Compound nutritional supplement based on Hu sheep active extract

Abstract

The invention discloses a compound nutritional supplement based on Hu sheep milk exosomes, which is characterized in that fresh Hu sheep milk is used as a raw material, the fresh Hu sheep milk is sterilized and then subjected to centrifugal pretreatment to remove milk fat and casein, and the high-purity Hu sheep milk exosome extract is obtained through ultracentrifugation and size exclusion chromatography purification; 1, 5-anhydride-D-sorbitol and alpha-D-mannose pentaacetate are compounded to form a freeze-drying protective agent, and freeze-dried exosome powder is prepared through ultralow-temperature pre-freezing and freeze-drying; and carrying out a chitosan and hyaluronic acid layer-by-layer coating process to obtain the composite nutritional supplement. The supplement can efficiently maintain the structural integrity and biological activity of the Hu sheep milk exosome, can significantly protect the barrier function of intestinal epithelial cells and inhibit inflammatory response, is excellent in biocompatibility, can play a role in regulating intestinal health in a targeted manner, and is suitable for the field of nutritional supplement related to intestinal inflammation protection and intestinal function improvement.

Description

Technical Field

[0001] This invention belongs to the field of nutritional health products, specifically relating to a compound nutritional supplement based on active extracts of Hu sheep. Background Technology

[0002] Inflammatory bowel disease (IBD) and other gut-related diseases have become prevalent chronic illnesses worldwide. Their core pathological features include impaired intestinal barrier function, immune dysregulation, and persistent inflammation. Clinical manifestations include diarrhea, abdominal pain, and bloody, mucous stools, severely impacting patients' quality of life. Furthermore, long-term illness increases the risk of complications such as colon cancer. Currently, there are numerous nutritional supplements related to gut health regulation, but they generally suffer from several core deficiencies: First, the active ingredients have poor stability, especially bioactive molecules which are easily inactivated during processing and storage; second, they lack intestinal targeting, as active ingredients are easily degraded by gastrointestinal fluids, making it difficult to efficiently accumulate at the lesion site; and third, their intestinal protective effects are limited, often targeting only a single pathological link and failing to simultaneously repair the intestinal barrier, suppress inflammatory responses, and regulate immune balance.

[0003] Milk-derived exosomes, as natural nanovesicles, are rich in bioactive molecules such as microRNA, proteins, and lipids. They exhibit unique advantages in intestinal development, immune regulation, and intestinal barrier protection, and have become a research hotspot in the fields of nutrition and medicine. Sheep milk exosomes can alleviate symptoms of inflammatory bowel disease by inhibiting excessive accumulation of reactive oxygen species in the intestine, reducing lipid peroxidation levels, and decreasing intestinal epithelial cell death; while bovine milk exosomes can upregulate the expression of intestinal tight junction proteins and improve intestinal permeability. However, the industrial application of milk exosomes still faces multiple technical bottlenecks: First, separation and purification are difficult. The nanomicelles formed by casein in milk and the exosomes have highly overlapping particle sizes, and conventional centrifugation methods easily lead to co-precipitation, seriously affecting the purity of exosomes. Second, storage stability is poor. Ice crystal formation and osmotic pressure fluctuations during freeze-drying can easily damage the exosome membrane structure and cause loss of biological activity. Existing single protective agents are difficult to meet the requirements of membrane stability and osmotic pressure regulation. Third, there is a lack of specific process design. Existing technologies are mostly for exosomes from cow's milk and ordinary goat's milk, and the separation and preparation process has not been optimized for the component characteristics of Hu sheep's milk, resulting in the failure to fully utilize the activity advantages of Hu sheep's milk exosomes. Fourth, intestinal delivery efficiency is low. After oral administration, exosomes are easily degraded by gastric acid and digestive enzymes, making it difficult to reach the site of colon inflammation to exert their effects.

[0004] Furthermore, existing freeze-drying protectants are mostly traditional sugars such as sucrose and trehalose. While they can provide some osmotic pressure protection, their effectiveness in maintaining the integrity of the exosome lipid bilayer and inhibiting particle aggregation is limited. The synergistic mechanism of action of compound protectants has not been fully explored, and there is a complete lack of specialized protectant formulations for exosomes from sheep milk. At the same time, intestinal-targeted delivery technology for exosomes still needs improvement; the lack of a delivery system that combines biocompatibility and specific degradation capabilities limits its practical application in the intervention of intestinal diseases.

[0005] Therefore, developing an efficient separation and purification process for exosomes from sheep milk, constructing a common and stable composite protection system, and combining it with targeted delivery technology to prepare a composite nutritional supplement with stable active ingredients and significant intestinal protection effects has important practical application value for filling the industry's technological gap and meeting the clinical and market needs for intestinal health regulation. Summary of the Invention

[0006] The purpose of this invention is to provide a compound nutritional supplement based on sheep milk exosomes that has stable active ingredients, significant intestinal protection effects, and high biosafety. It addresses the problems of existing milk exosomes, such as difficulty in isolation and purification, easy loss of activity during freeze-drying, and insufficient intestinal targeting, and achieves protection of the intestinal epithelial cell barrier and inhibition of inflammation.

[0007] The technical solution adopted by the present invention to achieve the above objectives is as follows: A method for preparing a compound nutritional supplement, comprising, Fresh milk from Hu sheep was sterilized to remove milk fat and cell debris, resulting in secondary whey. An aqueous acetic acid solution was added to the secondary whey, and after incubation, it was centrifuged to obtain pretreated whey. The pretreated whey was purified by ultracentrifugation and size exclusion chromatography to obtain exosome extract from sheep milk. The exosome extract from sheep milk was mixed with a freeze-drying protectant and then pre-frozen and freeze-dried at ultra-low temperature to obtain freeze-dried exosome powder. The lyophilized exosome powder was reconstituted with PBS buffer, mixed with chitosan solution and centrifuged, then coated with hyaluronic acid solution and centrifuged to obtain a compound nutritional supplement. Preferably, the freeze-drying protectant includes 1,5-anhydride-D-sorbitol and pentaacetic acid-α-D-mannose.

[0008] Preferably, the mass ratio of 1,5-anhydride-D-sorbitol to the exosome extract of sheep milk is 0.8-1.4:100.

[0009] Preferably, the mass ratio of α-D-mannose pentaacetic acid to the exosome extract of sheep milk is 0.6-1.2:100.

[0010] When 1,5-anhydride-D-sorbitol and pentaacetic acid-α-D-mannose work together, they can maintain the osmotic pressure balance around exosomes, reducing membrane damage caused by drastic osmotic pressure changes during freeze-drying. At the same time, they can interact with exosome membrane components to stabilize the lipid bilayer structure, prevent membrane fusion and rupture, provide a stable physical environment for exosomes, buffer external freezing pressure changes, reduce mechanical damage to vesicles caused by ice crystal formation, and thus inhibit the aggregation tendency between exosome particles, maintaining their dispersion. This ensures that exosomes retain good structural integrity and bioactivity during freeze-drying and subsequent preparation, allowing the functional biomolecules they carry to function normally.

[0011] Preferably, differential centrifugation is used to remove milk fat and cell debris, and differential centrifugation includes a first centrifugation and a second centrifugation.

[0012] Preferably, the conditions for the first centrifugation are 2-5℃ and 2000×g-4000×g for 15-25 minutes.

[0013] Preferably, the conditions for the second centrifugation are 2-5℃ and 5000×g-6000×g for 15-25 minutes.

[0014] Preferably, the concentration of the acetic acid aqueous solution is 9-11% v / v.

[0015] Preferably, the volume ratio of secondary whey to aqueous acetic acid solution is 10:0.5-2.

[0016] Preferably, the conditions for ultracentrifugation are 2-5℃, 125000×g-145000×g for 0.5-2h.

[0017] Preferably, the size exclusion chromatography column is a 2.6×25-40cm SephacrylS-500 column, and the eluent is a PBS buffer containing 15-25 mmol / L EDTA.

[0018] Preferably, the ultra-low temperature pre-freezing conditions are -85℃ to -75℃ for 1.5-2.5 hours.

[0019] Preferably, the freeze-drying conditions are -58℃ to -55℃ and 0.04 to 0.06 MPa for 45-50 hours.

[0020] A compound nutritional supplement.

[0021] The use of a compound nutritional supplement in regulating the gut.

[0022] Preferably, regulating the gut includes protecting the intestinal epithelial cell barrier function.

[0023] More preferably, the freeze-drying protectant includes diethyl 2-hydroxysuccinate, and the mass ratio of diethyl 2-hydroxysuccinate to the exosome extract of sheep milk is 0.2-0.8:100.

[0024] Diethyl 2-hydroxysuccinate, as an important component of freeze-drying protectant, works in conjunction with 1,5-anhydride-D-sorbitol and α-D-mannose pentaacetic acid to regulate the charge distribution on the surface of exosomes, thereby altering the electrostatic interactions between particles and reducing interparticle attraction. This further inhibits the aggregation of exosomes during freeze-drying and reconstitution. Simultaneously, its molecular structure can interact with exosome membrane components, enhancing membrane stability and reducing the loss of active ingredients, thus more efficiently exerting the protective and repairing effects of exosomes on the intestinal barrier.

[0025] This invention also provides a method for preparing pretreated whey, comprising: Fresh sheep milk was pasteurized at 71-73℃ for 13-17 seconds, then centrifuged at 2-5℃ and 2000×g-4000×g for 15-25 minutes to remove the supernatant and bottom cell debris. The middle layer was collected to obtain primary whey. The primary whey was centrifuged at 2-5℃ and 5000×g-6000×g for 15-25 minutes to remove the supernatant. The middle layer was collected to obtain secondary whey. The secondary whey was added to a 9-11% (v / v) acetic acid aqueous solution, the pH was adjusted to 4.0-4.2, and incubated at 200×g-400×g for 10-20 minutes. It was then centrifuged at 2-5℃ and 9000×g-11000×g for 15-25 minutes. The supernatant was collected and filtered to obtain pretreated whey.

[0026] Preferably, the volume ratio of secondary whey to 9-11% (v / v) acetic acid aqueous solution is 10:0.5-2.

[0027] This invention also provides a method for preparing lyophilized exosome powder, comprising: The pretreated whey was ultracentrifuged at 2-5℃ and 125000×g-145000×g- for 0.5-2h, the precipitate was collected, and resuspended in PBS buffer to obtain crude exosome extract from sheep milk. The crude exosome extract from sheep milk was purified by size exclusion chromatography, and the eluent was collected to obtain exosome extract from sheep milk. A freeze-drying protectant was added to the exosome extract from sheep milk, and the mixture was mixed evenly. The mixture was pre-frozen at -85℃ to -75℃ for 1.5-2.5h, and then freeze-dried at -58℃ to -55℃ and 0.04-0.06MPa for 45-50h to obtain freeze-dried exosome powder.

[0028] Preferably, the mass-to-volume ratio of the precipitate to the PBS buffer is 1 mg: 0.5-2 mL.

[0029] Preferably, the size exclusion chromatography column includes a 2.6×25-40cm Sephacryl S-500 column.

[0030] Preferably, the elution buffer is a PBS buffer containing 15-25 mmol / L EDTA.

[0031] Preferably, the mass-to-volume ratio of crude extract of exosomes from sheep milk to eluent is 1 mg: 10-30 mL.

[0032] Preferably, the flow rate of the eluent is 0.3-0.5 mL / min.

[0033] Preferably, the freeze-drying protectant includes 1,5-anhydride-D-sorbitol and pentaacetic acid-α-D-mannose.

[0034] Preferably, the mass ratio of 1,5-anhydride-D-sorbitol to the exosome extract of sheep milk is 0.8-1.4:100.

[0035] Preferably, the mass ratio of α-D-mannose pentaacetic acid to the exosome extract of sheep milk is 0.6-1.2:100.

[0036] More preferably, the freeze-drying protectant includes diethyl 2-hydroxysuccinate.

[0037] More preferably, the mass ratio of diethyl 2-hydroxysuccinate to the exosome extract of sheep milk is 0.2-0.8:100.

[0038] This invention also provides a method for preparing a compound nutritional supplement, comprising: Chitosan was dissolved in 0.05-0.15% (V / V) acetic acid aqueous solution to obtain a chitosan solution; hyaluronic acid was dissolved in deionized water to obtain a hyaluronic acid solution; lyophilized exosome powder was reconstituted with PBS buffer to obtain an exosome resuspension; the exosome resuspension was added to the chitosan solution, and the mixture was shaken at 2-5℃ and 400×g-500×g for 10-20 min, and then centrifuged at 2-5℃ and 20000×g-22000×g for 1 minute. Collect the precipitate after 5-25 min, wash with washing buffer and resuspend in deionized water to obtain a complex resuspension; add hyaluronic acid solution to the complex resuspension, shake at 2-5℃ and 450×g-550×g for 10-20 min, centrifuge at 2-5℃ and 20000×g-22000×g for 15-25 min, collect the precipitate, wash 1-3 times with washing buffer, resuspend in PBS buffer, and filter to obtain the complex nutritional supplement.

[0039] Preferably, in the chitosan solution, the mass-to-volume ratio of chitosan to 0.05-0.15% (V / V) acetic acid aqueous solution is 1-3 mg:1 mL.

[0040] Preferably, in the hyaluronic acid solution, the mass-to-volume ratio of hyaluronic acid to deionized water is 1-3 mg:1 mL.

[0041] Preferably, in the exosome resuspension, the mass-to-volume ratio of lyophilized exosome powder to PBS buffer is 1-10 mg: 1 mL.

[0042] Preferably, the mass of the chitosan solution is measured by the mass of the chitosan therein, and the mass of the exosome resuspension is measured by the mass of the exosomes therein, with a mass ratio of chitosan to exosomes of 1-3:5.

[0043] Preferably, the mass of the hyaluronic acid solution is measured by the mass of the hyaluronic acid therein, and the mass of the exosome resuspension is measured by the mass of the exosomes therein, with the mass ratio of hyaluronic acid to exosomes being 1-3:5.

[0044] Preferably, the washing solution is deionized water.

[0045] Preferably, in the complex resuspension, the mass-to-volume ratio of the precipitate to deionized water is 1 mg: 0.5-5 mL.

[0046] Preferably, in the compound nutritional supplement, the mass-to-volume ratio of the precipitate to the PBS buffer is 1 mg: 0.5-5 mL.

[0047] This invention employs a specific process for separating and purifying exosomes from Hu sheep milk, combined with a freeze-drying protectant composed of 1,5-anhydride-D-sorbitol and α-D-mannose pentaacetic acid, and a composite coating process using chitosan and hyaluronic acid. Therefore, it offers the following advantages: First, it efficiently separates and purifies Hu sheep milk exosomes, effectively removing impurities and obtaining high-purity active extracts. Second, the combined freeze-drying protectant significantly enhances the structural stability and bioactivity retention of exosomes, reducing aggregation and loss of active ingredients. Third, the prepared compound nutritional supplement effectively protects the intestinal epithelial cell barrier function, inhibits inflammatory responses, and exhibits good biocompatibility. Therefore, this invention is a compound nutritional supplement based on Hu sheep active extracts, characterized by advanced technology, stable active ingredients, significant intestinal protection effects, and high safety. Attached Figure Description

[0048] Figure 1 This is a schematic diagram showing the particle size distribution test results of lyophilized exosome powder.

[0049] Figure 2 This is a schematic diagram of the test results showing the protective effect of compound nutritional supplements on the intestinal epithelial cell barrier function.

[0050] Figure 3 This is a schematic diagram of the biosafety test results for a compound nutritional supplement. Detailed Implementation

[0051] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0053] Example 1: Preparation of pretreated whey: Fresh sheep milk was pasteurized at 72℃ for 15 seconds, centrifuged at 4℃ and 3000×g for 20 minutes to remove the supernatant and bottom cell debris, and the middle layer was collected to obtain primary whey. The primary whey was centrifuged at 4℃ and 5500×g for 20 minutes to remove the supernatant, and the middle layer was collected to obtain secondary whey. The secondary whey was added to a 10% (v / v) acetic acid aqueous solution, the pH was adjusted to 4.1, incubated at 300×g for 15 minutes, centrifuged at 4℃ and 10000×g for 20 minutes, the supernatant was collected, and filtered through a 0.22 μm sterile filter membrane to obtain pretreated whey. The volume ratio of secondary whey to 10% (v / v) acetic acid aqueous solution was 10:1.

[0054] Preparation of lyophilized exosome powder: The pretreated whey was ultracentrifuged at 135000×g for 1 h at 4 °C, the precipitate was collected, and resuspended in PBS buffer to obtain crude extract of exosomes from sheep milk; the crude extract of exosomes from sheep milk was purified by size exclusion chromatography, and the eluent was collected at 280 nm wavelength using a UV detector to obtain exosome extract from sheep milk; 1,5-anhydride-D-sorbitol and α-D-mannose pentaacetic acid were added to the exosome extract from sheep milk, mixed evenly, pre-frozen at -80 °C for 2 h, and freeze-dried at -56 °C and 0.05 MPa for 48 h to obtain lyophilized exosome powder. The mass-to-volume ratio of the precipitate to PBS buffer was 1 mg:1 mL; the size exclusion chromatography column was a 2.6 × 30 cm Sephacryl S-500 column, the eluent was PBS buffer containing 20 mmol / L EDTA, the mass-to-volume ratio of crude exosome extract from sheep milk to eluent was 1 mg:15 mL, and the flow rate of the eluent was 0.4 mL / min; the mass ratio of 1,5-anhydride-D-sorbitol to sheep milk exosome extract was 1:100, and the mass ratio of α-D-mannose pentaacetate to sheep milk exosome extract was 0.8:100.

[0055] Preparation of the compound nutritional supplement: Chitosan was dissolved in 0.1% (V / V) acetic acid aqueous solution to obtain a chitosan solution; hyaluronic acid was dissolved in deionized water to obtain a hyaluronic acid solution; lyophilized exosome powder was reconstituted with PBS buffer to obtain an exosome resuspension; the exosome resuspension was added to the chitosan solution, shaken at 4℃ and 500×g for 15 min, centrifuged at 4℃ and 21000×g for 20 min, the precipitate was collected, washed with washing buffer, and resuspended in deionized water to obtain a compound resuspension; hyaluronic acid solution was added to the compound resuspension, shaken at 4℃ and 500×g for 15 min, centrifuged at 4℃ and 21000×g for 20 min, the precipitate was collected, washed twice with washing buffer, resuspended with PBS buffer, and filtered through a 0.22 μm sterile filter membrane to obtain the compound nutritional supplement. In the chitosan solution, the mass-to-volume ratio of chitosan to 0.1% (v / v) acetic acid aqueous solution was 2 mg:1 mL; in the hyaluronic acid solution, the mass-to-volume ratio of hyaluronic acid to deionized water was 2 mg:1 mL; in the exosome resuspension, the mass-to-volume ratio of lyophilized exosome powder to PBS buffer was 5 mg:1 mL; the mass of the chitosan solution was measured by the mass of chitosan, the mass of the hyaluronic acid solution was measured by the mass of hyaluronic acid, and the mass of the exosome resuspension was measured by the mass of exosomes; the mass ratio of chitosan to exosomes was 2:5, and the mass ratio of hyaluronic acid to exosomes was 2:5; the washing solution was deionized water; in the complex resuspension, the mass-to-volume ratio of precipitate to deionized water was 1 mg:1 mL; in the complex nutritional supplement, the mass-to-volume ratio of precipitate to PBS buffer was 1 mg:1 mL.

[0056] Example 2: The only difference between this example and Example 1 is the preparation of the lyophilized exosome powder.

[0057] Preparation of lyophilized exosome powder: The pretreated whey was ultracentrifuged at 135000×g for 1 h at 4 °C, the precipitate was collected, and resuspended in PBS buffer to obtain crude extract of exosomes from sheep milk; the crude extract of exosomes from sheep milk was purified by size exclusion chromatography, and the eluent was collected at 280 nm wavelength using a UV detector to obtain exosome extract from sheep milk; 1,5-anhydride-D-sorbitol and α-D-mannose pentaacetic acid were added to the exosome extract from sheep milk, mixed evenly, pre-frozen at -80 °C for 2 h, and freeze-dried at -56 °C and 0.05 MPa for 48 h to obtain lyophilized exosome powder. The mass-to-volume ratio of the precipitate to PBS buffer was 1 mg:1 mL; the size exclusion chromatography column was a 2.6 × 30 cm Sephacryl S-500 column, the eluent was PBS buffer containing 20 mmol / L EDTA, the mass-to-volume ratio of crude exosome extract from Hu sheep milk to eluent was 1 mg:15 mL, and the flow rate of the eluent was 0.4 mL / min; the mass ratio of 1,5-anhydride-D-sorbitol to Hu sheep milk exosome extract was 1:100, and the mass ratio of α-D-mannose pentaacetate to Hu sheep milk exosome extract was 1:100.

[0058] Example 3: The only difference between this example and Example 1 is the preparation of the lyophilized exosome powder.

[0059] Preparation of lyophilized exosome powder: The pretreated whey was ultracentrifuged at 135000×g for 1 h at 4 °C, the precipitate was collected, and resuspended in PBS buffer to obtain crude extract of exosomes from sheep milk; the crude extract of exosomes from sheep milk was purified by size exclusion chromatography, and the eluent was collected at 280 nm wavelength using a UV detector to obtain exosome extract from sheep milk; 1,5-anhydride-D-sorbitol, α-D-mannose pentaacetate, and diethyl 2-hydroxysuccinate were added to the exosome extract from sheep milk, mixed evenly, pre-frozen at -80 °C for 2 h, and freeze-dried at -56 °C and 0.05 MPa for 48 h to obtain lyophilized exosome powder. The mass-to-volume ratio of the precipitate to PBS buffer was 1 mg:1 mL; the size exclusion chromatography column was a 2.6 × 30 cm Sephacryl S-500 column, the eluent was PBS buffer containing 20 mmol / L EDTA, the mass-to-volume ratio of crude exosome extract from sheep milk to eluent was 1 mg:15 mL, and the flow rate of the eluent was 0.4 mL / min; the mass ratio of 1,5-anhydride-D-sorbitol to sheep milk exosome extract was 1:100, the mass ratio of α-D-mannose pentaacetate to sheep milk exosome extract was 0.8:100, and the mass ratio of diethyl 2-hydroxysuccinate to sheep milk exosome extract was 0.4:100.

[0060] Example 4: The only difference between this example and Example 1 is the preparation of the lyophilized exosome powder.

[0061] Preparation of lyophilized exosome powder: The pretreated whey was ultracentrifuged at 135000×g for 1 h at 4 °C, the precipitate was collected, and resuspended in PBS buffer to obtain crude extract of exosomes from sheep milk; the crude extract of exosomes from sheep milk was purified by size exclusion chromatography, and the eluent was collected at 280 nm wavelength using a UV detector to obtain exosome extract from sheep milk; 1,5-anhydride-D-sorbitol, α-D-mannose pentaacetate, and diethyl 2-hydroxysuccinate were added to the exosome extract from sheep milk, mixed evenly, pre-frozen at -80 °C for 2 h, and freeze-dried at -56 °C and 0.05 MPa for 48 h to obtain lyophilized exosome powder. The mass-to-volume ratio of the precipitate to PBS buffer was 1 mg:1 mL; the size exclusion chromatography column was a 2.6 × 30 cm Sephacryl S-500 column, the eluent was PBS buffer containing 20 mmol / L EDTA, the mass-to-volume ratio of crude exosome extract from Hu sheep milk to eluent was 1 mg:15 mL, and the flow rate of the eluent was 0.4 mL / min; the mass ratio of 1,5-anhydride-D-sorbitol to Hu sheep milk exosome extract was 1:100, the mass ratio of α-D-mannose pentaacetate to Hu sheep milk exosome extract was 0.8:100, and the mass ratio of diethyl 2-hydroxysuccinate to Hu sheep milk exosome extract was 0.6:100.

[0062] Comparative Example 1: The only difference between this comparative example and Example 1 is the preparation of the lyophilized exosome powder.

[0063] Preparation of lyophilized exosome powder: The pretreated whey was ultracentrifuged at 135000×g for 1 h at 4 °C, the precipitate was collected, and resuspended in PBS buffer to obtain crude extract of exosomes from sheep milk; the crude extract of exosomes from sheep milk was purified by size exclusion chromatography, and the eluent was collected at 280 nm wavelength using a UV detector to obtain exosome extract from sheep milk; 1,5-anhydride-D-sorbitol was added to the exosome extract from sheep milk, mixed evenly, pre-frozen at -80 °C for 2 h, and then freeze-dried at -56 °C and 0.05 MPa for 48 h to obtain lyophilized exosome powder. The mass-to-volume ratio of the precipitate to PBS buffer was 1 mg:1 mL; the size exclusion chromatography column was a 2.6 × 30 cm Sephacryl S-500 column, the eluent was PBS buffer containing 20 mmol / L EDTA, the mass-to-volume ratio of crude exosome extract from sheep milk to eluent was 1 mg:15 mL, the flow rate of the eluent was 0.4 mL / min; the mass ratio of 1,5-anhydride-D-sorbitol to sheep milk exosome extract was 1:100.

[0064] Comparative Example 2: The only difference between this comparative example and Example 1 is the preparation of the lyophilized exosome powder.

[0065] Preparation of lyophilized exosome powder: The pretreated whey was ultracentrifuged at 135000×g for 1 h at 4 °C, the precipitate was collected, and resuspended in PBS buffer to obtain crude extract of exosomes from sheep milk; the crude extract of exosomes from sheep milk was purified by size exclusion chromatography, and the eluent was collected at 280 nm wavelength using a UV detector to obtain exosome extract from sheep milk; α-D-mannose pentaacetic acid was added to the exosome extract from sheep milk, mixed evenly, pre-frozen at -80 °C for 2 h, and then freeze-dried at -56 °C and 0.05 MPa for 48 h to obtain lyophilized exosome powder. The mass-to-volume ratio of the precipitate to PBS buffer was 1 mg:1 mL; the size exclusion chromatography column was a 2.6 × 30 cm Sephacryl S-500 column, the eluent was PBS buffer containing 20 mmol / L EDTA, the mass-to-volume ratio of crude exosome extract from Hu sheep milk to eluent was 1 mg:15 mL, the flow rate of the eluent was 0.4 mL / min; the mass ratio of α-D-mannose pentaacetate to Hu sheep milk exosome extract was 0.8:100.

[0066] Comparative Example 3: The only difference between this comparative example and Example 1 is the preparation of the lyophilized exosome powder.

[0067] Preparation of lyophilized exosome powder: The pretreated whey was ultracentrifuged at 135000×g for 1 h at 4 °C, the precipitate was collected, and resuspended in PBS buffer to obtain crude extract of exosomes from sheep milk; the crude extract of exosomes from sheep milk was purified by size exclusion chromatography, and the eluent was collected at 280 nm wavelength using a UV detector to obtain exosome extract from sheep milk; sucrose was added to the exosome extract from sheep milk, mixed evenly, pre-frozen at -80 °C for 2 h, and then freeze-dried at -56 °C and 0.05 MPa for 48 h to obtain lyophilized exosome powder. The mass-to-volume ratio of precipitate to PBS buffer was 1 mg:1 mL; the size exclusion chromatography column was a 2.6 × 30 cm Sephacryl S-500 column, the eluent was PBS buffer containing 20 mmol / L EDTA, the mass-to-volume ratio of crude exosome extract from Hu sheep milk to eluent was 1 mg:15 mL, the flow rate of the eluent was 0.4 mL / min, and the mass ratio of sucrose to Hu sheep milk exosome extract was 1:100.

[0068] Comparative Example 4: The only difference between this comparative example and Example 1 is the preparation of the lyophilized exosome powder.

[0069] Preparation of lyophilized exosome powder: The pretreated whey was ultracentrifuged at 135000×g for 1 h at 4 °C, the precipitate was collected, and resuspended in PBS buffer to obtain crude extract of exosomes from sheep milk; the crude extract of exosomes from sheep milk was purified by size exclusion chromatography, and the eluent was collected at 280 nm wavelength using a UV detector to obtain exosome extract from sheep milk; sorbitol was added to the exosome extract from sheep milk, mixed evenly, pre-frozen at -80 °C for 2 h, and then freeze-dried at -56 °C and 0.05 MPa for 48 h to obtain lyophilized exosome powder. The mass-to-volume ratio of the precipitate to PBS buffer was 1 mg:1 mL; the size exclusion chromatography column was a 2.6 × 30 cm Sephacryl S-500 column, the eluent was PBS buffer containing 20 mmol / L EDTA, the mass-to-volume ratio of crude exosome extract from Hu sheep milk to eluent was 1 mg:15 mL, the flow rate of the eluent was 0.4 mL / min; the mass ratio of sorbitol to Hu sheep milk exosome extract was 1:100.

[0070] Comparative Example 5: The only difference between this comparative example and Example 1 is the preparation of the lyophilized exosome powder.

[0071] Preparation of lyophilized exosome powder: The pretreated whey was ultracentrifuged at 135000×g for 1 h at 4 °C, the precipitate was collected, and resuspended in PBS buffer to obtain crude extract of exosomes from sheep milk; the crude extract of exosomes from sheep milk was purified by size exclusion chromatography, and the eluent was collected at 280 nm wavelength using a UV detector to obtain exosome extract from sheep milk; mannose was added to the exosome extract from sheep milk, mixed evenly, pre-frozen at -80 °C for 2 h, and then freeze-dried at -56 °C and 0.05 MPa for 48 h to obtain lyophilized exosome powder. The mass-to-volume ratio of precipitate to PBS buffer was 1 mg:1 mL; the size exclusion chromatography column was a 2.6 × 30 cm Sephacryl S-500 column, the eluent was PBS buffer containing 20 mmol / L EDTA, the mass-to-volume ratio of crude exosome extract from Hu sheep milk to eluent was 1 mg:15 mL, the flow rate of the eluent was 0.4 mL / min; the mass ratio of mannose to Hu sheep milk exosome extract was 1:100.

[0072] Comparative Example 6: The only difference between this comparative example and Example 1 is the preparation of the lyophilized exosome powder.

[0073] Preparation of lyophilized exosome powder: The pretreated whey was ultracentrifuged at 135000×g for 1 h at 4 °C, the precipitate was collected, and resuspended in PBS buffer to obtain crude exosome extract from sheep milk. The crude exosome extract from sheep milk was purified by size exclusion chromatography, and the eluent was collected at 280 nm wavelength using a UV detector to obtain exosome extract from sheep milk. The exosome extract from sheep milk was pre-frozen at -80 °C for 2 h, and then freeze-dried at -56 °C and 0.05 MPa for 48 h to obtain lyophilized exosome powder. The mass-to-volume ratio of precipitate to PBS buffer was 1 mg:1 mL. The size exclusion chromatography column was a 2.6×30 cm Sephacryl S-500 column, the eluent was PBS buffer containing 20 mmol / L EDTA, the mass-to-volume ratio of crude exosome extract from sheep milk to eluent was 1 mg:15 mL, and the flow rate of the eluent was 0.4 mL / min.

[0074] Experimental Example 1: Particle size distribution test of lyophilized exosome powder.

[0075] Test samples: lyophilized exosome powders prepared in each example and comparative example.

[0076] Test method: 5 mg of lyophilized exosome powder was reconstituted in 1 mL of PBS buffer, ultrasonically dispersed for 10 min, and an appropriate amount of dispersion was added to the sample cell. The particle size was measured using a dynamic light scattering instrument at 25 °C with the scattering angle set to 90 ° to obtain the average particle size of the lyophilized exosome powder.

[0077] The particle size distribution test results of the lyophilized exosome powder prepared in this invention are as follows: Figure 1 As shown, in Example 1, 1,5-anhydride-D-sorbitol and pentaacetic acid α-D-mannose were used as freeze-drying protectants. The combined use of these two protectants maintained the osmotic pressure balance around the exosomes, stabilized the membrane structure, provided a stable physical environment for the exosomes, buffered changes in external freezing pressure, reduced aggregation, and resulted in a lower average particle size. In Example 2, the amount of pentaacetic acid α-D-mannose was increased, further enhancing membrane structure stability, improving aggregation inhibition, and decreasing the average particle size compared to Example 1. In Examples 3 and 4, diethyl 2-hydroxysuccinate was introduced to form a composite freeze-drying protectant, which could further reduce the average particle size by adjusting the surface charge distribution of the exosomes and decreasing interparticle attraction. Comparative Examples 1 and 2 used a single protectant. The first example could not simultaneously maintain osmotic pressure and stabilize the membrane structure, resulting in limited inhibition of aggregation and a larger average particle size than Example 1. The second example used sucrose in Comparative Example 3, which provided some osmotic pressure protection, but the average particle size was still lower than that of Example 1. The third example used ordinary sorbitol, which lacked the cyclic structure of 1,5-anhydride-D-sorbitol, resulting in weaker binding to the exosome membrane and insufficient stabilization of the membrane structure. The fourth example used ordinary mannose in Comparative Example 5, which was not acetylated, and its aggregation inhibition effect was even worse than that of Example 1. The fifth example did not add any protective agent, and the exosomes aggregated extensively during freeze-drying due to osmotic pressure imbalance and membrane structure damage, resulting in the largest average particle size, significantly higher than all other examples, fully demonstrating the superiority of the freeze-drying protective agent combination of the present invention.

[0078] Experimental Example 2: Protective test of compound nutritional supplements on intestinal epithelial cell barrier function.

[0079] Test samples: compound nutritional supplements prepared in each example and comparative example.

[0080] Test method: Caco-2 cells were seeded onto polycarbonate membranes in Transwell chambers and DMEM medium containing 10% fetal bovine serum was added. The cells were cultured in a 37°C, 5% CO2 incubator for 21 days to form a dense monolayer epithelial barrier. After detecting the initial resistance value using a transepithelial resistance meter, lipopolysaccharide was added to the apical side of the chamber to construct an intestinal barrier damage model. At the same time, each experimental group was given a compound nutritional supplement diluted to the same concentration with sterile PBS, while the blank control group was given an equal amount of sterile PBS. After culturing for another 24 hours, the transepithelial resistance (TEER) value of each group was measured using a transepithelial resistance meter.

[0081] The protective test results of the compound nutritional supplement prepared in this invention on the intestinal epithelial cell barrier function are as follows: Figure 2As shown, in Example 1, the combined protective effect of 1,5-anhydride-D-sorbitol and pentaacetic acid α-D-mannose allowed the exosomes from Hu sheep milk to maintain good activity during freeze-drying and subsequent preparation. The bioactive components carried by these exosomes effectively inhibited lipopolysaccharide-induced barrier damage and maintained the integrity of tight intercellular junctions. In Example 2, by increasing the amount of pentaacetic acid α-D-mannose, the structural stability of the exosomes was further enhanced, making it easier for their active components to exert their barrier protective effect. In Examples 3 and 4, the newly added diethyl 2-hydroxysuccinate, in conjunction with the existing protective agents, not only reduced the loss of exosome active components but also promoted the interaction between exosomes and intestinal epithelial cells, thereby more efficiently repairing the damaged barrier. This effect was more significant with increased dosage. In contrast, Comparative Examples 1 and 2, using only a single freeze-drying protective agent, could not fully maintain the activity of the exosomes. The protective effect of the exosomes in Comparative Example 1 is weaker than that in Comparative Example 2. Comparative Example 3 uses sucrose as a protectant, but its protective effect on highly active exosomes is inferior to that of the composite protectant system of this invention. Exosomes cannot effectively perform their barrier protection function after their activity is damaged. In Comparative Example 4, a large amount of active ingredients are lost during the freeze-drying process. Comparative Example 5 shows weak interaction with the exosome membrane, making it difficult to inhibit membrane rupture and leakage of active molecules, resulting in poor protection of the intestinal epithelial cell barrier and a significantly lower transepithelial resistance value than in Example 1. Comparative Example 6, without any freeze-drying protectant, shows severe structural damage to the exosomes during freeze-drying, resulting in a large loss of active ingredients and almost no protective effect on the intestinal epithelial cell barrier. It exhibits the lowest resistance value, fully demonstrating the intestinal protection advantages brought by the design of the freeze-drying protectant as a composite nutritional supplement.

[0082] Experimental Example 3: Biosafety Test of Compound Nutritional Supplements.

[0083] Test samples: compound nutritional supplements prepared in each example and comparative example.

[0084] Test method: Each compound nutritional supplement was freeze-dried under vacuum and ground into powder. An extract with a concentration of 20 mg / mL was prepared by adding sterile physiological saline and then filtered through a 0.22 μm filter for sterilization. L929 cells were seeded in DMEM medium containing 10% fetal bovine serum and cultured at 37℃ in a 5% CO2 incubator until the logarithmic growth phase. Cells were then cultured at a rate of 1×10⁻⁶ cells / mL. 4 Cells were seeded at a density of 100 μL / well in 96-well plates, and 100 μL of cell suspension was added to each well. The cells were cultured for 24 h to allow them to adhere. The original culture medium was discarded, and 100 μL of each extract was added to the experimental group, while 100 μL of sterile physiological saline was added to the negative control group. After culturing for another 24 h, 20 μL of 5 mg / mL MTT solution was added to each well, and the cells were incubated in the dark for 4 h. After discarding the liquid in the wells, 150 μL of DMSO was added to each well, and the cells were shaken for 10 min to dissolve the crystals. The absorbance of each well was measured at 490 nm using a microplate reader, and the relative cell viability was calculated.

[0085] The biosafety test results of the compound nutritional supplement prepared in this invention are as follows: Figure 3 As shown, the relative cell viability of the compound nutritional supplement samples prepared in each embodiment was consistently higher than 90% when tested by the MTT assay, with small differences between groups and uniform viability. The compound nutritional supplement prepared by this invention through a specific separation and purification process, a compound freeze-drying protective agent system, and a chitosan-hyaluronic acid layer-by-layer coating process has excellent biocompatibility and biosafety, with no obvious cytotoxicity, and fully meets the food safety standards in the field of nutritional health products. This provides direct and reliable experimental support for its subsequent industrial application, safety in oral intake scenarios, and clinical translation feasibility.

[0086] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0087] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A method for preparing a compound nutritional supplement, characterized in that: include, Fresh milk from Hu sheep was sterilized to remove milk fat and cell debris, resulting in secondary whey. An aqueous acetic acid solution was added to the secondary whey, and after incubation, it was centrifuged to obtain pretreated whey. The pretreated whey was purified by ultracentrifugation and size exclusion chromatography to obtain exosome extract from sheep milk. The exosome extract from sheep milk was mixed with a freeze-drying protectant and then pre-frozen and freeze-dried at ultra-low temperature to obtain freeze-dried exosome powder. The lyophilized exosome powder was reconstituted with PBS buffer, mixed with chitosan solution and centrifuged, then coated with hyaluronic acid solution and centrifuged to obtain a compound nutritional supplement. The freeze-drying protectant comprises 1,5-anhydride-D-sorbitol and pentaacetic acid-α-D-mannose.

2. The method for preparing a compound nutritional supplement according to claim 1, characterized in that: The mass ratio of 1,5-anhydride-D-sorbitol to the exosome extract of sheep milk is 0.8-1.4:

100.

3. The method for preparing a compound nutritional supplement according to claim 1, characterized in that: The mass ratio of α-D-mannose pentaacetic acid to the exosome extract of sheep milk is 0.6-1.2:

100.

4. The method for preparing a compound nutritional supplement according to claim 1, characterized in that: Milk fat and cell debris are removed by differential centrifugation, which includes a first centrifugation and a second centrifugation. The conditions for the first centrifugation are 2-5℃ and 2000×g-4000×g for 15-25 min, and the conditions for the second centrifugation are 2-5℃ and 5000×g-6000×g for 15-25 min.

5. The method for preparing a compound nutritional supplement according to claim 1, characterized in that: The concentration of the acetic acid aqueous solution is 9-11% v / v, and the volume ratio of the secondary whey to the acetic acid aqueous solution is 10:0.5-2.

6. The method for preparing a compound nutritional supplement according to claim 1, characterized in that: The conditions for ultracentrifugation are 2-5℃, 125000×g-145000×g for 0.5-2h.

7. A method for preparing a compound nutritional supplement according to claim 1, characterized in that: The size exclusion chromatography column is a 2.6×25-40cm Sephacryl S-500 column, and the eluent is PBS buffer containing 15-25 mmol / L EDTA.

8. The method for preparing a compound nutritional supplement according to claim 1, characterized in that: The conditions for ultra-low temperature pre-freezing are -85℃ to -75℃ for 1.5 to 2.5 hours; the conditions for freeze drying are -58℃ to -55℃ and 0.04 to 0.06 MPa for 45 to 50 hours.

9. A compound nutritional supplement, characterized in that: Prepared by any of the preparation methods described in claims 1-8.

10. The use of the compound nutritional supplement according to claim 9 in regulating the intestines, characterized in that: The regulation of the gut includes protecting the barrier function of intestinal epithelial cells.

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