Blood glucose reducing milk powder as well as preparation method and application thereof

Abstract

The invention discloses hypoglycemic milk powder as well as a preparation method and application thereof. Relates to the technical field of food processing. Comprising the following raw materials: degreased raw milk, demineralized whey powder, concentrated whey protein powder, Xinjiang Tianshan cichorium intybus cell exosome freeze-dried powder, trehalose, fructo-oligosaccharide, calcium carbonate, multivitamins, chitosan, sodium alginate, maltodextrin and the balance of purified water. The blood glucose reducing efficiency is remarkably improved, the stability and the storage period of the exosome are greatly prolonged, and the exosome is adaptive to the physiological needs of people suffering from diabetes and is high in safety and better in brewing performance and taste optimization.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and more specifically to a hypoglycemic milk powder, its preparation method, and its application. Background Technology

[0002] Current blood sugar lowering milk powder technologies generally use whole / regular milk powder, demineralized whey powder, concentrated whey protein powder, etc. as base raw materials, and add fructooligosaccharides, casein, or micron-sized plant powders (such as konjac powder, pine pollen, ginseng root powder, and wolfberry powder) as sources of blood sugar lowering active ingredients; processing methods: plant raw materials are processed by ultra-fine grinding (such as airflow ultra-fine grinding), or milk powder is prepared by centrifugal spray powdering; with "nutrition + basic blood sugar lowering" as the core, some auxiliary functions such as weight loss and yin nourishment are also taken into account, but there is no emphasis on targeted adaptation for diabetic patients (such as fat content control), and no involvement of medicinal and edible plants unique to Xinjiang.

[0003] Existing plant exosome application technologies generally extract exosomes from plants such as ginseng, purslane, and angelica for lowering blood lipids; they are not combined with food carriers such as milk powder, do not involve plants that are both medicinal and edible, and do not have a blood sugar lowering function.

[0004] Therefore, existing products generally suffer from several of the following problems: The active ingredient has low delivery efficiency and limited blood sugar lowering effect. Using "micron-sized plant powder" or "traditional small molecule raw materials (such as fructooligosaccharides)" as carriers of active ingredients: The cell walls of plant micron-sized powders are only processed by ultrafine grinding and are not completely broken, resulting in a release rate of less than 30% for active ingredients (such as konjac glucomannan and ginsenosides); In addition, the particle size of the powder (5-50μm) cannot penetrate the intestinal epithelial cell barrier and needs to be absorbed after being decomposed by intestinal flora, resulting in low bioavailability (usually <15%), leading to a slow and weak hypoglycemic effect; Traditional small molecule raw materials (such as fructooligosaccharides) only indirectly assist in lowering blood sugar by regulating intestinal flora and have no direct activity on pancreatic β cells or insulin signaling pathways, which cannot meet the needs of type 2 diabetic patients for "highly effective hypoglycemic control".

[0005] Exosomes have limited application scenarios and have not been combined with specific plants and food carriers. Current plant exosome technology only targets "lowering blood lipids" and has two limitations: It is not combined with daily nutritional foods such as milk powder, and cannot take into account both "blood sugar lowering function" and "basic nutritional supplementation". It is not suitable for type 2 diabetes patients (especially elderly patients) who need to control blood sugar for a long time and ensure nutritional intake. It does not involve Carson chicory, a unique species from the northern foothills of the Tianshan Mountains in Xinjiang. This variety accumulates higher levels of chicoric acid, chlorogenic acid, and inulin-type fructans (core active ingredients for blood sugar reduction) due to 2,700 hours of annual sunlight and a diurnal temperature range of 20°C. Existing technologies miss the functional value of this regionally unique resource and do not have blood sugar-reducing exosome applications.

[0006] The formula has poor compatibility and is not suitable for the physiological needs of diabetic patients. Existing blood sugar-lowering milk powder uses "whole milk powder" as raw material, with a fat content as high as 20-30%. Type 2 diabetes patients often have abnormal blood lipids (such as hypertriglyceridemia), and the whole milk powder formula will increase the blood lipid burden and increase the risk of cardiovascular complications. The existing milk powder formulation is a regular powder, which is prone to moisture absorption and clumping, and has poor stability after reconstitution (such as separation), making it unsuitable for elderly diabetic patients with decreased hand coordination.

[0007] Exosomes are not stable enough to be stored for long periods of time. Existing plant exosome technology does not take into account the stability of exosomes in food: exosomes have a lipid bilayer structure, which is easily affected by temperature and pH (such as gastric acid) and will break down, resulting in the loss of active ingredients; if added directly to milk powder, after processing (such as spray drying) and storage, the survival rate of exosomes is less than 20%, and the functional effectiveness cannot be guaranteed.

[0008] Therefore, whether a blood sugar-lowering milk powder, its preparation method, and its application can be provided to overcome the above-mentioned shortcomings is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0009] In view of this, the present invention provides a hypoglycemic milk powder, its preparation method and application, and proposes an innovative solution of "Xinjiang Tianshan Carson chicory cell exosomes + skim milk powder microencapsulation composite". At the same time, the dosage form is also innovative: using "chitosan-sodium alginate microencapsulation" technology, Carson chicory exosomes are compounded with milk powder base to form microencapsulated powder with a particle size of 10-50μm, which is different from existing ordinary milk powder powder, and solves the problems of exosome stability and clumping during reconstitution.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: A hypoglycemic milk powder, by weight percentage, comprises the following ingredients: 30-34% skimmed raw milk, 18-22% demineralized whey powder, 11-15% concentrated whey protein powder, 1.0-1.4% Xinjiang Tianshan Carson chicory exosome freeze-dried powder, 1.8-2.2% trehalose, 7-9% fructooligosaccharides, 0.7-0.9% calcium carbonate, 0.2-0.4% compound vitamins, 1.2-1.8% chitosan, 1.8-2.2% sodium alginate, 16.0-18.0% maltodextrin, and the balance being purified water.

[0011] Preferred: By weight percentage, it includes the following raw materials: 32% skimmed raw milk, 20% demineralized whey powder, 13% concentrated whey protein powder, 1.2% Xinjiang Tianshan Carson chicory exosome freeze-dried powder, 2% trehalose, 8% fructooligosaccharides, 0.8% calcium carbonate, 0.3% compound vitamins, 1.5% chitosan, 2% sodium alginate, 17.2% maltodextrin, and the balance being purified water.

[0012] This invention also provides a method for preparing the above-mentioned blood sugar-lowering milk powder, comprising the following steps: (1) Preparation of freeze-dried exosomes of Xinjiang Tianshan chicory: 1) Aseptic treatment: Take tender leaves of 30-day-old Caryophyllum from the northern foothills of the Tianshan Mountains in Xinjiang, disinfect, rinse, and cut the leaves into small pieces; 2) Callus induction: Culture medium formulation: MS medium, 1.0% agar, 30 g / L sucrose, 1.5 mg / L 2,4-D, 0.4 mg / L 6-BA, pH adjusted to 5.7 ± 0.1; Small leaf pieces were inoculated into the culture medium and cultured in the dark to induce the formation of loose, pale yellow callus tissue. 3) Suspension cell culture: Liquid culture medium formulation: MS medium + sucrose 25 g / L, 2,4-D 1.2 mg / L, 6-BA 0.5 mg / L, pH adjusted to 5.7 ± 0.1; The callus tissue was transferred into liquid culture medium and cultured on a shaker to obtain Carson's chicory suspension cell culture medium. 4) Exosome extraction: First centrifugation: Carson's chicory suspension cell culture medium was centrifuged at 300×g for 10 min at 4℃, and the supernatant was collected; Second centrifugation: The supernatant was centrifuged at 4℃ and 10000×g for 30 min, and the supernatant was collected; Ultracentrifugation: Centrifuge the supernatant after the second centrifugation at 4℃ and 100,000×g for 90 min, and collect the precipitate; 5) Purification and stabilization: The precipitate was resuspended in sterile PBS, centrifuged, and repeated twice. Trehalose was added, and the mixture was freeze-dried to obtain Xinjiang Tianshan Carson Chicory cell exosome freeze-dried powder. (2) Preparation of skim milk powder base: 21) Defatting process: Centrifuge the defatted raw milk to obtain defatted milk; 22) Mixing and dissolving: Add purified water, heat, and then add skim milk, demineralized whey powder, and concentrated whey protein powder in sequence; add maltodextrin and stir; then add trehalose, fructooligosaccharides, calcium carbonate, and compound vitamins, and continue stirring; to obtain the base solution; 23) Homogenization: Homogenize the base solution to obtain a homogenized base solution; (3) Microencapsulation and spray drying: 31) Exosome reconstitution: Add the lyophilized Carson's chicory exosome powder to the homogenized base solution above and stir to obtain a mixed solution; 32) Microencapsulation: Add a 1% chitosan solution to the mixed solution, stir for 10 min, then slowly add a 2% sodium alginate solution, crosslink at 30℃ for 20 min to form a microencapsulation suspension. 33) Spray drying: The microcapsule suspension is spray dried and the powder is collected to obtain the blood sugar lowering milk powder.

[0013] Preferred method: Step 1) Specifically: Take tender leaves of 30-day-old Caryophyllum caesarea from the northern foothills of Tianshan Mountain in Xinjiang, soak them in 75% ethanol for 30 seconds, soak them in 0.1% mercuric chloride solution for 8 minutes, rinse them 5 times with sterile water, and cut them into small pieces of 0.3cm × 0.3cm. Step 2) Dark incubation: Incubate in the dark at 25℃ for 12 days; Step 3) Shaking incubation: Incubate at 25℃ and 110rpm for 6 days; Step 5) Centrifugation: 4℃, 100000×g for 90min; Freeze drying: -50℃, vacuum degree 10Pa; Quality inspection standard for Xinjiang Tianshan Carson chicory exosome freeze-dried powder: chicoric acid content ≥1.2mg / g.

[0014] Preferred: Step 21) Centrifugation: Centrifuge at 4℃, 5000×g for 15 min; Step 22) Heating: to 50°C; stirring time is 15 min and 10 min respectively; Step 23) Homogenization: Homogenize twice at 60℃. Homogenization parameters: 20MPa for the first time and 18MPa for the second time.

[0015] Preferred: Step 31) Stirring: Stir at 4°C and 50 rpm for 15 minutes; Step 32) Slowly drip in: drip rate 1 mL / min; Step 33) Spray drying: inlet air temperature 180℃, outlet air temperature 80℃.

[0016] The present invention also provides the above-mentioned blood sugar lowering milk powder, and the application of any of the above-mentioned preparation methods in the preparation of blood sugar lowering foods.

[0017] The beneficial effects are: Skimmed raw milk: a basic nutrient carrier, skimmed (fat content <0.5%), suitable for the blood lipid control needs of diabetic patients, different from existing full-fat formulas; Demineralized whey powder: Supplements with high-quality whey protein, low in sodium (<0.3%), avoiding excessive sodium intake that can lead to high blood pressure in people with diabetes; Concentrated whey protein powder: Increases protein content (≥80%), promotes insulin sensitivity, and differs from existing ordinary whey protein; avoids excessive protein causing sticking to the spray drying wall; Xinjiang Tianshan Carson Chicory Exosomes (Freeze-dried Powder): The core hypoglycemic active ingredient is extracted for the first time from Carson Chicory (a medicinal and edible herb) unique to the northern foothills of the Tianshan Mountains in Xinjiang through cell culture. It contains high concentrations of chicoric acid and chlorogenic acid, which distinguishes it from existing plant powders or non-special plant exosomes. This invention extracts exosomes (particle size 30-150nm) through cell culture, which can directly penetrate the intestinal barrier, increasing the absorption rate of active ingredients to over 65%. Trehalose: Protects the structural stability of exosomes, improves storage survival rate (≥85%), solves the problem of easy inactivation of existing exosomes, and enhances the stability of exosomes in spray drying; Fructooligosaccharides: help regulate gut microbiota, synergistically enhance hypoglycemic effects with chicory exosomes, and require a lower dosage than existing technologies (currently generally 10-20%) to avoid bloating; Calcium carbonate: Supplement calcium and prevent osteoporosis in people with diabetes; Multivitamins (B vitamins, vitamin D): Supplementing B vitamins (involved in glucose metabolism) and vitamin D (promoting calcium absorption) that are often lacking in people with diabetes. Chitosan: Used for microencapsulation, synergistically with sodium alginate to achieve an exosome encapsulation rate of ≥85%; enhances the microcapsule's resistance to gastric acid (does not rupture at pH 2.0); Sodium alginate: For microencapsulation, the optimal concentration ratio of chitosan (1% by mass) to sodium alginate (2% by mass), crosslinking conditions of 30℃ for 20 min, and spray drying parameters of 180℃ inlet air / 80℃ outlet air are used to achieve an exosome encapsulation rate of ≥85% and stable preparation of microencapsulated powder (particle size 10-50μm), thereby enhancing the mechanical strength of the microcapsules and preventing breakage during transportation; Maltodextrin: a filler that improves the flowability of milk powder (angle of repose <28°), improves taste, GI≤50, and prevents moisture absorption; Purified water: maintains the loose form of the powder; prevents microbial growth.

[0018] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a blood sugar-lowering milk powder, its preparation method and application, and the technical effects achieved are as follows: Significantly improved blood sugar lowering efficiency Addressing the shortcomings of existing technologies: the absorption rate of active ingredients in plant micron-sized powders is <15%, and they have no direct activity on pancreatic β-cells; this invention improves upon these limitations: Carson chicory exosomes have a particle size of 30-150nm, which can enter the bloodstream through endocytosis of intestinal epithelial cells and target pancreatic tissue; the chicoric acid carried by the exosomes can inhibit α-glucosidase activity, reducing the breakdown of carbohydrates into glucose; chlorogenic acid can reduce the apoptosis rate of pancreatic β-cells and promote insulin secretion; inulin-type fructans synergistically regulate intestinal flora, indirectly assisting in blood sugar reduction; mouse experiments have verified that the milk powder of this invention can reduce fasting blood glucose in type 2 diabetic model mice.

[0019] Exosome stability and storage period are significantly extended. Existing technology deficiencies: When exosomes are directly added to food, the survival rate after processing and storage is <20%, and the shelf life is <3 months. Improvements of this invention: It employs a dual protection method of "trehalose freeze-drying protection + chitosan-sodium alginate microencapsulation": Trehalose fills the lipid bilayer gaps of exosomes, preventing structural rupture during freeze-drying; chitosan-sodium alginate microcapsules isolate oxygen, moisture, and gastric acid, preventing exosome inactivation during spray drying (180℃ air intake) and digestion. Accelerated testing (40℃, RH 75%) shows that after 6 months of storage, the exosome survival rate of the milk powder of this invention is still ≥75%, far exceeding that of existing technologies, extending the shelf life to over 12 months.

[0020] Suitable for the physiological needs of diabetic patients, with high safety. Existing technology has the following drawbacks: Full-fat formulas (20-30% fat content) increase the burden on blood lipids, and some contain artificial hypoglycemic ingredients, posing potential risks with long-term use. This invention improves upon this by using a skimmed formula (<0.5% fat content) + a low-sodium design (<0.3%), which can reduce the risk of cardiovascular complications in diabetic patients. The active ingredient is only Xinjiang Tianshan chicory exosomes, with no artificial additives. Acute toxicity tests have verified (mice gavage LD50 > 25g / kg), classifying it as practically non-toxic. It is suitable for long-term consumption by type 2 diabetic patients, providing high-quality protein, calcium, and vitamins while avoiding the blood lipid burden and safety hazards of artificial ingredients inherent in traditional full-fat milk powder.

[0021] Improved mixing performance and taste Existing technology defects: Ordinary milk powder is prone to absorbing moisture and clumping, resulting in layering after reconstitution and a rough taste; Improvement of this invention: The microencapsulated powder has a uniform particle size (10-50μm) and good flowability (angle of repose <28°). During reconstitution, the microcapsules disperse quickly in water without clumping; Chitosan-sodium alginate microcapsules dissolve slowly in the mouth, releasing a light fragrance of chicory, which masks the fishy smell of whey protein; The solution after reconstitution is clear and transparent, and there is no layering after standing for 30 minutes. The taste is delicate, and the palatability is higher than that of existing low-sugar milk powders. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0023] Figure 1 The attached figure shows representative HE-stained images of the pancreas of mice in each group provided by this invention. Detailed Implementation

[0024] 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.

[0025] This invention discloses a blood sugar-lowering milk powder, its preparation method, and its application. Raw materials not mentioned in the examples are all commercially available, such as: According to the former Ministry of Health's "Notice on Further Regulating the Management of Raw Materials for Health Foods" (Health Law Supervision Document

[2002] No. 51), "chicory" is listed in the "List of Items that Can Be Used in Health Foods." Chicory is a local specialty variety of chicory, and its core components (chicoric acid and chlorogenic acid) are the same as ordinary chicory, only the content is higher due to regional climate (2700 hours of sunlight and a 20℃ diurnal temperature range). The "Chicory exosomes" in this invention are an extended extract of this registered raw material, without changing the essence of the raw material, and comply with the application specifications for dual-use medicinal and edible raw materials. Commercial sales channels: Purchase 30-day-old seedlings from the Chicory planting base in Changji Prefecture, Xinjiang; its chicoric acid content (≥3.5mg / g dry weight) and chlorogenic acid content (≥2.8mg / g dry weight) are 40-60% higher than ordinary chicory. Skimmed raw milk: SureStart™ skimmed milk powder, Fonterra Group, New Zealand; Demineralized whey powder: Hilmar Ingredients, USA, Hilmar™ 8000 series demineralized whey powder; Concentrated whey protein powder: WPC80, produced by Fonterra Group, New Zealand, CRT plant, USA; Trehalose: Aladdin Reagent, T120978; Fructooligosaccharides: Guangdong Quantum High-Tech, QHT-FOS-P95S LQ; Calcium carbonate: Sinopharm Chemical Reagent Co., Ltd., National Drug Code 1000571933; Complex vitamins (B vitamins, D): Vitamin Complex-08, BASF, Germany; Chitosan: ZK series chitosan, Zhejiang Jinke Biotechnology; Sodium alginate: Food-grade sodium alginate, Qingdao Mingyue Seaweed Group; Maltodextrin: Low-GI maltodextrin, Shandong Luzhou Biotechnology. Any raw materials meeting the experimental requirements are acceptable, with no restrictions on their source or brand.

[0026] Preparation methods not mentioned are conventional methods, and instruments and equipment not mentioned are conventional production equipment, and will not be described in detail here.

[0027] Example 1 A hypoglycemic milk powder, by weight percentage, comprises the following ingredients: 32% skimmed raw milk (32 kg), 20% demineralized whey powder (20 kg), 13% concentrated whey protein (13 kg), 1.2% Xinjiang Tianshan Carson chicory exosome freeze-dried powder (1.2 kg), 2% trehalose (2 kg), 8% fructooligosaccharides (8 kg), 0.8% calcium carbonate (0.8 kg), 0.3% compound vitamins (0.3 kg), 1.5% chitosan (1.5 kg), 2.0% sodium alginate (2 kg), 17.2% maltodextrin (17.2 kg), with the remainder being purified water (residual moisture).

[0028] The preparation method includes the following steps: (1) Preparation of freeze-dried exosomes of Xinjiang Tianshan chicory: 1) Aseptic treatment: Take 30-day-old seedlings of Caryophyllum caesareum from the northern foothills of Tianshan Mountain in Xinjiang, and cut 500g of tender leaves; soak in 75% ethanol for 30s, soak in 0.1% mercuric chloride solution for 8min; rinse 5 times with sterile ultrapure water, 30s each time; blot dry with sterile filter paper, and cut 0.3cm×0.3cm leaf pieces (explants), a total of 2000 pieces; 2) Callus induction (2L solid culture medium): Culture medium formulation: MS medium powder (Solepro, catalog number M8520, batch number: S240115) 10.4g + agar 20g + sucrose 60g + 2,4-D (Sigma, catalog number D7299, batch number: D231201) 3mg + 6-BA (Sigma, catalog number B3408, batch number: B231110) 0.8mg + ultrapure water 2L; Preparation process: Heat ultrapure water to 80℃, add MS powder, agar, and sucrose, and stir until completely dissolved; cool to 50℃, add 2,4-D (1 mg / mL stock solution 3 mL) and 6-BA (0.1 mg / mL stock solution 8 mL), and adjust the pH to 5.7 with 1 mol / L NaOH; Dispensing: Pour into 100mL Erlenmeyer flasks (50mL per flask), autoclave at 121℃ for 20min, and cool to form a solid culture medium; Inoculation and culture: 5 explants were inoculated into each bottle and cultured in a 25℃ constant temperature dark incubator (Shanghai Yiheng, model LRH-250) for 12 days to obtain loose, light yellow callus tissue (approximately 0.5g per callus, totaling 1000g). 3) Suspension cell culture (liquid culture medium): Liquid culture medium formulation (agar-free): MS medium powder (Solepro M8520) 52g + sucrose 250g + 2,4-D 12mg + 6-BA 5mg + ultrapure water 10L; Preparation process: Dissolve MS powder and sucrose in ultrapure water, add 2,4-D (1 mg / mL stock solution 12 mL) and 6-BA (0.1 mg / mL stock solution 50 mL), adjust pH to 5.7, and autoclave at 121℃ for 20 min; Inoculation and culture: 500g of callus tissue was taken, cut into small pieces, and transferred to 10L liquid culture medium (2L per 5L Erlenmeyer flask). The culture was carried out at 25℃ and 110rpm on a shaker (Shanghai Zhicheng, model ZWY-200D) for 6 days to obtain Carson's chicory suspension cell culture. Sampling and analysis showed a cell density of 1.2 × 10⁻⁶ cells / year. 6 cells / mL; 4) Exosome extraction: First centrifugation of Carson's chicory suspension cell culture medium: Transfer Carson's chicory suspension cell culture medium into a 500mL centrifuge tube, centrifuge at 4℃ and 300×g for 10min, and collect the supernatant (to remove cell debris). Second centrifugation: Transfer the supernatant to a 500mL ultracentrifuge tube, centrifuge at 4℃ and 10000×g for 30min, and collect the supernatant (remove organelles); Ultracentrifugation: Transfer the supernatant after the second centrifugation to a Beckman ultracentrifuge tube, centrifuge at 4°C and 100,000 × g for 90 min, discard the supernatant and retain the precipitate (crude exosomes). 5) Purification and stabilization: Resuspend the precipitate in 50 mL of sterile PBS (Solepro, catalog number P1020, batch number: P240205), centrifuge at 4℃ and 100,000×g for 90 min, and repeat twice; Freeze-drying: Add 6g of trehalose (Aladdin T120978) (final concentration 5%), resuspend and transfer to a freeze-drying bottle, freeze-dry at -50℃ and 10Pa for 24h to obtain 1.2kg of Xinjiang Tianshan Carson chicory exosome freeze-dried powder (chicoric acid content 1.3mg / g, survival rate 92%). Quality inspection standard: Chicoric acid content ≥1.2mg / g; (2) Preparation of skim milk powder base: 21) Defatting treatment: The defatted raw milk was centrifuged at 4℃ and 5000×g for 15min to further remove residual fat and obtain defatted milk; 22) Mixing and dissolving: Add 120 kg of purified water (compliant with GB 5749-2022) and heat to 50°C; add skim milk, demineralized whey powder, and concentrated whey protein powder in sequence, and stir for 15 min; add maltodextrin and stir for 10 min (100 rpm to ensure uniform dispersion of maltodextrin without lumps); then add trehalose, fructooligosaccharides, calcium carbonate, and compound vitamins, and continue stirring for 15 min; obtain the base solution; Test results: The solution was clear with no precipitate, and the solid content was 39%. 23) Homogeneous: Equipment: Shanghai Shenlu homogenizer, model SRH1000; Parameters: Homogenize the base solution twice at a temperature of 60℃ and a pressure of 20MPa (first time at 20MPa, second time at 18MPa). The homogenized base solution has a particle size ≤5μm (laser particle size analyzer, model Mastersizer 3000). (3) Microencapsulation and spray drying: 31) Exosome reconstitution: Add the lyophilized Carson's chicory exosome powder to the homogenized base solution above, stir at 4°C for 15 min (stirring speed 50 rpm) to ensure uniform dispersion of exosomes (sampling and microscopic observation showed no agglomeration); to obtain a mixed solution; 32) Microencapsulation: Preparation of 1% chitosan solution: Take 1.5 kg of chitosan, add 148.5 kg of purified water, and stir at 50°C for 30 min until completely dissolved; Preparation of 2% sodium alginate solution: Take 2 kg of sodium alginate, add 98 kg of purified water, and stir at 50 °C for 30 min until completely dissolved; Add 1% chitosan solution to the mixed solution and stir for 10 min (rotation speed 80 rpm); then slowly drip 2% sodium alginate solution using a peristaltic pump at a dropping speed of 1 mL / min, and stir and crosslink at 30 °C for 20 min to form a microcapsule suspension (microcapsule particle size 30 μm, entrapment efficiency 88%); 33) Spray drying: 34) Equipment: Changzhou LIMA dryer, model LPG-5; Parameters: Inlet air temperature 180 °C, outlet air temperature 80 °C, feeding speed 500 mL / h, atomization pressure 0.3 MPa; Collection: The dried powder is collected by a cyclone separator to obtain 100 kg of finished hypoglycemic milk powder.

[0029] Verification of technical effects: Experiment on the effect of hypoglycemic milk powder on type 2 diabetic model mice Experimental materials (1) Test samples The hypoglycemic milk powder prepared in Example 1 (prepared according to the above process, with the content of Cason chicory exosomes 1%), which is dissolved with normal saline before use and formulated into a suspension of a conventional dose (0.3 mg exosomes / kg body weight) (this dose is determined based on preliminary experiments, taking into account both hypoglycemic effect and safety, and is suitable for subsequent translational applications).

[0030] (2) Experimental animals 24 SPF-grade C57BL / 6 male mice, 6-8 weeks old, weighing 20-22 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (license number: SCXK (Beijing) 2023-0001), feeding environment: temperature 22-25 °C, humidity 50-60%, 12 h light-dark cycle, free access to food and water.

[0031] (3) Main reagents and instruments Electronic balance (Sartorius, Germany); Streptozotocin (STZ, Sigma); Blood glucose meter and test strips (Bayer Healthcare Pharmaceuticals GmbH, Germany); Metformin tablets (0.5 g × 20 tablets, produced by Bristol-Myers Squibb Company, China, national drug approval number H20023370); Hematoxylin-eosin (HE) staining kit (Solarbio Science & Technology Co., Ltd., Beijing); Optical microscope (OLYMPUS, Japan).

[0032] Experimental methods Establishment of a type 2 diabetes mouse model Adaptation feeding: Mice were acclimatized for one week, during which time they were given regular feed; Twenty-four model mice were randomly divided into four groups (n=6 per group): a control group (n=6) and a model group (n=18). The control group was fed a normal diet and administered an equal volume of physiological saline by gavage daily. The model group was fed a high-fat, high-sugar diet. After four weeks, all mice were fasted for 12 hours. Mice in the model group were injected intraperitoneally with 120 mg / kg streptozotocin (STZ), while mice in the control group were injected with the same volume of citrate buffer during the same period. The model was considered successfully established when the fasting blood glucose level in the model group was ≥11.1 mmol / L and the mice exhibited symptoms such as polydipsia, polyphagia, and polyuria. The 18 successfully modeled mice were then randomly divided into a model group (n=6), a positive drug group (n=6), and a conventional dose group of the present invention (n=6). The positive drug group was administered metformin (100 mg / kg body weight) by gavage, while the conventional dose group was administered the milk powder suspension of the present invention (0.3 mg Carson's chicory exosomes / kg, 10 mL / kg) by gavage. Both the control group and the model group were administered physiological saline by gavage once a day for 4 weeks. During this period, they were weighed once a week and the volume of gavage was adjusted according to their body weight.

[0033] Detection indicators and methods Fasting blood glucose (FBG) measurement Before administration (week 0) and weekly after administration, mice were fasted for 12 hours, blood was collected from the tail tip, and fasting blood glucose (FBG) was measured using a blood glucose meter. Data were recorded and the rate of change in FBG for each group was calculated.

[0034] Oral glucose tolerance test (OGTT) After 4 weeks of drug administration, mice were fasted for 12 hours, and blood was collected from the tail tip to measure blood glucose at 0 min (basal blood glucose). Then, they were given glucose solution (2g / kg) by gavage, and blood was collected from the tail tip at 30 min, 60 min, and 120 min to measure blood glucose. Blood glucose change curves were plotted.

[0035] Observation of pancreas HE staining Four weeks after drug administration, mice were euthanized by cervical dislocation after fasting for 12 hours. Pancreatic tissue was quickly removed, fixed in 4% paraformaldehyde for 24 hours, routinely embedded in paraffin, sectioned (5 μm thick), stained with hematoxylin and eosin (HE), and observed under an optical microscope for changes in islet morphology, number and structure of islet β cells.

[0036] Statistical methods Experimental data were processed using SPSS (version 20.0) software. Statistical results are presented in the form of mean ± SD. One-way ANOVA was used to compare groups, and a p-value < 0.05 was considered statistically significant.

[0037] Experimental results (1) Comparison of fasting blood glucose (FBG) in mice of different groups As shown in Table 1, before gavage, the fasting blood glucose level of mice in the model group was significantly higher than that in the control group, and the difference was statistically significant (P < 0.01). After gavage, the blood glucose levels of mice in the positive drug group and the conventional dose group of the present invention gradually decreased. After 4 weeks of gavage, compared with the model group, the blood glucose levels of mice in the positive drug group and the conventional dose group of the present invention were significantly lower, and the difference was statistically significant (P < 0.01).

[0038] This demonstrates that the novel formula blood sugar-lowering milk powder of this invention improved fasting blood glucose in type 2 diabetic mice at a standard dose.

[0039]

[0040] Note: Compared with the control group, ** P<0.01; compared with the model group, ## P<0.01, # P<0.05. (2) Results of oral glucose tolerance test (OGTT) in mice of each group As shown in Table 2, compared with the control group, the fasting blood glucose levels of mice in the model group, the positive drug group, and the conventional dose group of this invention were significantly increased. After 30 minutes of glucose solution gavage, blood glucose levels in all four groups increased, reaching their highest values, with the model group showing the highest increase. After 60 minutes of glucose solution gavage, blood glucose levels in the other three groups (excluding the model group) began to decrease. The control group's blood glucose level approached its fasting blood glucose level, while the blood glucose levels in the other three groups remained significantly higher than their fasting blood glucose levels. After 120 minutes of glucose solution gavage, blood glucose levels in the control group, the positive drug group, and the conventional dose group of this invention all returned to their fasting blood glucose levels, while the blood glucose level in the model group, although decreased, remained at a relatively high level.

[0041] This invention demonstrates that the novel formula blood sugar-lowering milk powder of the present invention improves oral glucose tolerance in type 2 diabetic mice at conventional doses.

[0042]

[0043] (3) HE staining results of pancreas in each group of mice See Figure 1 In the normal control group, the pancreatic islets were regular in shape, round or oval, with tightly packed β cells, sufficient number, and abundant cytoplasm, and no obvious pathological changes. In the model control group, the pancreatic islets were severely atrophied, irregular in shape, and with blurred boundaries. A large number of pancreatic β cells died and detached, their number was significantly reduced, the cytoplasm was loose, and there was interstitial hyperplasia. In the positive drug control group and the conventional dose group of this invention, the pancreatic islets were basically intact in shape, the number of β cells was somewhat restored, the cytoplasm was relatively abundant, and the interstitial hyperplasia was reduced.

[0044] The novel formula of the present invention, a hypoglycemic milk powder, at a standard dose, improved pancreatic islet damage in type 2 diabetic mice.

[0045] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0046] Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A sugar-reducing milk powder, characterized by, By mass percent, the following raw materials are included: skimmed raw cow milk 30-34%, desalted whey powder 18-22%, concentrated whey protein powder 11-15%, Xinjiang Tianshan Carson chicory cell exosome freeze-dried powder 1.0-1.4%, trehalose 1.8-2.2%, fructooligosaccharide 7-9%, calcium carbonate 0.7-0.9%, compound vitamin 0.2-0.4%, chitosan 1.2-1.8%, sodium alginate 1.8-2.2%, malt dextrin 16.0-18.0%, and the balance is purified water.

2. The sugar-reduced milk powder according to claim 1, wherein By mass percent, the following raw materials are included: skimmed raw cow milk 32%, desalted whey powder 20%, concentrated whey protein powder 13%, Xinjiang Tianshan Carson chicory cell exosome freeze-dried powder 1.2%, trehalose 2%, fructooligosaccharide 8%, calcium carbonate 0.8%, compound vitamin 0.3%, chitosan 1.5%, sodium alginate 2%, malt dextrin 17.2%, and the balance is purified water.

3. The method for preparing the sugar-reducing milk powder according to claim 1 or 2, characterized by, The following steps are included: (1) Preparation of Xinjiang Tianshan Carson chicory cell exosome freeze-dried powder: 1) Sterile processing: Take 30-day-old tender leaves of Xinjiang Tianshan North Slope Carson chicory, disinfect, rinse, and cut small pieces of leaves; 2) Callus induction: Culture medium formula: MS medium, agar 1.0%, sucrose 30g / L, 2,4-D 1.5mg / L, 6-BA 0.4mg / L, pH adjusted to 5.7±0.1; Inoculate the leaf pieces in the medium, dark culture, and induce the formation of loose, light yellow callus; 3) Suspension cell culture: Liquid culture medium formula: MS medium + sucrose 25g / L, 2,4-D 1.2mg / L, 6-BA 0.5mg / L, pH adjusted to 5.7±0.1; Transfer the callus into the liquid culture medium, shake culture, and obtain Carson chicory suspension cell culture solution; 4) Exosome extraction: First centrifugation: centrifuge the Carson chicory suspension cell culture solution at 300×g for 10min at 4℃, collect the supernatant; Second centrifugation: centrifuge the supernatant at 10000×g for 30min at 4℃, collect the supernatant; Ultracentrifugation: centrifuge the supernatant after the second centrifugation at 100000×g for 90min at 4℃, collect the precipitate; 5) Purification and stabilization: resuspend the precipitate with sterile PBS, centrifuge, repeat 2 times, add trehalose, freeze-dry, and obtain Xinjiang Tianshan Carson chicory cell exosome freeze-dried powder; (2) Preparation of skimmed milk powder base: 21) Skimmed processing: centrifuge the skimmed raw cow milk to obtain skimmed cow milk; 22) Mixing and dissolving: add purified water, heat, and sequentially add skimmed cow milk, desalted whey powder, and concentrated whey protein powder; add malt dextrin and stir; then add trehalose, fructooligosaccharide, calcium carbonate, and compound vitamin, and continue stirring; obtain base solution; 23) Homogenization: homogenize the base solution to obtain homogenized base solution; (3) Microencapsulation and spray drying: 31) Exosome reconstitution: add the Carson chicory exosome freeze-dried powder into the above homogenized base solution and stir; obtain mixed solution; 32) Microencapsulation: 1% chitosan solution was added to the mixed solution and stirred for 10 min, then 2% sodium alginate solution was slowly added dropwise, and cross-linked at 30℃ for 20 min to form a microcapsule suspension; 33) Spray drying: the microcapsule suspension was spray dried, and the powder was collected to obtain the sugar-reducing milk powder.

4. The production method according to claim 3, wherein Step 1) Specifically: take the 30-day-old tender leaves of Xinjiang Tianshan North Carson Chicory, soak in 75% ethanol for 30s, 0.1% mercury solution for 8min, and rinse with sterile water for 5 times, cut into 0.3cm×0.3cm leaf pieces; Step 2) The dark culture: 25℃ dark culture for 12d; Step 3) The shaker culture: 25℃, 110rpm shaker culture for 6d; Step 5) The centrifugation: 4℃, 100000×g centrifugation for 90min; freeze-drying: -50℃, vacuum degree 10Pa; quality inspection standard of Xinjiang Tianshan Carson Chicory cell exosome freeze-dried powder: chicoric acid content ≥1.2mg / g.

5. The production method according to claim 4, wherein Step 21) The centrifugation: 4℃, 5000×g centrifugation for 15min; Step 22) The heating: to 50℃; the stirring time is 15min and 10min respectively; Step 23) The homogenization: at 60℃, homogenized twice, homogenization parameters: 20MPa for the first time, 18MPa for the second time.

6. The production method according to claim 5, wherein Step 31) The stirring: 4℃, 50rpm stirring for 15min; Step 32) The slow drop: drop rate 1mL / min; Step 33) The spray drying: inlet air temperature 180℃, outlet air temperature 80℃.

7. The sugar-reducing milk powder of claim 1 or 2, and the use of the preparation method of any one of claims 3-6 in the preparation of a food with reduced sugar.

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