent is a chloroform and n-butanol solution with a volume ratio of 4:1. The volume of the Sevage reagent is 1 / 3 of the volume of the aqueous solution of Ganoderma lucidum extract. The volume of distilled water in the aqueous solution of Ganoderma lucidum extract is 10 mL / g based on the mass of Ganoderma lucidum extract.

[0019] Further, in step (1), the volume of distilled water used is 50-150 mL / g (preferably 100 mL / g) based on the mass of Ganoderma lucidum polysaccharide; the molar ratio of Ganoderma lucidum polysaccharide to sodium periodate is 1:0.1-1 (preferably 1:0.6); and the volume of ethylene glycol used is 1-10 mL / g (preferably 5 mL / g) based on the mass of Ganoderma lucidum polysaccharide.

[0020] Furthermore, in step (1), the molecular weight cutoff of the dialysis bag is 3.5 kDa.

[0021] Further, in step (2), the carboxymethyl chitosan (CMCS) aqueous solution has a mass concentration of 5%, and the polypeptide has a mass concentration of 0.1%. The oxidized Ganoderma lucidum polysaccharide aqueous solution has a mass concentration of 3%. The oxidized Ganoderma lucidum polysaccharide aqueous solution and the polypeptide solution are mixed at a volume ratio of 1:1.

[0022] Furthermore, the polypeptide in step (2) includes a pearl polypeptide, which is prepared according to the method of patent application CN112390848A.

[0023] The present invention also provides the application of the polypeptide hydrogel in the preparation of polypeptide intestinal sustained-release drugs, wherein the drugs enable 71% of the polypeptides to be released in the intestine, and the drug dosage form includes a solid dosage form, and the administration method includes oral administration.

[0024] The present invention also provides an application of the polypeptide hydrogel in the preparation of antibacterial drugs, wherein the polypeptide in the hydrogel is a pearl polypeptide, and the antibacterial drug is a drug that inhibits the activity of Escherichia coli or Staphylococcus aureus.

[0025] Compared with existing methods, the beneficial effects of this invention are mainly reflected in:

[0026] (1) The preparation method of the polypeptide hydrogel of the present invention is simple and the gelation is rapid. The present invention improves the gelation speed by using oxidized Ganoderma lucidum polysaccharide and carboxymethyl chitosan.

[0027] (2) The polypeptide hydrogel of the present invention has good stability. The polypeptide hydrogel has a porous structure and good mechanical properties, thermal stability and swelling characteristics.

[0028] (3) The polypeptide hydrogel of the present invention has antioxidant activity and tyrosinase inhibition activity; the scavenging rate of 1 mg / mL Ganoderma lucidum polysaccharide hydrogel extract against DPPH, ABTS and hydroxyl radicals reached 25%, 30% and 48%, respectively, while the addition of pearl polypeptide further enhanced its antioxidant capacity, reaching 27%, 33% and 54%, respectively. The 1 mg / mL pearl polypeptide hydrogel extract inhibited tyrosinase by 19%, inhibited Escherichia coli by more than 99%, and inhibited Staphylococcus aureus by more than 98%.

[0029] (4) The polypeptide hydrogel of the present invention provides a physical barrier for bioactive peptides, enabling them to overcome the influence of the gastrointestinal environment, allowing 71% of the polypeptides to be released in the intestine, improving the bioavailability of peptides, and effectively solving the problem of limitations on the use of polypeptides. (iv) Description of the attached drawings

[0031] Figure 1 Photos of Ganoderma lucidum polysaccharide hydrogels at different oxidation levels.

[0032] Figure 2 Antioxidant activity of Ganoderma lucidum polysaccharides with different oxidation levels.

[0033] Figure 3 Scanning electron micrographs of Ganoderma lucidum polysaccharide hydrogels and pearl polypeptide hydrogels with different volume ratios.

[0034] Figure 4 Infrared spectra of Ganoderma lucidum polysaccharides and oxidized Ganoderma lucidum polysaccharides.

[0035] Figure 5 Infrared spectra of Ganoderma lucidum polysaccharide hydrogel, pearl polypeptide hydrogel, and CMCS.

[0036] Figure 6 G'-G'' curves of Ganoderma lucidum polysaccharide hydrogel and pearl polypeptide hydrogel.

[0037] Figure 7 Swelling rates of Ganoderma lucidum polysaccharide hydrogel and pearl polypeptide hydrogel.

[0038] Figure 8 1. In vitro simulation of gastrointestinal digestion and release of pearl polypeptide hydrogel pearl polypeptides.

[0039] Figure 9 Antioxidant and antityrosinase activities of extracts of Ganoderma lucidum polysaccharide hydrogel and pearl polypeptide hydrogel.

[0040] Figure 10 Antibacterial activity of Ganoderma lucidum polysaccharide hydrogel and pearl polypeptide hydrogel. (V) Detailed Implementation Methods

[0042] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0043] The Ganoderma lucidum extract used in this embodiment of the invention was purchased from Zhejiang Huihe Health Technology Co., Ltd., and its polysaccharide content is ≥10%. The pearl polypeptide was prepared according to the method of Example 3 in patent application CN112390848A.

[0044] The room temperature described in this invention is 25-30℃.

[0045] Example 1: Preparation and performance testing of Ganoderma lucidum polysaccharide hydrogel

[0046] 1. Preparation of Ganoderma lucidum polysaccharide hydrogel

[0047] (1) Preparation of Ganoderma lucidum polysaccharides

[0048] Add 1 / 3 volume of Sevage reagent (a chloroform and n-butanol solution in a 4:1 volume ratio) to 40 g of Ganoderma lucidum extract dissolved in 400 mL of distilled water. Mix and shake to remove the lower aqueous layer and denatured proteins at the interface. Repeat this process several times until no denatured proteins precipitate. Collect the clear polysaccharide solution. Add anhydrous ethanol with a final volume concentration of 80%, let stand overnight at room temperature, centrifuge, and collect the precipitate. Redissolve the precipitate in water, remove the organic solvent by rotary evaporation, and freeze-dry at -80℃ for 48 h to obtain 15 g of Ganoderma lucidum polysaccharide, denoted as GLP.

[0049] (2) Preparation of oxidized Ganoderma lucidum polysaccharides

[0050] Weigh 1 g (0.0056 mol) of the Ganoderma lucidum polysaccharide sample prepared in step (1) and add it to 100 mL of distilled water. Stir until completely dissolved. Add 0.7 g (0.0032 mol) of sodium periodate under light-protected conditions and react at room temperature in the dark for 6 h. Add 5 mL of ethylene glycol and stir for 30 min to terminate the reaction. Place the reaction mixture into a dialysis bag (3.5 kDa) and dialyze with pure water for 2 days. Take the retentate and freeze-dry it at -80℃ for 48 h to obtain 0.7 g of oxidized Ganoderma lucidum polysaccharide, denoted as OGLP.

[0051] (3) Preparation of Ganoderma lucidum polysaccharide hydrogel

[0052] A 3% (w / w) aqueous solution of oxidized Ganoderma lucidum polysaccharide was prepared by adding water to the oxidized Ganoderma lucidum polysaccharide. This solution was then mixed with a 5% (w / w) aqueous solution of carboxymethyl chitosan (CMCS) at a volume ratio of 1:1. The mixture was allowed to stand at room temperature to form a gel, resulting in a Ganoderma lucidum polysaccharide hydrogel, denoted as G-GLP2. (See photo below.) Figure 1 .

[0053] 2. Performance testing of Ganoderma lucidum polysaccharide hydrogel

[0054] (1) gelation time

[0055] The gelation time was determined using the vial inversion method (Wang Bulei. Preparation and characterization of Bletilla striata polysaccharide hydrogel, 2021), see Table 1.

[0056] (2) Oxidation degree

[0057] The oxidation degree of Ganoderma lucidum polysaccharides was determined by titration (Wang Kuntang. Preparation, performance study and application of Bletilla striata polysaccharide-based hydrogel in wound repair, 2022), see Table 1.

[0058] 3. The antioxidant activity of oxidized Ganoderma lucidum polysaccharides was evaluated.

[0059] (1) DPPH removal rate

[0060] Add 50 µL of 0.1–5 mg / mL (0.1, 0.2, 0.5, 1, 2, 5 mg / mL) OGLP aqueous solution prepared in step 1 to a 96-well plate, followed by 150 µL of 0.2 mg / mL DPPH ethanol solution (sample group); mix 50 µL of OGLP aqueous solution (0.1, 0.2, 0.5, 1, 2, 5 mg / mL) with 150 µL of anhydrous ethanol solution (blank control group); mix 150 µL of 0.2 mmol / L DPPH ethanol solution with 50 µL of distilled water (substrate group). Incubate at room temperature for 30 min and measure absorbance (517 nm). Under the same conditions, replace OGLP with V... C GLP was used as a control. Results are shown below. Figure 2 .

[0061]

[0062] In the formula: A0 is the absorbance of the blank control group; A i A represents the absorbance of the sample group. j ν represents the absorbance of the substrate group.

[0063] (2) ABTS clearance rate

[0064] Dissolve 40 mg of ABTS (2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt) and 6.88 mg of potassium persulfate in 10 ml of distilled water, and let stand at room temperature in the dark for 12 h to obtain the ABTS stock solution. Add PBS buffer (pH 7.4) to the ABTS stock solution until its absorbance at 734 nm is 0.70 ± 0.02 to obtain the ABTS working solution.

[0065] Add 50 µL of 0.1–5 mg / mL (0.1, 0.2, 0.5, 1, 2, 5 mg / mL) OGLP aqueous solution prepared in step 1 to a 96-well plate, followed by 150 µL of ABTS working solution to form the sample group; mix 50 µL of OGLP aqueous solution (0.1, 0.2, 0.5, 1, 2, 5 mg / mL) with 150 µL of PBS buffer to form the blank control group; and mix 50 µL of distilled water with 150 µL of ABTS working solution to form the substrate group. After mixing, react at room temperature for 5 min and measure the absorbance at 734 nm. Under the same conditions, replace OGLP with V C GLP was used as a control. Results are shown below. Figure 2 .

[0066]

[0067] In the formula: A t The absorbance of the blank control group; A bA represents the absorbance of the sample group. s ν represents the absorbance of the substrate group.

[0068] (3) Hydroxyl radical scavenging rate

[0069] Add 300 μL of 9 mmol / L FeSO4 aqueous solution and 300 μL of 9 mmol / L salicylic acid ethanol solution to a test tube, then add 100 μL of OGLP aqueous solution prepared in step 1 at concentrations of 0.1-5 mg / mL (0.1, 0.2, 0.5, 1, 2, 5 mg / mL), followed by 300 μL of 8.8 mmol / L H2O2 aqueous solution. This constitutes the sample group. Distilled water is used instead of H2O2 as the substrate group, and distilled water is used instead of OGLP aqueous solution as the blank control group. React in a 37℃ water bath for 30 min, and then measure the absorbance at 510 nm. Under the same conditions, replace OGLP with V C GLP was used as a control. Results are shown in […]. Figure 2 .

[0070]

[0071] In the formula: A k The absorbance of the blank control group; A q A represents the absorbance of the sample group. h ν represents the absorbance of the substrate group.

[0072] Comparative Example 1:

[0073] In Example 1, step (1), the amount of sodium periodate added was changed to 20% of the molar amount of Ganoderma lucidum polysaccharide. All other operations remained the same. The gelation time and oxidation degree are shown in Table 1. Photographs of the Ganoderma lucidum polysaccharide hydrogel are shown in [Table 1]. Figure 1 .

[0074] Comparative Example 2:

[0075] In Example 1, step (1), the amount of sodium periodate added was changed to 40% of the molar amount of Ganoderma lucidum polysaccharide. All other operations remained the same. The gelation time and oxidation degree are shown in Table 1. Photographs of the Ganoderma lucidum polysaccharide hydrogel are shown in [Table 1]. Figure 1 .

[0076] Comparative Example 3:

[0077] In Example 1, step (1), the amount of sodium periodate added was changed to 80% of the molar amount of Ganoderma lucidum polysaccharide. All other operations remained the same. The gelation time and oxidation degree are shown in Table 1. Photographs of the Ganoderma lucidum polysaccharide hydrogel are shown in [Table 1]. Figure 1 Antioxidant activity is shown in Figure 2 .

[0078] Comparative Example 4:

[0079] In Example 1, step (1), the amount of sodium periodate added was changed to 100% of the molar amount of Ganoderma lucidum polysaccharide. All other operations remained the same. The gelation time and oxidation degree are shown in Table 1. Photographs of the Ganoderma lucidum polysaccharide hydrogel are shown in [Table 1]. Figure 1 Antioxidant activity is shown in Figure 2 .

[0080] Table 1. Gel formation time of each hydrogel

[0081]

[0082] Table 1 shows that the Ganoderma lucidum polysaccharide hydrogel prepared by the molar ratio of Ganoderma lucidum polysaccharide to sodium periodate of 1:0.6 as described in Example 1 has more ideal characterization results.

[0083] Figure 2 The results showed that the aqueous solution of Ganoderma lucidum polysaccharide had good antioxidant activity. Comparing the antioxidant activities of different concentrations of GLP and OGLP, the oxidation degree of OGLP was 25%, 26%, and 29%. OGLP with an oxidation degree of 25% showed the best antioxidant activity, but it was slightly lower than that of GLP. Therefore, the oxidized Ganoderma lucidum polysaccharide with an oxidation degree of 25% prepared by the molar ratio of Ganoderma lucidum polysaccharide and sodium periodate in Example 1 (1:0.6) was selected as the raw material for the hydrogel.

[0084] Example 2: Preparation of Pearl Polypeptide Hydrogel

[0085] (1) Preparation of oxidized Ganoderma lucidum polysaccharide

[0086] Weigh 1 g (0.0056 mol) of the Ganoderma lucidum polysaccharide sample prepared by the method in Example 1 and add it to 100 mL of distilled water. Stir until completely dissolved. Add 0.7 g (0.0032 mol) of sodium periodate under light-protected conditions and react at room temperature in the dark for 6 h. Add 5 mL of ethylene glycol and stir for 30 min to terminate the reaction. Place the reaction mixture into a dialysis bag (3.5 kDa) and dialyze with pure water for 2 days. Take the retentate and freeze-dry it at -80℃ for 48 h to obtain 0.7 g of oxidized Ganoderma lucidum polysaccharide.

[0087] (2) Preparation of pearl polypeptide hydrogel

[0088] Prepare a 3% (w / w) aqueous solution of oxidized Ganoderma lucidum polysaccharide by adding a certain amount of water to the oxidized Ganoderma lucidum polysaccharide.

[0089] Pearl polypeptide was added to a 5% (w / w) aqueous solution of carboxymethyl chitosan (CMCS) to achieve a pearl polypeptide concentration of 0.1%, thus obtaining a pearl polypeptide solution.

[0090] An aqueous solution of oxidized Ganoderma lucidum polysaccharide and a pearl polypeptide solution were mixed at a volume ratio of 1:1 and allowed to stand at room temperature to form a gel, resulting in a pearl polypeptide hydrogel, denoted as G-GLP-PP.

[0091] Comparative Example 5:

[0092] In step 1(3) of Example 1, the volume ratio of the oxidized Ganoderma lucidum polysaccharide solution to the 5% carboxymethyl chitosan (CMCS) aqueous solution was changed to 1:2, and the rest of the operation was the same, to obtain Ganoderma lucidum polysaccharide hydrogel, which was denoted as G-GLP1.

[0093] Comparative Example 6:

[0094] In step 1(3) of Example 1, the volume ratio of oxidized Ganoderma lucidum polysaccharide solution to 5% carboxymethyl chitosan (CMCS) aqueous solution was changed to 1:0.5, and the rest of the operation was the same, to obtain Ganoderma lucidum polysaccharide hydrogel, which was denoted as G-GLP3.

[0095] Example 3: Detection of different gel properties

[0096] 1. Scanning electron microscopy (SEM) detection

[0097] The gels prepared in Examples 1 and 2 and Comparative Examples 5 and 6 were examined by scanning electron microscopy, and the results are shown in the figure. Figure 3 The Ganoderma lucidum polysaccharide-based hydrogels all exhibited irregular porous network structures. Furthermore, in Examples 1, 5, and 6, the pore size of the G-GLP hydrogels gradually decreased and the dispersibility gradually became more uniform with increasing proportion of Ganoderma lucidum polysaccharides. However, the addition of pearl polypeptides in Example 2 did not disrupt the three-dimensional network structure of the hydrogel.

[0098] 2. Fourier transform infrared spectroscopy detection

[0099] Fourier transform infrared spectroscopy was performed on the oxidized Ganoderma lucidum polysaccharide (OGLP) and Ganoderma lucidum polysaccharide (GLP) prepared in Example 1. The results are shown in the figure. Figure 4 Compared to the spectrum of Ganoderma lucidum polysaccharides, the OGLP spectrum shows a higher concentration at 1702 cm⁻¹ due to the stretching vibration of -CHO. -1 A new absorption peak appeared at the OGLP, confirming the presence of -CHO.

[0100] Fourier transform infrared spectroscopy was performed on carboxymethyl chitosan (CMCS), Ganoderma lucidum polysaccharide hydrogel prepared in Example 1 (G-GLP2), and pearl polypeptide hydrogel prepared in Example 2 (G-GLP-PP). The results are shown in the figure. Figure 5 1702cm in G-GLP and G-GLP-PP -1 The stretching vibrations associated with the C=O group of the aldehyde group disappear at 1640 cm⁻¹, while at 1640 cm⁻¹... -1 The left and right sides show characteristic absorption peaks of imine bonds, indicating that a Schiff base reaction has occurred.

[0101] 3. Rheological test

[0102] Parallel plates with a 1.0 mm gap and a 25 mm diameter were used at 25°C. 0.6 mL of gel samples G-GLP1, G-GLP-PP, G-GLP2, and G-GLP3 prepared in Examples 1 and 2 and Comparative Examples 5 and 6 were fixed on the sample stage of a rheometer (HR20, TA Instruments Waters Corporation, USA) for dynamic frequency and stress-strain scanning. The storage modulus (G') and loss modulus (G'') of the samples were recorded under different scanning modes. The frequency scanning range was 0.1–100 Hz with a stress of 1%; the stress scanning range was 0.1–1000% with a frequency of 1 Hz.

[0103] See results Figure 6 Oscillatory strain scanning spectroscopy revealed that all three hydrogels possessed a long linear viscoelastic region with solid and stable mechanical properties. With increasing proportions of oxidized Ganoderma lucidum polysaccharides, the provided aldehyde groups gradually increased, leading to tighter cross-linking and a later collapse point. Specifically, G-GLP1 showed G' and G'' intersection at 332% strain, while G-GLP2 and 3 had collapse points of 403% and 440%, respectively. This indicates that increased cross-linking strength enhances the mechanical properties of the hydrogel. Frequency scanning results showed that all three hydrogels remained stable within the 0.1-10 Hz range. The hydrogel with added peptides exhibited a slightly later collapse point (425%), indicating that the addition of peptides resulted in more stable mechanical properties.

[0104] 4. Swelling experiment

[0105] 1g of the hydrogels G-GLP1, G-GLP-PP, G-GLP2, and G-GLP3 prepared in Examples 1 and 2 and Comparative Examples 5 and 6 were immersed in PBS buffer (pH 7.4) at room temperature. After swelling, the hydrogel samples were removed from the buffer at different times, the water on the sample surface was wiped off with filter paper, and the samples were weighed until the weight of the hydrogel no longer increased. This weight was recorded as W. S The weight of the sample before soaking is recorded as W0. Swelling data for each hydrogel were tested in parallel with three samples, and the average value was taken.

[0106]

[0107] The results are as follows Figure 7As shown, after soaking in PBS at room temperature for 21 h, G-GLP 1, G-GLP 2, and G-GLP 3 hydrogels all reached swelling equilibrium, with swelling ratios of 71.69 ± 2.0%, 57.84 ± 4.5%, and 34.11 ± 1.6%, respectively. With increasing proportions of oxidized Ganoderma lucidum polysaccharides, the aldehyde content increased, and the swelling ratio of the hydrogel gradually decreased. This is related to the cross-linking density of the hydrogel. As the aldehyde content increases, the cross-linking density increases, leading to increased structural stability of the hydrogel network, thus reducing the hydrogel's ability to absorb liquid. The addition of peptides decreased the swelling ratio of the hydrogel, possibly because the peptides enhanced the structural stability of the hydrogel.

[0108] 5. In vitro simulated gastrointestinal digestion experiment

[0109] Gastric juice: Add 2.0 g NaCl to 70 mL of 1.0 N HCl and bring the volume to 1 L. Adjust the pH to 2.0 ± 0.1 with 0.2 N HCl or 0.2 N NaOH. After autoclaving, add 3.2 g of pure pepsin to obtain simulated gastric juice (SGF).

[0110] Intestinal fluid: Add 6.8 g of potassium dihydrogen phosphate to 250 mL of distilled water, then add 77 mL of 0.2 N NaOH, and bring the volume to 1 L. Adjust the pH to 6.8 ± 0.1 with 0.2 N NaOH or 0.2 N HCl, and then sterilize the solution. Add 10 g of trypsin (porcine pancreas) and 1% bile salts to obtain simulated intestinal fluid (SIF).

[0111] Take 0.5 g of the G-GLP-PP hydrogel prepared according to the method in Example 2 and place it in 5 mL of simulated gastric fluid. Shake the hydrogel at 37°C and 100 rpm for 2 h. Then, transfer the hydrogel to 5 mL of simulated intestinal fluid and shake for 6 h. Take 1 mL of simulated gastric or intestinal fluid every 20 min and immediately replenish with an equal volume of release medium (simulated gastric or intestinal fluid). Detect the protein concentration in the sample solution using the BCA method and calculate the peptide release rate.

[0112]

[0113] Where: n i n is the protein content of the sample solution at the i-th time point; n0 is the protein content of the simulated gastric juice or simulated intestinal juice; w is the content of peptides in the hydrogel.

[0114] See results Figure 8During the 120-minute incubation period of SGF with G-GLP-PP hydrogel, only 29% of the peptides were slowly released. Conversely, when the hydrogel was transferred to SIF, the release rate of peptides increased significantly, with the remaining peptides (PP, 71%) released within 240 minutes. This indicates that the G-GLP-PP hydrogel effectively maintains the activity of PP after passing through SGF, allowing it to reach the intestines as a whole and gradually release PP.

[0115] 6. In vitro antioxidant and antityrosinase experiments

[0116] 0.1 g of G-GLP2 and G-GLP-PP were soaked in 100 mL of pure water for 24 h, and filtered to obtain a 1 mg / mL hydrogel extract. The antioxidant activity and tyrosinase inhibitory activity were detected using the hydrogel extract, 1 mg / mL CMCS aqueous solution, 1 mg / mL OGLP aqueous solution, and 1 mg / mL PP aqueous solution as test samples. The antioxidant activity detection method was the same as that described in Example 1.

[0117] To test the absorbance of mushroom tyrosinase (800 U / mL) and test sample solution, 30 μL of mushroom tyrosinase and 30 μL of test sample solution were added to a 96-well plate. After incubation at room temperature for 10 min, 50 μL of L-DOPA aqueous solution (1 mM) and 90 μL of phosphate buffer (0.2 M, pH=6.8) were added. The plate was then incubated at 37°C for 15 min, and the absorbance was measured at 475 nm and recorded as A. The absorbance of the plate with 30 μL of phosphate buffer instead of test sample solution was recorded as B. The absorbance of the plate with 30 μL of phosphate buffer instead of mushroom tyrosinase was recorded as A0. The absorbance of the plate with a mixture of 150 μL of phosphate buffer and 50 μL of L-DOPA aqueous solution was recorded as B0. Kojic acid was used as a positive control.

[0118]

[0119] The results are as follows Figure 9 As shown, compared with G-GLP2 hydrogel, the hydrogel with added PP exhibits improved antioxidant activity, with significantly enhanced scavenging activity of ABTS and hydroxyl radicals. However, the DPPH scavenging activity remained largely unchanged compared to G-GLP2, possibly due to the lower DPPH scavenging activity of PP. CMCS and OGLP tyrosinase inhibition rates were low, resulting in a lower tyrosinase inhibition rate for G-GLP; however, the addition of PP significantly improved the tyrosinase inhibition rate of the hydrogel.

[0120] 7. Antibacterial test

[0121] The antibacterial properties of the hydrogels were studied using *Escherichia coli* (CMCC 44102) and *Staphylococcus aureus* (CMCC 26003). 600 μL of the mixture from Examples 1 and 2 before gelation was prepared in 24-well plates, allowed to stand at room temperature to form hydrogels G-GLP2 and G-GLP-PP, and then sterilized under UV irradiation for 24 h. Bacterial suspensions (10 μL, 1 × 10⁻⁶) were then... 6 CFU / mL was uniformly added to the surface of the hydrogel, with bacteria alone serving as a control group. After incubation at 37 °C for 4 h, 1 mL of sterile PBS was added, and the mixture was sonicated for 5 min. 50 μL of the bacterial suspension was then coated onto LB agar plates. After incubation upside down at 37 °C for 18 hours, photographs were taken and colonies were counted. The results are as follows: Figure 10 As shown, G-GLP2 hydrogel exhibited antibacterial activity against *Escherichia coli* and *Staphylococcus aureus*. Compared with G-GLP2 hydrogel, the addition of PP further improved the antibacterial effect. Almost no colonies formed in the G-GLP-PP hydrogel group plates, and the inhibition rates against both tested bacteria were significantly improved. Furthermore, only G-GLP2 hydrogel caused bacterial death exceeding 96%. The antibacterial performance of G-GLP-PP hydrogel was significantly better than that of hydrogel without PP, with an inhibition rate exceeding 99% against *Escherichia coli* and exceeding 98% against *Staphylococcus aureus*.

[0122] The results showed that the Ganoderma lucidum polysaccharide hydrogel possessed good mechanical stability and swelling properties, and hydrogels with different properties could be obtained by adjusting the proportion of oxidized Ganoderma lucidum polysaccharide in the hydrogel. The addition of pearl polypeptides improved the mechanical stability of the hydrogel. Furthermore, in vitro digestion experiments demonstrated that the Ganoderma lucidum polysaccharide hydrogel could provide a physical barrier for pearl polypeptides, enabling their release in the intestine and improving their bioavailability.

Claims

1. A polypeptide hydrogel based on Ganoderma applanatum polysaccharide, characterized in that, The polypeptide hydrogel is prepared by the following method: (1) Preparation of oxidized Ganoderma lucidum polysaccharide Ganoderma lucidum polysaccharide is added to distilled water and stirred until completely dissolved. Sodium periodate is added under light shielding conditions, and the reaction is carried out at room temperature for 5-10 hours under light shielding. The reaction is terminated by adding ethylene glycol and stirring. The reaction mixture is loaded into a dialysis bag and dialyzed in pure water for 1-3 days. The dialysate is collected and freeze-dried to obtain oxidized Ganoderma lucidum polysaccharide. The molar ratio of Ganoderma lucidum polysaccharide to sodium periodate is 1:0.

6. (2) Preparation of polypeptide hydrogel A polypeptide solution is obtained by adding polypeptide to a 2-10% carboxymethyl chitosan aqueous solution to obtain a polypeptide mass concentration of 0.05-1%. An oxidized Ganoderma lucidum polysaccharide aqueous solution with a mass concentration of 1-5% is mixed with the polypeptide solution at a volume ratio of 1:0.5-2 to form a gel at room temperature, thereby obtaining a polypeptide hydrogel. The polypeptide is a pearl polypeptide.

2. The polypeptide hydrogel of claim 1, wherein, In step (1), Ganoderma lucidum polysaccharide is obtained by removing protein from Ganoderma lucidum extract, and the mass content of polysaccharide is 55-65%.

3. The polypeptide hydrogel of claim 1, wherein In step (1), Ganoderma lucidum polysaccharide is prepared by the following method: Sevage reagent is added to a Ganoderma lucidum extract aqueous solution. After mixing and shaking, the lower water layer and denatured protein at the junction are removed. The operation is repeated multiple times until there is no denatured protein sedimentation. The clear polysaccharide solution is collected. Anhydrous ethanol with a final volume concentration of 80% is then added, and the mixture is left to stand at room temperature overnight. The mixture is centrifuged, and the precipitate is collected. The precipitate is redissolved in water, and the organic solvent is removed by rotary evaporation. The mixture is freeze-dried at -80°C for 48 hours to obtain Ganoderma lucidum polysaccharide. The Sevage reagent is a solution of chloroform and n-butanol at a volume ratio of 4:

1. The volume of the Sevage reagent is 1 / 3 of the volume of the Ganoderma lucidum extract aqueous solution. The volume of distilled water in the Ganoderma lucidum extract aqueous solution is 10 mL / g based on the mass of Ganoderma lucidum extract.

4. The polypeptide hydrogel of claim 1, wherein In step (1), the volume of distilled water is 50-150 mL / g based on the mass of Ganoderma lucidum polysaccharide. The volume of ethylene glycol is 1-10 mL / g based on the mass of Ganoderma lucidum polysaccharide.

5. The polypeptide hydrogel of claim 1, wherein In step (1), the molecular weight cut-off of the dialysis bag is 3.5 kDa.

6. The polypeptide hydrogel of claim 1, wherein In step (2), the mass concentration of the carboxymethyl chitosan aqueous solution is 5%, the mass concentration of the polypeptide is 0.1%, the mass concentration of the oxidized Ganoderma lucidum polysaccharide aqueous solution is 3%, and the oxidized Ganoderma lucidum polysaccharide aqueous solution is mixed with the polypeptide solution at a volume ratio of 1:

1.

7. Use of the polypeptide hydrogel of claim 1 in the preparation of a polypeptide intestinal sustained-release drug.

8. Use of the polypeptide hydrogel of claim 1 in the preparation of an antibacterial drug.

9. Use according to claim 8, wherein the compound is ###0002### The antibacterial drug is a drug that inhibits the activity of Escherichia coli or Staphylococcus aureus.

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