Targeting complex for treating fatty liver in cats and method of making same

Abstract

The application discloses a targeted compound for treating feline fatty liver and a preparation method thereof, belongs to the technical field of biotechnology, and aims at solving the problem of treating feline fatty liver.The compound comprises astaxanthin and a composite carrier, wherein the composite carrier comprises sea cucumber oligopeptide, gelatin oligopeptide and galactosylated bacterial cellulose carrier; the mass ratio of astaxanthin to the composite carrier is 1:(10-100); and the mass ratio of the sea cucumber oligopeptide, the gelatin oligopeptide and the galactosylated bacterial cellulose carrier in the composite carrier is (1-3):(1-3):1.The compound can improve and treat feline fatty liver and has liver targeting property.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a liver-targeting microcapsule with an effect on improving fatty liver in cats and its preparation method. Background Technology

[0002] Feline hepatic liposis (FHL), also known as feline fat deposition syndrome, is a hepatobiliary disease specific to cats. The accumulation of triglycerides in hepatocytes leads to increased liver weight and volume, resulting in secondary liver dysfunction. Without effective treatment, it can cause considerable mortality. FHL is more common in middle-aged cats, with a generally higher incidence in females than males, and secondary FHL is slightly more common than primary FHL. With the continuous improvement of living standards, the proportion of pet cats suffering from fatty liver is also increasing. Long-term overnutrition is the main cause of feline fatty liver. In addition, stress factors and digestive tract diseases can also induce fatty liver, significantly impacting the health of pet cats. Patent application CN105250411B discloses a pharmaceutical composition for treating feline fatty liver syndrome, using a combination of more than twenty kinds of traditional Chinese medicine, including cassia seed, to treat cats with fatty liver. However, this method has drawbacks such as complex composition and slow onset of action. Furthermore, clinically used drug treatments often have significant side effects.

[0003] Multiple studies have shown that sea cucumber peptides obtained through enzymatic hydrolysis can improve liver enlargement, oxidative stress, inflammatory infiltration, and fatty degeneration in cases of acute liver injury caused by carbon tetrachloride, drug-induced liver injury caused by rifampin and isoniazid, and alcoholic liver injury. Meanwhile, fish maw peptides obtained through hydrolysis of fish maw possess strong antioxidant activity, capable of scavenging intracellular ROS, reducing intracellular lipid accumulation and oxidative stress, and alleviating lipid peroxidation, showing promise for the prevention and treatment of fatty liver. However, due to the instability of bioactive peptides, gastrointestinal physiological barriers, and limitations in physicochemical properties, sea cucumber peptides are difficult to deliver intact to the liver and exert their effects. On the other hand, astaxanthin (3,3'-dihydroxy-4,4'-diketoyl-β,β'-carotene) is distributed in marine algae, fungi, and crustaceans, with Haematococcus pluvialis being the most ideal natural source of astaxanthin. Multiple studies have confirmed that astaxanthin, as a natural and potent antioxidant, plays an important role in inhibiting liver fibrosis, preventing liver tumors, alleviating hepatic insulin resistance, and treating non-alcoholic fatty liver disease. However, astaxanthin also suffers from problems such as low water solubility and poor stability, which seriously affect its application in the treatment of liver injury.

[0004] Therefore, if the bioavailability of sea cucumber peptides, fish maw peptides and astaxanthin can be improved, they can achieve good liver protection and liver damage relief after oral administration. Constructing a suitable delivery system that can resist the influence of the digestive tract environment, enhance the permeability of mucus and epithelial barriers and stabilize the targeting of hepatocytes is a very effective technical means. When applied to the treatment of feline fatty liver, it has the advantages of no toxic side effects and rapid effect. Summary of the Invention

[0005] In view of the above objectives, in a first aspect, in order to solve the problem of liver-targeted delivery and treatment of feline fatty liver, the targeted complex for treating feline fatty liver in some embodiments of this application includes astaxanthin and a composite carrier, wherein the composite carrier includes sea cucumber oligopeptide, fish maw oligopeptide and galactosylated bacterial cellulose carrier.

[0006] The mass ratio of astaxanthin to the composite carrier is 1:(10-100), and the mass ratio of sea cucumber oligopeptides, fish maw oligopeptides and galactosylated bacterial cellulose carrier in the composite carrier is (1-3):(1-3):1.

[0007] In some embodiments of this application, the targeted complex for treating feline fatty liver has an astaxanthin to complex carrier mass ratio of 1:10.

[0008] In some embodiments of this application, a targeted complex for treating feline fatty liver is provided, wherein the mass ratio of sea cucumber oligopeptide, fish maw oligopeptide, and galactosylated bacterial cellulose carrier is 2:2:1.

[0009] In some embodiments of this application, the targeted complex for treating feline fatty liver contains sea cucumber oligopeptides with an amino acid sequence including Tyr-Pro-Asp-Cys-Pro-Gly-Pro.

[0010] In some embodiments of this application, the targeted complex for treating feline fatty liver, the amino acid sequence of the fish maw oligopeptide includes Tyr-Cys-Tyr-Cys-Pro-Arg.

[0011] In a second aspect, the method for preparing a targeted complex for treating feline fatty liver in some embodiments of this application includes...

[0012] S41. Dissolve astaxanthin in an ethanol solution and dissolve the composite carrier in water;

[0013] S42. Add an ethanol solution containing astaxanthin oil to a solution of the composite carrier, heat to react, evaporate to remove the ethanol, and obtain a targeted complex for treating feline fatty liver.

[0014] In some embodiments of this application, the method for preparing a targeted complex for treating feline fatty liver includes step S41, in which astaxanthin oil with a content of 10% is dissolved in an ethanol solution and stirred evenly, and the composite carrier is dissolved in ultrapure water. The mass-volume ratio of astaxanthin oil to ethanol solution is 1-10%, and the mass ratio of astaxanthin oil to composite carrier is 1:(1-10).

[0015] In step S42, an ethanol solution containing astaxanthin oil is slowly added dropwise to the composite carrier solution. During the dropwise addition, the mixture is heated and stirred to carry out the reaction. The ethanol is evaporated to remove the ethanol, and the mixture is freeze-dried to obtain a powder of the composite. The stirring speed is 500–1500 r / min, the reaction temperature is 40–60℃, and the reaction time is 2–4 h.

[0016] The method for preparing a targeted complex for treating feline fatty liver in some embodiments of this application includes...

[0017] Sea cucumber oligopeptides, fish maw oligopeptides, and galactosylated bacterial cellulose carrier were dissolved in ultrapure water, stirred at 80–100°C for 2–6 hours, and then freeze-dried to obtain a composite carrier. The galactosylated bacterial cellulose carrier was prepared as follows:

[0018] In some embodiments of this application, the method for preparing a targeted complex for treating feline fatty liver includes, S21: dissolving bacterial cellulose in a dimethyl sulfoxide solution containing tetrabutylammonium acetate and stirring until no gel aggregates, to obtain a homogeneous bacterial cellulose solution with a mass-volume ratio of 1-5%.

[0019] In S22, lactobionic acid is dissolved in a dimethyl sulfoxide (DMSO) solution containing 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and the carboxyl groups are activated by stirring for 1-3 hours to obtain a lactobionic acid solution with a mass-to-volume ratio of 1-10%; wherein, the ratio of lactobionic acid:1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC):N-hydroxysuccinimide (NHS) is 1-5:1:1;

[0020] S23, add bacterial cellulose solution to lactobionic acid solution, heat and stir, remove impurities, freeze dry to obtain galactosylated bacterial cellulose carrier powder, wherein the heating and stirring temperature is 40-50℃ and the heating and stirring time is 10-24h.

[0021] In some embodiments of this application, the method for preparing a targeted complex for treating feline fatty liver preferably includes an activation time of 2 hours in step S22.

[0022] Preferably, in step S22, the ratio of lactobionic acid: 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC): N-hydroxysuccinimide (NHS) is 1:1:1;

[0023] Preferably, in step S23, removing impurities includes dialysis with distilled water in a dialysis tube to remove unreacted lactobionic acid, bacterial cellulose, and a dimethyl sulfoxide (DMSO) solution containing tetrabutylammonium acetate.

[0024] Preferably, in step S23, the dialysis time is 24 to 72 hours, more preferably 72 hours.

[0025] In a second aspect, some embodiments of this application provide a method for preparing the sea cucumber oligopeptides in the above-mentioned complex, including...

[0026] S11. Add sea cucumber to water of 2 to 3 times its mass volume to make a homogenate and place it in an enzymatic hydrolysis tank;

[0027] S12. Add 2-5‰ of the weight of the sea cucumber to the enzymatic hydrolysis tank, and then heat up to inactivate the enzyme to obtain sea cucumber protein hydrolysate. The complex protease includes alkaline protease, papain and flavor protease, with the ratio of alkaline protease:papain:flavor protease = 3-5:1-2:1-3.

[0028] S13. Centrifuge the sea cucumber protein hydrolysate to obtain a clear liquid;

[0029] S14. Perform membrane separation on the supernatant, with a molecular weight cutoff of 3000 Da, to obtain a membrane-separated sample;

[0030] S15. Concentrate the membrane-separated sample, freeze-dry it, and obtain sea cucumber oligopeptide powder;

[0031] S16. Dissolve sea cucumber oligopeptide powder in water to a concentration of 20-30 mg / mL;

[0032] S17. The dissolved substance was separated and purified by gel chromatography column, with deionized water as the elution solvent and a flow rate of 0.3-0.5 mL / min. The absorbance was detected at 220 nm, and the elution peak with a retention time of 10 min was collected.

[0033] S18. Further purification was performed using a reversed-phase column under the following chromatographic conditions: mobile phase: 5-80% (v / v) methanol containing 0.1% (v / v) trifluoroacetic acid; flow rate: 0.8 mL / min; linear elution time: 35 min; absorbance was detected at 220 nm; and the chromatographic peak with a retention time of 16 min was collected to obtain sea cucumber oligopeptides.

[0034] Methods for preparing sea cucumber oligopeptides in the above-described complexes according to some embodiments of this application:

[0035] Preferably, in step S17, a 20mm×100mm Sephadex G10 gel chromatography column is used for separation and purification;

[0036] Preferably, in step S18, a 300SSB-C18RP-HPLC reverse-phase column is used for further purification;

[0037] Preferably, in step S12, the enzymatic hydrolysis temperature is 50℃~60℃, the enzymatic hydrolysis pH is 8.0~9.0, and the enzymatic hydrolysis time is 3~6h;

[0038] Preferably, in step S12, after enzymatic hydrolysis, the temperature is raised to 90°C for 10 minutes to inactivate the enzyme, thereby obtaining sea cucumber protease hydrolysate;

[0039] Preferably, the sea cucumber protease hydrolysate is centrifuged at 8000 rpm for 10 minutes;

[0040] Preferably, in step S15, the membrane-separated sample is concentrated under vacuum at 60°C and freeze-dried to obtain sea cucumber oligopeptide powder.

[0041] Preferably, in step S16, the sea cucumber oligopeptide powder is dissolved in water to a concentration of 25 mg / mL;

[0042] Preferably, in step S18, the sea cucumber oligopeptides are concentrated and freeze-dried to obtain sea cucumber oligopeptide powder.

[0043] Preferably, in step S11, the sea cucumber includes any one or a combination of sea cucumbers such as sea cucumber, sea cucumber, sea cucumber, sea cucumber, and sea cucumber.

[0044] On a third-party level, some embodiments of this application provide methods for preparing the fish maw oligopeptides in the aforementioned complex, including...

[0045] S11. Add 10 to 20 times its weight volume of water to make a homogenate and place it in an enzymatic hydrolysis tank;

[0046] S12. Add 2-5% of the weight of the fish maw to the enzymatic hydrolysis tank with a complex protease. After enzymatic hydrolysis, heat up to inactivate the enzyme and obtain fish maw protease hydrolysate. The complex protease includes neutral protease, trypsin, bromelain and flavor protease. The ratio of neutral protease:trypsin:bromelain:flavor protease is 1-3:2-4:3-5:2-5.

[0047] S13. Centrifuge the fish maw enzyme hydrolysate to obtain the supernatant;

[0048] S14. Perform membrane separation on the supernatant, with a molecular weight cutoff of 3000 Da;

[0049] S15. The membrane solution was separated and purified by gel chromatography column, with deionized water as the elution solvent and an elution flow rate of 0.5–0.8 mL / min. The absorbance was detected at 220 nm, and the elution peak with a retention time of 14–15 min was collected.

[0050] S16. Further purification was performed using a chromatographic column under the following chromatographic conditions: mobile phase A was water containing 0.05–0.1% trifluoroacetic acid by volume, mobile phase B was acetonitrile, and the gradient elution conditions were: 0–5 min, 10% B, 5–15 min, 10%–15% B, 15–25 min, 15% B–25% B, 25–40 min, 25% B–35% B, with a flow rate of 0.8 mL / min. The elution peak with a retention time of 12–13 min was collected to obtain fish maw oligopeptides.

[0051] Preparation method of fish maw oligopeptides in the above-described complex according to some embodiments of this application:

[0052] Preferably, in step S12, the enzymatic hydrolysis temperature is 50–60°C, the enzyme reaction pH is 8.0–9.0, and the enzymatic hydrolysis time is 4 hours.

[0053] Preferably, in step S12, after enzymatic hydrolysis, the temperature is raised to 80-90°C for 10 minutes to inactivate the enzyme, thereby obtaining the fish maw proteolytic solution;

[0054] Preferably, in step S13, the fish maw protein hydrolysate is centrifuged at 8000 rpm for 10 minutes;

[0055] Preferably, in step S15, a 20mm×100mm Sephadex G10 gel chromatography column is used for separation and purification;

[0056] Preferably, in step S16, a C18 chromatographic column is used for further purification;

[0057] Preferably, in step S16, the fish maw oligopeptides are concentrated and freeze-dried to obtain fish maw oligopeptide powder.

[0058] Beneficial effects:

[0059] This invention utilizes galactose-modified bacterial cellulose as a liver-targeted delivery carrier for sea cucumber oligopeptides, fish maw oligopeptides, and astaxanthin. On one hand, the active peptides are linked to the galactose grafted onto the bacterial cellulose via a Maillard reaction; on the other hand, the active peptides and astaxanthin are encapsulated through emulsification. Furthermore, the active peptides and astaxanthin are simultaneously coated by the unique three-dimensional network structure of bacterial cellulose, prolonging the release time of the sea cucumber peptides, fish maw peptides, and astaxanthin. This method exhibits extremely high biocompatibility and biodegradability. The presence of numerous hydroxyl groups also effectively counteracts the structural effects of extreme pH changes on the core material. Compared to galactose-modified chitosan delivery systems, the three-dimensional network structure of bacterial cellulose has significantly higher porosity, resulting in a more effective coating of the core material that protects the active peptides from hydrolytic digestion by various proteases in the gastrointestinal tract and improves the stability of astaxanthin in the gastrointestinal tract. Therefore, the sea cucumber oligopeptide-fish maw oligopeptide-astaxanthin-galactosylated bacterial cellulose microcapsules have strong flexibility and high adhesion. They can not only cope with the peristalsis of gastrointestinal muscles and the shear stress caused by the flow rate of gastric juice in the lumen to reduce the mechanical degradation of active peptides and astaxanthin, but also enhance the adhesion of the strong adhesion layer, effectively prolonging the residence time in the gastrointestinal tract and enhancing absorption. This flexibility also reduces the probability of the core material being phagocytosed by macrophages in the reticuloendothelial system after entering the systemic circulation, thereby improving the efficiency of delivery to target cells.

[0060] In summary, the sea cucumber oligopeptide-fish maw oligopeptide-astaxanthin-galactosylated bacterial cellulose prepared by the method provided in this invention protects the molecular structure of the active peptides and astaxanthin in the gastrointestinal tract, enhances their absorption, delays release, and ultimately delivers them precisely to the hepatocytes of cats. The sea cucumber oligopeptide alleviates hepatocyte swelling and inflammatory infiltration caused by fat accumulation, the fish maw oligopeptide reduces liver fat accumulation, and the astaxanthin reduces the peroxidation of proteins and fats. These three components synergistically regulate the oxidative stress system of hepatocytes and improve fatty degeneration, resulting in better therapeutic effects on feline fatty liver compared to using them alone. This method is simple and easy to operate, suitable for industrial-scale preparation, and has promising applications in the prevention and treatment of fatty liver in pet cats. Attached Figure Description

[0061] Figure 1 Changes in liver lipid accumulation.

[0062] Figure 2 The three-dimensional network structure of bacterial cellulose. Detailed Implementation

[0063] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments do not constitute a limitation on the scope of protection of the present invention.

[0064] This invention provides a microcapsule containing sea cucumber oligopeptides, fish maw oligopeptides, and astaxanthin as core materials, which can protect these components in the cat's gastrointestinal tract by reducing their inactivation by digestive hydrolytic enzymes such as pH and proteases. Simultaneously, it enhances adhesion to the mucosa to prolong their time in the gastrointestinal tract and improve absorption. Furthermore, after absorption, it is directly and targeted to lipid-accumulated hepatocytes for repair and improvement.

[0065] The technical solution adopted by this invention to solve its technical problem is: a liver-targeting microcapsule with an effect of improving fatty liver in cats, which undergoes the following process steps:

[0066] Step 1. Preparation of sea cucumber oligopeptides: Fresh sea cucumber was mixed with 2-3 times its weight volume of water to form a homogenate, which was then placed in an enzymatic hydrolysis tank. A complex protease at 2-5‰ of the sea cucumber's weight was added. After enzymatic hydrolysis, the temperature was raised to 90℃ for 10 minutes to inactivate the enzyme, yielding a sea cucumber protease hydrolysate. The hydrolysate was centrifuged at 8000 rpm for 10 minutes to remove particulate matter. Molecular weight separation was then performed using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da to obtain a membrane-separated sample. This sample was concentrated to a certain volume under vacuum at 60℃ and freeze-dried to obtain sea cucumber oligopeptide powder. The sea cucumber oligopeptide powder was dissolved in water to a concentration of 25 mg / mL. Separation and purification were performed using a Sephadex G 10 (20 mm × 100 mm) gel chromatography column with deionized water as the elution solvent and a flow rate of 0.3-0.5 mL / min. The absorbance was measured at 220 nm, and the elution peak with a retention time of 10 min was collected. A 300 SSB-C18 column was used for further analysis. Further purification was performed using an RP-HPLC reversed-phase column under the following chromatographic conditions: mobile phase: 5-80% (v / v) methanol containing 0.1% (v / v) trifluoroacetic acid (TFA); flow rate: 0.8 mL / min; linear elution time: 35 min; absorbance was measured at 220 nm; the chromatographic peak with a retention time of 16 min was collected; and the product was concentrated and freeze-dried to obtain sea cucumber oligomeric active peptide powder.

[0067] Furthermore, the sea cucumber mentioned in step 1 is a type of sea cucumber, such as the imitation sea cucumber, the flower sea cucumber, the rice sea cucumber, the ball sea cucumber, or the jade-foot sea cucumber, with the imitation sea cucumber and the rice sea cucumber being preferred.

[0068] Furthermore, in step 1, the enzymatic hydrolysis uses a complex protease with an alkaline protease:papain:flavor protease ratio of 3–5:1–2:1–3, and the mass ratio of the complex protease to the added sea cucumber raw material is 1:10–15. The enzymatic hydrolysis reaction temperature is 50℃–60℃, the hydrolysis pH is 8.0–9.0, and the hydrolysis time is 3–6 hours.

[0069] Furthermore, the amino acid sequence of the sea cucumber oligopeptide mentioned in step 1 is: Tyr-Pro-Asp-Cys-Pro-Gly-Pro.

[0070] Step 2. Preparation of fish maw oligopeptides: After processing the fish maw sample, add 10-20 times its weight volume of water to make a homogenate and place it in an enzymatic hydrolysis vessel. Then add 2-5% of the fish maw's weight of a complex protease and hydrolyze for 4 hours. After hydrolysis, heat to 80-90℃ to inactivate the enzyme for 10 minutes to obtain the fish maw protease hydrolysate. Centrifuge the protease hydrolysate at 8000 rpm for 10 minutes to remove particulate matter. Then, use membrane separation technology to separate the sample, with a molecular weight cutoff of 3000 Da. The membrane solution is purified by Sephadex G10 (20 mm × 100 mm) gel chromatography column with deionized water as the elution solvent and an elution flow rate of 0.5-0.8 mL / min. The absorbance is detected at 220 nm, and the elution peak with a retention time of 14-15 min is collected. A C18 column is used, with mobile phase A being water containing 0.05-0.1% trifluoroacetic acid by volume and mobile phase B being acetonitrile. The gradient elution conditions are: 0-5 min, 10%B, 5-15 min, 10%-15%B, 15-25 min, 15%B-25%B, 25-40 min, 25%B-35%B, flow rate of 0.8 mL / min, collect the elution peak with a retention time of 12-13 min, concentrate and freeze-dry to obtain fish maw oligopeptides.

[0071] Furthermore, the fish maw mentioned in step 1 is sourced from yellow croaker.

[0072] Furthermore, in step 2, a complex protease with a ratio of neutral protease:trypsin:bromelain:flavor protease = 1-3:2-4:3-5:2-5 is used for enzymatic hydrolysis. The hydrolysis temperature is 50-60℃, and the pH value of the enzyme reaction is controlled at 8.0-9.0.

[0073] Furthermore, the amino acid sequence of the fish maw oligopeptide mentioned in step 1 is: Tyr-Cys-Tyr-Cys-Pro-Arg.

[0074] Step 3. Preparation of galactosylated bacterial cellulose carrier: Bacterial cellulose was dissolved in a dimethyl sulfoxide (DMSO) solution containing tetrabutylammonium acetate and stirred until no gelatinous aggregate was formed to prepare a homogeneous bacterial cellulose solution; lactobionic acid was dissolved in a DMSO solution containing 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) (1:1) for carboxyl activation; the bacterial cellulose solution was added to the lactobionic acid solution and heated and stirred, and then dialyzed with distilled water in a dialysis tube to remove unreacted lactobionic acid, bacterial cellulose and DMSO solvent containing tetrabutylammonium acetate and other impurities, and freeze-dried to obtain galactosylated bacterial cellulose powder.

[0075] Furthermore, the bacterial cellulose solution in step 3 has a mass-to-volume ratio of 1–5%.

[0076] Furthermore, the mass-to-volume ratio of the lactobionic acid solution in step 3 is 1–10%.

[0077] Furthermore, in step 3, the mass ratio of lactobionic acid, EDC and NHS is 1 to 5:1:1, preferably 1:1:1.

[0078] Furthermore, the activation time in step 3 is 1 to 3 hours, preferably 2 hours.

[0079] Furthermore, the heating and stirring temperature in step 3 is 40–50°C, and the stirring time is 10–24 hours.

[0080] Furthermore, the dialysis time in step 3 is 24 to 72 hours, preferably 72 hours.

[0081] Step 4. Preparation of sea cucumber oligopeptide-fish maw oligopeptide-galactosylated bacterial cellulose complex carrier: Take the sea cucumber oligopeptide powder, fish maw oligopeptide powder and galactosylated bacterial cellulose powder prepared in steps 1 and 2, redissolve them in 10 times the volume of ultrapure water, stir and react at 80-100℃ for 2-6 hours, and freeze dry to obtain sea cucumber oligopeptide-fish maw oligopeptide-galactosylated bacterial cellulose complex carrier powder.

[0082] Furthermore, in step 4, the mass ratio of sea cucumber oligopeptide powder, fish maw oligopeptide, and galactosylated bacterial fiber powder is 1-3:1-3:1, preferably 2:2:1.

[0083] Step 5. Preparation of the sea cucumber oligopeptide-fish maw oligopeptide-astaxanthin-galactosylated bacterial cellulose complex carrier: Dissolve astaxanthin oil with 10% astaxanthin content in ethanol solution and stir evenly. Redissolve sea cucumber oligopeptide-fish maw oligopeptide-astaxanthin-galactosylated bacterial cellulose powder in 10 times its volume of ultrapure water. Then, slowly drop the astaxanthin oil ethanol solution into the stirred sea cucumber oligopeptide-fish maw oligopeptide-astaxanthin-galactosylated bacterial cellulose solution and heat to react. After evaporating to remove ethanol, freeze-dry to obtain sea cucumber oligopeptide-fish maw oligopeptide-astaxanthin-galactosylated bacterial cellulose microcapsules (SCP-FMP-AST-GBC) powder.

[0084] Furthermore, in step 5, the mass-volume ratio of astaxanthin oil to ethanol solution is 1-10%, preferably 5%.

[0085] Furthermore, in step 5, the mass ratio of astaxanthin oil to sea cucumber oligopeptide-fish maw oligopeptide astaxanthin-galactosylated bacterial cellulose is 1:1 to 10, preferably 1:1.

[0086] Furthermore, in step 5, the stirring speed is 500–1500 r / min, the reaction temperature is 40–60℃, and the reaction time is 2–4 hours.

[0087] While there are numerous precedents for galactose-mediated targeted delivery systems to desialylate glycoprotein receptors on the cell membrane of hepatocytes, most studies focus on directly modifying drugs with galactose or constructing galactose-modified liposomes or polymeric micelle carriers to achieve good targeted delivery performance. However, it is unclear whether cats with fatty liver can tolerate these delivery systems. Furthermore, the high-pressure homogenization and ultrasound processes involved in emulsion delivery systems may affect the structure of astaxanthin, thereby impacting encapsulation efficiency, stability, and bioavailability.

[0088] This invention utilizes galactose-modified bacterial cellulose as a liver-targeted delivery carrier for sea cucumber oligopeptides, fish maw oligopeptides, and astaxanthin. On one hand, the active peptides are linked to the galactose grafted onto the bacterial cellulose via a Maillard reaction; on the other hand, the active peptides and astaxanthin are encapsulated through emulsification. Furthermore, the active peptides and astaxanthin are simultaneously coated by the unique three-dimensional network structure of bacterial cellulose, prolonging the release time of the sea cucumber peptides, fish maw peptides, and astaxanthin. This method exhibits extremely high biocompatibility and biodegradability. The presence of numerous hydroxyl groups also effectively counteracts the structural effects of extreme pH changes on the core material. Compared to galactose-modified chitosan delivery systems, the three-dimensional network structure of bacterial cellulose has significantly higher porosity, resulting in a more effective coating of the core material that protects the active peptides from hydrolytic digestion by various proteases in the gastrointestinal tract and improves the stability of astaxanthin in the gastrointestinal tract. Therefore, the sea cucumber oligopeptide-fish maw oligopeptide-astaxanthin-galactosylated bacterial cellulose microcapsules have strong flexibility and high adhesion. They can not only cope with the peristalsis of gastrointestinal muscles and the shear stress caused by the flow rate of gastric juice in the lumen to reduce the mechanical degradation of active peptides and astaxanthin, but also enhance the adhesion of the strong adhesion layer, effectively prolonging the residence time in the gastrointestinal tract and enhancing absorption. This flexibility also reduces the probability of the core material being phagocytosed by macrophages in the reticuloendothelial system after entering the systemic circulation, thereby improving the efficiency of delivery to target cells.

[0089] In summary, the sea cucumber oligopeptide-fish maw oligopeptide-astaxanthin-galactosylated bacterial cellulose prepared by the method provided in this invention protects the molecular structure of the active peptides and astaxanthin in the gastrointestinal tract, enhances their absorption, delays release, and ultimately delivers them precisely to the hepatocytes of cats. The sea cucumber oligopeptide alleviates hepatocyte swelling and inflammatory infiltration caused by fat accumulation, the fish maw oligopeptide reduces liver fat accumulation, and the astaxanthin reduces the peroxidation of proteins and fats. These three components synergistically regulate the oxidative stress system of hepatocytes and improve fatty degeneration, resulting in better therapeutic effects on feline fatty liver compared to using them alone. This method is simple and easy to operate, suitable for industrial-scale preparation, and has promising applications in the prevention and treatment of fatty liver in pet cats.

[0090] Example 1:

[0091] Step 1. Add 1000g of fresh sea cucumber body wall to 3 times its weight of water to make a homogenate, place it in an enzymatic hydrolysis tank, and then add 20g of a complex protease with a composition of alkaline protease:papain:flavor protease = 5:2:3. Adjust the pH to 9.0, enzymatically hydrolyze at 50℃ for 4 hours, then heat to 90℃ for 10 minutes to inactivate the enzyme, obtaining sea cucumber protease hydrolysate. Centrifuge the sea cucumber protease hydrolysate at 8000 rpm for 10 minutes, then perform ultrafiltration at 3000 Da, and concentrate under vacuum at 60℃ to 25% of the total mass. Freeze-dry to obtain 58g of sea cucumber oligopeptide powder. Dissolve the sea cucumber oligopeptide powder in water to a concentration of 25 mg / mL, and separate and purify it using a Sephadex G 10 (20 mm × 100 mm) gel chromatography column with deionized water as the eluent and a flow rate of 0.5 mL / min. Detect the absorbance at 220 nm and collect the sample with a retention time of 10.11. The elution peak was min; further purification was performed using a 300 SSB-C18 RP-HPLC reversed-phase column under the following chromatographic conditions: mobile phase: 40% (v / v) methanol containing 0.1% (v / v) trifluoroacetic acid (TFA), flow rate: 0.8 mL / min, linear elution time: 35 min, and the chromatographic peak with a retention time of 16.25 min was collected. After concentration, the chromatographic peak was freeze-dried to obtain 7.8 g of sea cucumber oligomeric active peptide powder.

[0092] Step 2. Add 1000g of fish maw to 20 times its volume of water to make a homogenate, place it in an enzymatic hydrolysis tank, and then add 25g of a complex protease. The protease composition is neutral protease:trypsin:bromelain:flavor protease = 3:4:5:3. Adjust the pH to 8.5, enzymatically hydrolyze at 50℃ for 4 hours, then heat to 90℃ for 10 minutes to inactivate the enzyme, obtaining the fish maw protease hydrolysate. Centrifuge at 8000 rpm for 10 minutes, then perform ultrafiltration at 3000 Da. The membrane solution is then purified using a Sephadex G 10 (20 mm × 100 mm) gel chromatography column with deionized water as the elution solvent and a flow rate of 0.6 mL / min. The absorbance is measured at 220 nm, and the elution peak with a retention time of 14.61 min is collected. A C18 column is used, with mobile phase A being water containing 0.05% trifluoroacetic acid by volume and mobile phase B being acetonitrile. The gradient elution conditions are: 0–5 min, 10%B, 5-15 min, 10%-15%B, 15-25 min, 15%B-25%B, 25-40 min, 25%B-35%B, flow rate of 0.8 mL / min, collect the elution peak with a retention time of 12.41 min, concentrate and freeze-dry to obtain 7.5 g of fish maw oligopeptides;

[0093] Step 3. Dissolve 20g of bacterial cellulose in 1000ml of DMSO solution containing tetrabutylammonium acetate and stir until no gelatinous material coagulates to prepare a homogeneous bacterial cellulose solution; dissolve 10g of lactobionic acid in 1000ml of DMSO solution containing 5g of EDC and 5g of NHS and stir to activate the carboxyl groups for 2 hours; add the bacterial cellulose solution to the lactobionic acid solution, heat to 40℃ and stir for 12 hours, then dialyze for 24 hours and freeze dry to obtain 30g of galactosylated bacterial cellulose powder.

[0094] Step 4. Take 6g of sea cucumber oligopeptide powder, 6g of fish maw oligopeptide and 3g of galactosylated bacterial cellulose powder and redissolve them in 10 times the volume of ultrapure water. Stir and react at 90℃ for 4 hours, and freeze dry to obtain 15g of sea cucumber oligopeptide-fish maw oligopeptide-galactosylated bacterial cellulose complex carrier powder.

[0095] Step 5. Dissolve 10g of astaxanthin oil in 100ml of ethanol solution and stir well. Dissolve 10g of sea cucumber oligopeptide-fish maw oligopeptide-galactosylated bacterial cellulose powder in 10 times its volume of ultrapure water. Then slowly drop the astaxanthin oil ethanol solution into the stirring sea cucumber oligopeptide-fish maw oligopeptide-galactosylated bacterial cellulose solution and heat to 50℃. React at a stirring speed of 1500r / min for 2 hours. After evaporating and removing the ethanol, freeze dry to obtain 20g of sea cucumber oligopeptide-fish maw oligopeptide-astaxanthin-galactosylated bacterial cellulose microparticle powder.

[0096] Example 2:

[0097] Step 1. Add 1000g of fresh sea cucumber body wall to twice its weight of water to make a homogenate, place it in an enzymatic hydrolysis tank, and then add 5g of a complex protease with a composition of alkaline protease:papain:flavor protease = 3:1:3. Adjust the pH to 8.5, enzymatically hydrolyze at 55℃ for 4 hours, then heat to 90℃ for 10 minutes to inactivate the enzyme, obtaining sea cucumber protease hydrolysate. Centrifuge the sea cucumber protease hydrolysate at 8000 rpm for 10 minutes, then perform ultrafiltration at 3000 Da, and concentrate under vacuum at 60℃ to 20% of the total mass. Freeze-dry to obtain 63g of sea cucumber oligopeptide powder. Dissolve the sea cucumber oligopeptide powder in water to a concentration of 25 mg / mL, and separate and purify it using a Sephadex G 10 (20 mm × 100 mm) gel chromatography column with deionized water as the eluent and a flow rate of 0.3 mL / min at 220 °C. The absorbance was measured at nm, and the elution peak with a retention time of 10.15 min was collected. Further purification was performed using a 300 SSB-C18 RP-HPLC reversed-phase column under the following chromatographic conditions: mobile phase: 40% (v / v) methanol containing 0.1% (v / v) trifluoroacetic acid (TFA), flow rate: 0.8 mL / min, linear elution time: 35 min, and the chromatographic peak with a retention time of 16.29 min was collected. After concentration, the product was freeze-dried to obtain 8.1 g of sea cucumber oligomeric active peptide powder.

[0098] Step 2. Add 1000g of fish maw to 20 times its volume of water to make a homogenate and place it in an enzymatic hydrolysis tank. Then add 30g of a complex protease with a composition of neutral protease:trypsin:bromelain:flavor protease = 3:2:5:5. Adjust the pH to 8.0, enzymatically hydrolyze at 55℃ for 4 hours, then heat to 90℃ for 10 minutes to inactivate the enzyme, obtaining the fish maw protease hydrolysate. Centrifuge at 8000 rpm for 10 minutes, then perform ultrafiltration at 3000 Da. The membrane solution is then purified using a Sephadex G 10 (20 mm × 100 mm) gel chromatography column with deionized water as the elution solvent and a flow rate of 0.8 mL / min. The absorbance is measured at 220 nm, and the elution peak with a retention time of 14.31 min is collected. A C18 column is used with mobile phase A being water containing 0.1% trifluoroacetic acid by volume and mobile phase B being acetonitrile. The gradient elution conditions are: 0–5 min, 10%B, 5-15 min, 10%-15%B, 15-25 min, 15%B-25%B, 25-40 min, 25%B-35%B, flow rate of 0.8 mL / min, collect the elution peak with a retention time of 12.50 min, concentrate and freeze-dry to obtain 7.2 g of fish maw oligopeptides;

[0099] Step 3. Dissolve 10g of bacterial cellulose in 1000ml of DMSO solution containing tetrabutylammonium acetate and stir until no gelatinous material coagulates to prepare a homogeneous bacterial cellulose solution; dissolve 4g of lactobionic acid in 1000ml of DMSO solution containing 5g of EDC and 5g of NHS and stir to activate the carboxyl groups for 2 hours; add the bacterial cellulose solution to the lactobionic acid solution, heat to 50℃ and stir for 24 hours, then dialyze for 72 hours and freeze dry to obtain 13g of galactosylated bacterial cellulose powder.

[0100] Step 4. Take 6g of sea cucumber oligopeptide powder, 6g of fish maw oligopeptide powder and 2g of galactosylated bacterial cellulose powder and redissolve them in 10 times the volume of ultrapure water. Stir and react at 100℃ for 3 hours, and freeze dry to obtain 14g of sea cucumber oligopeptide-fish maw oligopeptide-galactosylated bacterial cellulose complex carrier powder.

[0101] Step 5. Dissolve 5g of astaxanthin oil in 100ml of ethanol solution and stir well. Dissolve 10g of sea cucumber oligopeptide-fish maw oligopeptide-galactosylated bacterial cellulose powder in 10 times its volume of ultrapure water. Then slowly drop the astaxanthin oil ethanol solution into the stirring sea cucumber oligopeptide-fish maw oligopeptide-galactosylated bacterial cellulose solution and heat to 60℃. React at 1000r / min for 4 hours. After evaporating and removing the ethanol, freeze dry to obtain 15g of sea cucumber oligopeptide-fish maw oligopeptide-astaxanthin-galactosylated bacterial cellulose (SCP-FMP-AST-GBC) powder.

[0102] Comparative Example 1:

[0103] Following steps 1 to 4 of Example 1, 92g of sea cucumber oligopeptide-fish maw oligopeptide-galactosylated bacterial cellulose complex carrier (SCP-FMP-GBC) powder was obtained.

[0104] Comparative Example 2:

[0105] Step 1. Dissolve 20g of bacterial cellulose in 1000ml of DMSO solution containing tetrabutylammonium acetate and stir until no gelatinous material coagulates to prepare a homogeneous bacterial cellulose solution; dissolve 10g of lactobionic acid in 1000ml of DMSO solution containing 5g of EDC and 5g of NHS and stir to activate the carboxyl groups for 2 hours; add the bacterial cellulose solution to the lactobionic acid solution, heat to 40℃ and stir for 24 hours, then dialyze for 48 hours and freeze dry to obtain 56g of galactosylated bacterial cellulose powder.

[0106] Step 2. Dissolve 10g of astaxanthin oil in 100ml of ethanol solution and stir well. Dissolve 10g of galactosylated bacterial cellulose powder in 10 times its volume of ultrapure water. Then slowly add the astaxanthin oil ethanol solution to the stirred galactosylated bacterial cellulose solution and heat to 50℃. React at 1000r / min for 3 hours. After evaporating to remove the ethanol, freeze dry to obtain 20g of astaxanthin-galactosylated bacterial cellulose (AST-GBC) microparticle powder.

[0107] Comparative Example 3:

[0108] Step 1: Obtain 8.0g of sea cucumber oligopeptides by following Step 1 of Example 1.

[0109] Step 2: Obtain 7.6g of fish maw oligopeptide by following Step 2 of Example 1.

[0110] Step 3: Dissolve 20 g of chitosan in 1000 mL of 0.1 mol / L MES buffer. After it is completely dissolved, adjust the pH to 5.7. Then add 8 g of lactobionic acid, 10 g of NHS and 10 g of EDC. Stir and react for 30 min. After it is completely dissolved, place it at 4 ℃ for 12 h. Then transfer it to room temperature for 12 h. Finally, dialyze it with a dialysis bag for 3 days. Take out the dialysate and freeze-dry it to obtain 30 g of galactosylated chitosan powder.

[0111] Step 4. Take 6g of sea cucumber oligopeptide powder, 6g of fish maw oligopeptide and 3g of galactosylated chitosan powder and redissolve them in 10 times the volume of ultrapure water. Stir and react at 90℃ for 4 hours, and freeze dry to obtain 15g of sea cucumber oligopeptide-fish maw oligopeptide-galactosylated chitosan powder.

[0112] Step 5. Dissolve 10g of astaxanthin oil in 100ml of ethanol solution and stir well. Dissolve 10g of sea cucumber oligopeptide-fish maw oligopeptide-galactosylated chitosan powder in 10 times its volume of ultrapure water. Then slowly drop the astaxanthin oil ethanol solution into the stirring sea cucumber oligopeptide-fish maw oligopeptide-galactosylated chitosan solution and heat to 50℃. React at 1000r / min for 3 hours. After evaporating and removing the ethanol, freeze dry to obtain 20g of sea cucumber oligopeptide-fish maw oligopeptide-astaxanthin-galactosylated chitosan (SCP-FMP-AST-GC) powder.

[0113] Experiment 1: Liver targeting efficiency

[0114] Male SD rats were acclimatized for 3-5 days and randomly divided into three groups: SCP-FMP-AST-GBC (5 mg / kg), AST-GBC (3 mg / kg), and SCP-FMP-AST-GC (5 mg / kg), with 12 rats in each group. Rats were fasted for 12 hours prior to administration and were administered the dosage via gavage. At 0.5, 1, 4, 12, and 24 hours post-administration (12 rats per time point, 3 rats per group), the rats were euthanized by decapitation. After blood was drained, liver, lung, and kidney samples were obtained. Twice the volume of physiological saline was added, and the samples were homogenized using a tissue homogenizer. Accurately pipette 200 μL of tissue homogenate into a 1.5 mL centrifuge tube, add 600 μL of methanol, vortex for 2 min, centrifuge at 10,000 rpm for 5 min, accurately pipette 500 μL of the supernatant, dry under nitrogen, reconstitute with 200 μL of methanol, vortex for 1 min, and inject 20 μL for HPLC analysis to determine the astaxanthin content. The liver targeting ability of SCP-FMP-AST-GBC was evaluated using targeting efficiency (Te) according to Gupta's method. The results are shown in the table below:

[0115] Table 1. AUC and targeting efficiency of different tissues

[0116]

[0117] As can be seen from Table 1, compared with the AST-GBC group and the SCP-FMP-AST-GC group, the SCP-FMP-AST-GBC group showed the strongest liver tropism.

[0118] Experiment 2: Improvement of Fat Accumulation in NAFLD Mice

[0119] After one week of acclimatization, 10 mice were randomly selected and fed a normal diet (blank control group), while the remaining mice were given a high-fat diet for free feeding for 8 weeks. The mice fed the high-fat diet were then randomly divided into three groups (n=12 per group) and administered the diet by gavage once daily for 6 weeks: (1) normal diet + saline (model group); (2) normal diet + SCP-FMP-AST-GBC; (3) normal diet + FMP; (4) normal diet + SCP; (5) normal diet + AST; (6) normal diet + SCP-FMP-AST-GC. At the end of week 14, all mice were sacrificed after a 4-hour fast. The livers were removed and frozen sectioned using standard methods. Oil Red O staining was performed according to the kit instructions, and the intracellular lipid droplets were extracted with isopropanol. The absorbance was measured at 570 nm. Figure 1As shown, compared with the control group, the oil red absorbance of lipid droplets in the model group was significantly increased, indicating that the NAFLD model was successfully established. SCP-FMP-AST-GBC intervention significantly reduced lipid accumulation in the liver of NAFLD mice, approaching the level of the control group, and the effect was superior to feeding sea cucumber oligopeptides, fish maw oligopeptides, or astaxanthin alone, as well as the sea cucumber oligopeptide-fish maw oligopeptide-astaxanthin-galactosylated chitosan transport system.

[0120] Experiment 3: Therapeutic effect on cats with fatty liver

[0121] Thirty cats with fatty liver disease were collected from a local veterinary hospital, with a female-to-male ratio of 1:1. The cats were divided into three groups: the SCP-FMP-AST-GBC group (5 mg / kg), the silymarin group (10 mg / kg), and a control group. They were fed twice daily for four weeks. At week 5, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the blood of the cats were measured, and symptoms such as vomiting, anorexia, and decreased activity were observed. The results are shown in Table 2. Compared with the control group, both the SCP-FMP-AST-GBC group and the silymarin group showed good therapeutic effects. In the SCP-FMP-AST-GBC group, the improvement in ALT and AST, important indicators of liver function, was most significant. Furthermore, the cure rate for vomiting and anorexia was higher in the SCP-FMP-AST-GBC group (90%) than in the silymarin group (50%).

[0122] Table 2. Changes in liver function and cure status before and after drug administration

[0123]

[0124] The above experimental results indicate that sea cucumber oligopeptides-fish maw oligopeptides-astaxanthin-galactosylated bacterial cellulose microcapsules exhibit strong liver-targeting properties after administration to cats with fatty liver disease. They also demonstrate high stability to gastrointestinal fluids, improving the bioavailability of the core material and requiring only a low dose to achieve good results. The sea cucumber oligopeptides, fish maw oligopeptides, and astaxanthin work together to reduce hepatocyte inflammation, decrease fat accumulation, and alleviate the peroxidation of biomolecules, effectively treating feline fatty liver syndrome.

[0125] Experiment 4: The therapeutic effect of sea cucumber oligopeptides on liver injury

[0126] HepG2 cells were co-cultured with lipopolysaccharide (LPS) (1 µg / mL) for 24 hours, and then with an aqueous solution of sea cucumber oligoactive peptides (2 µg / mL) for 24 hours. The secretion level of TNF-α inflammatory factor was detected. The results showed that the TNF-α content of the HepG2 cell LPS inflammation model without sea cucumber oligoactive peptide treatment reached 59.18±3.72 pg / ml, which was much higher than that of ordinary HepG2 cells in the blank group (4.45±0.83 pg / ml). After treatment with sea cucumber oligoactive peptide solution, the TNF-α level was significantly improved (10.3±1.27 pg / ml), showing that sea cucumber oligoactive peptides (Tyr-Pro-Asp-Cys-Pro-Gly-Pro) had a significant effect on alleviating hepatocyte inflammation.

[0127] Experiment 5: The effect of fish maw oligopeptides on improving adipose-derived hepatocytes

[0128] HepG2 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and incubated at 37°C in a 5% CO2 incubator. When the cell density in the 6-well plates reached 60%–70%, they were divided into three groups: control group: cultured in normal medium (containing a solution of equal concentration); model group: cultured in 0.25 mmol / L palmitic acid for 24 h, then replaced with normal medium; and fish maw oligopeptide group: cultured in 0.25 mmol / L palmitic acid for 24 h, then treated with 16 μg / mL fish maw oligopeptide solution for 12 h. Oil Red O staining solution (Oil Red stock solution: deionized water = 3:2) was added for staining for 15 min, rinsed several times with distilled water, and observed and photographed under a microscope. After dissolving the dye in isopropanol, the absorbance of each group was measured at 570 nm using a microplate reader. 570 The results showed that the D group of fish maw oligopeptides... 570 (0.28±0.08) is much lower than the D of the model group. 570 (0.49±0.06), close to the D of the blank group. 570 (0.21±0.08) indicates that the intervention of fish maw oligopeptide solution effectively reduced fat accumulation in fatty hepatocytes.

[0129] Finally, it should be noted that the above examples are merely a few specific embodiments of the present invention. All derivatives that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. A targeted complex for treating fatty liver in cats, characterized in that, It includes astaxanthin and a complex carrier, wherein the complex carrier includes sea cucumber oligopeptides, fish maw oligopeptides and galactosylated bacterial cellulose carrier. The mass ratio of astaxanthin to the composite carrier is 1:10 to 100, and the mass ratio of sea cucumber oligopeptides, fish maw oligopeptides and galactosylated bacterial cellulose carrier in the composite carrier is 1 to 3:1 to 3:

1. The amino acid sequence of the sea cucumber oligopeptide is Tyr-Pro-Asp-Cys-Pro-Gly-Pro; The amino acid sequence of fish maw oligopeptides includes Tyr-Cys-Tyr-Cys-Pro-Arg.

2. The targeted complex for treating feline fatty liver according to claim 1, characterized in that, The mass ratio of astaxanthin to the composite carrier is 1:

10.

3. The targeted complex for treating feline fatty liver according to claim 1, characterized in that, in, The mass ratio of sea cucumber oligopeptides, fish maw oligopeptides, and galactosylated bacterial cellulose carrier is 2:2:

1.

4. A method for preparing the targeted complex for treating feline fatty liver as described in claim 1, characterized in that, include S41. Dissolve astaxanthin in an ethanol solution and dissolve the composite carrier in water; S42. Add an ethanol solution containing astaxanthin oil to a solution of the composite carrier, heat to react, evaporate to remove the ethanol, and obtain a targeted complex for treating feline fatty liver.

5. The preparation method according to claim 4, characterized in that, In step S41, astaxanthin oil with a content of 10% is dissolved in an ethanol solution and stirred evenly. The composite carrier is dissolved in ultrapure water. The mass-volume ratio of astaxanthin oil to ethanol solution is 1% to 10%, and the mass ratio of astaxanthin oil to composite carrier is 1:(1 to 10). In step S42, an ethanol solution containing astaxanthin oil is slowly added dropwise to the composite carrier solution. During the dropwise addition, the mixture is heated and stirred to carry out the reaction. The ethanol is evaporated to remove the ethanol, and the mixture is freeze-dried to obtain a powder of the composite. The stirring speed is 500–1500 r / min, the reaction temperature is 40–60℃, and the reaction time is 2–4 h.

6. The preparation method according to claim 4, characterized in that, Methods for preparing composite carriers, including Sea cucumber oligopeptides, fish maw oligopeptides, and galactosylated bacterial cellulose carrier were dissolved in ultrapure water, stirred at 80-100℃ for 2-6 hours, and then freeze-dried to obtain a composite carrier.

7. The preparation method according to claim 6, characterized in that, in, Galactosylated bacterial cellulose carrier was prepared based on the following method: S21. Dissolve bacterial cellulose in a dimethyl sulfoxide solution containing tetrabutylammonium acetate and stir until no gelatinous material coagulates, to obtain a homogeneous bacterial cellulose solution with a mass-volume ratio of 1% to 5%. S22. Dissolve lactobionic acid in a dimethyl sulfoxide (DMSO) solution containing 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and stir to activate the carboxyl groups for 1-3 hours to obtain a lactobionic acid solution with a mass-to-volume ratio of 1%-10%; wherein, the ratio of lactobionic acid:1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC):N-hydroxysuccinimide (NHS) is 1-5:1:1; S23. Add the bacterial cellulose solution to the lactobionic acid solution, heat and stir to remove impurities, freeze dry to obtain galactosylated bacterial cellulose carrier powder, wherein the heating and stirring temperature is 40-50℃ and the heating and stirring time is 10-24h.

8. The preparation method according to claim 7, characterized in that, In step S22, the activation time is 2 hours.

9. The preparation method according to claim 7, characterized in that, In step S22, the ratio of lactobionic acid: 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC): N-hydroxysuccinimide (NHS) is 1:1:

1.

10. The preparation method according to claim 7, characterized in that, In step S23, the removal of impurities includes dialysis with distilled water in a dialysis tube to remove unreacted lactobionic acid, bacterial cellulose, and dimethyl sulfoxide (DMSO) solution containing tetrabutylammonium acetate.

11. The preparation method according to claim 10, characterized in that, In step S23, the dialysis time is 24–72 hours.

12. The preparation method according to claim 11, characterized in that, The dialysis time is 72 hours.

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