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CN-121970891-A - Composite nutritional supplement based on Hu sheep active extract

CN121970891ACN 121970891 ACN121970891 ACN 121970891ACN-121970891-A

Abstract

The invention discloses a composite nutritional supplement based on a Hu sheep milk exosome, which is prepared by taking fresh milk of a Hu sheep as a raw material, sterilizing, performing centrifugal pretreatment to remove milk fat and casein, performing ultracentrifugation and size exclusion chromatography purification to obtain a high-purity Hu sheep milk exosome extract, compounding 1, 5-anhydride-D-sorbitol and pentaacetic acid alpha-D-mannose to form a freeze-drying protective agent, preparing freeze-dried exosome powder through ultralow-temperature prefreezing and freeze-drying, and performing a layer-by-layer coating process on chitosan and hyaluronic acid. The supplement can efficiently maintain the structural integrity and biological activity of the exosome of the Hu sheep, can obviously protect the barrier function of intestinal epithelial cells and inhibit inflammatory reaction, has excellent biocompatibility, can target and play the role of regulating intestinal health, and is suitable for the field of nutrition supplement related to intestinal inflammation protection and intestinal function improvement.

Inventors

  • ZHANG BIN
  • ZENG LIN

Assignees

  • 蚌埠学院

Dates

Publication Date
20260505
Application Date
20260226

Claims (10)

  1. 1. A preparation method of a composite nutritional supplement is characterized by comprising the steps of, Sterilizing fresh milk of Hu sheep, removing milk fat and cell debris to obtain secondary whey, adding aqueous solution of acetic acid into the secondary whey, incubating, and centrifuging to obtain pretreated whey; Purifying the pretreated whey liquid through ultracentrifugation and size exclusion chromatography to obtain a Hu sheep milk exosome extract, mixing the Hu sheep milk exosome extract with a freeze-drying protective agent, and performing ultralow temperature pre-freezing and freeze-drying to obtain freeze-dried exosome powder; re-dissolving the freeze-dried exosome powder by using PBS buffer solution, mixing with chitosan solution, centrifuging, mixing with hyaluronic acid solution for coating, and centrifuging to obtain the composite nutritional supplement; The freeze drying protective agent comprises 1, 5-anhydride-D-sorbitol and pentaacetic acid alpha-D-mannose.
  2. 2. The method of claim 1, wherein the mass ratio of 1, 5-anhydride-D-sorbitol to Hu sheep milk exosome extract is 0.8-1.4:100.
  3. 3. The method of claim 1, wherein the ratio of the pentaacetic acid alpha-D-mannose to the extract of the exosome of Hu sheep milk is 0.6-1.2:100.
  4. 4. The method of claim 1, wherein the milk fat and cell debris are removed by differential centrifugation, the differential centrifugation comprises a first centrifugation at 2-5 ℃ and 2000 Xg-4000 Xg for 15-25min and a second centrifugation at 2-5 ℃ and 5000 Xg-6000 Xg for 15-25min.
  5. 5. The method of claim 1, wherein the concentration of the aqueous acetic acid solution is 9-11% v/v, and the volume ratio of the secondary whey to the aqueous acetic acid solution is 10:0.5-2.
  6. 6. The method of claim 1, wherein the ultracentrifugation is performed at a temperature of 2-5 ℃ for 0.5-2h at 125000Xg-145000Xg.
  7. 7. The method of claim 1, wherein the size exclusion chromatography column is a SEPHACRYL S-500 column of 2.6X25-40 cm, and the eluent is PBS buffer containing 15-25 mmol/LEDTA.
  8. 8. The preparation method of the composite nutritional supplement according to claim 1, wherein the ultralow temperature pre-freezing condition is pre-freezing at-85 ℃ to-75 ℃ for 1.5-2.5 hours, and the freeze-drying condition is freezing at-58 ℃ to-55 ℃ and 0.04-0.06 MPa for 45-50 hours.
  9. 9. A composite nutritional supplement characterized by being prepared by the preparation method as claimed in any one of claims 1 to 8.
  10. 10. The use of a complex nutritional supplement according to claim 9 for regulating the intestinal tract, wherein the regulating the intestinal tract comprises protecting the intestinal epithelial cell barrier function.

Description

Composite nutritional supplement based on Hu sheep active extract Technical Field The invention belongs to the field of nutritional health products, and particularly relates to a compound nutritional supplement based on Hu sheep active extracts. Background Intestinal related diseases such as inflammatory bowel disease are chronic diseases with high global incidence, which take intestinal barrier function damage, immune disorder and persistent inflammation as core pathological characteristics, clinical manifestations include diarrhea, abdominal pain, mucopurulent bloody stool and the like, seriously influence the life quality of patients, and increase the risk of complications such as colon cancer after long-term illness. At present, nutritional supplements related to intestinal health regulation are various, but have the core defects that firstly, the stability of active ingredients is poor, particularly bioactive molecules are easy to inactivate in the processing and storage processes, secondly, the targeting of the intestinal tract is insufficient, the active ingredients are easy to be degraded by gastrointestinal fluid and difficult to be efficiently enriched at focus positions, thirdly, the intestinal tract protection effect is limited, and the intestinal tract protection effect is limited only aiming at a single pathological link, so that the intestinal tract barrier cannot be synchronously repaired, the inflammatory reaction can not be inhibited and the immune balance can not be regulated. Milk-derived exosomes are used as natural nano vesicles, are rich in bioactive molecules such as micrornas, proteins, lipids and the like, exhibit unique advantages in intestinal development, immunoregulation and intestinal barrier protection, and have become research hotspots in the fields of nutrition and medicine. The goat milk exosome can relieve inflammatory bowel disease by inhibiting excessive accumulation of active oxygen in intestinal tract, reducing lipid peroxidation level, reducing death of intestinal epithelial cells, and can up regulate intestinal tract tight junction protein expression and improve intestinal tract permeability. However, the industrialized application of the milk exosomes still faces multiple technical bottlenecks, namely, the separation and purification difficulty is high, nano micelles formed by casein in milk are overlapped with the grain size of the exosomes, the co-precipitation of the nano micelles and the exosomes is easily caused by a conventional centrifugal method, the purity of the exosomes is seriously affected, the storage stability is poor, the membrane structure of the exosomes is easily damaged and the bioactivity is lost due to ice crystal formation and osmotic pressure fluctuation in the freeze-drying process, the membrane stability and the osmotic pressure regulation requirement are difficult to be simultaneously met by the existing single protective agent, the specific process design is lacking, the existing technology is mainly aimed at the exosomes of cow milk and common sheep, the separation and preparation process is not optimized aiming at the component characteristics of the Hu sheep milk, the activity advantage of the Hu sheep exosomes is not fully exerted, the intestinal delivery efficiency is low, the exosomes are easily degraded by gastric acid and digestive enzymes after being orally taken, and the colon inflammation part is difficult to be exerted. In addition, most of the existing freeze-drying protective agents are conventional saccharides such as sucrose and trehalose, and although a certain osmotic pressure protection can be provided, the conventional freeze-drying protective agents have limited effects in maintaining the integrity of exosome lipid bilayer membranes and inhibiting particle aggregation. The combined action mechanism of the composite protective agent is not fully excavated, and the formula of the special protective agent for the exosomes of the Hu sheep milk is in a blank state. Meanwhile, the intestinal targeted delivery technology of exosomes still needs to be perfected, and a delivery system with biocompatibility and specific degradability is lacking, so that the practical application of the exosomes in intestinal disease intervention is limited. Therefore, a high-efficiency separation and purification process for the exosomes of the Hu sheep milk is developed, a jointly-stable composite protection system is constructed, and a targeting delivery technology is combined to prepare the composite nutritional supplement with stable active ingredients and remarkable intestinal protection effect, so that the composite nutritional supplement has important practical application value for filling technical blank of industry and meeting clinical and market demands for intestinal health adjustment. Disclosure of Invention The invention aims to provide a compound nutritional supplement based on a Hu sheep milk exosome, which has stable active in