Mogroside delivery system and preparation method thereof

Abstract

The invention relates to a mogroside delivery system and a preparation method thereof, the mindset that multiple wall materials are mixed / compounded and then directly embedded or embedded layer by layer is broken, and the delivery system with excellent delivery performance is prepared in a mixed system of whey protein isolate, hyaluronic acid and mogroside in cooperation with a urea-induced self-assembly technology. The problems that in an existing mogroside delivery technology, the bioavailability is low, stability in a gastric acid environment is poor, and intestinal tract targeting is insufficient are solved, and efficient embedding, precise delivery, high bioavailability and physiological effects are achieved. The delivery system can be used for preparing food beneficial to controlling in-vivo fat, maintaining the healthy level of blood sugar and maintaining the healthy level of blood fat, and can also be used for preparing drugs for resisting obesity, reducing blood sugar and resisting inflammation. The preparation process is simple, the production cost is low, and the product can be widely applied to the fields of functional foods, special dietary foods and medicines, and has remarkable market competitiveness and industrial development potential.

Description

Technical Field

[0001] This application belongs to the field of functional food processing technology, specifically relating to a mogroside delivery system and its preparation method, as well as the application of the delivery system in the preparation of foods that help control body fat, maintain healthy blood sugar levels, maintain healthy blood lipid levels, and help regulate intestinal flora. The delivery system can also be used to prepare drugs with anti-obesity, hypoglycemic, and anti-inflammatory effects. Background Technology

[0002] Mogrosides are natural triterpenoid saponins extracted from monk fruit. They are not only high in sweetness and low in calories, but also possess various physiological functions such as anti-oxidation, anti-inflammation, and regulation of blood sugar and lipids, showing great promise in the development of functional foods. However, their practical application faces serious bottlenecks: on the one hand, mogrosides are easily hydrolyzed and inactivated in the acidic environment of the stomach, and due to their strong molecular polarity and poor membrane permeability, their oral bioavailability is extremely low. Studies have shown that the absolute bioavailability of mogroside V is only 8.73% ± 1.46%. On the other hand, their bitter aftertaste and metallic aftertaste also affect the palatability of the product. Encapsulation technology is commonly used to protect the active ingredients while allowing them to exert their effects; however, due to their structure and properties, no excellent delivery system for mogrosides has yet been found. Therefore, constructing a delivery system that can protect the active ingredients, achieve targeted release into the intestine, and improve flavor characteristics has become a key technological challenge for promoting the high-value utilization of mogrosides.

[0003] Among various delivery carriers, food-based protein nanoparticles are highly favored due to their high biocompatibility, biodegradability, and safety. Whey protein isolate (WPI) is a promising wall material, possessing excellent amphiphilicity, emulsifying properties, and film-forming ability. It can encapsulate bioactive substances through hydrophobic interactions, hydrogen bonds, and electrostatic interactions. Theoretically, WPI can encapsulate mogrosides within its hydrophobic core, providing some physical protection. However, previous preliminary experiments have shown that single WPI nanoparticle systems have significant limitations: they are prone to aggregation, precipitation, or degradation when encountering extreme pH levels (especially near their isoelectric point pI=4.5-5.2), high ionic strength, or gastrointestinal enzymes, leading to premature leakage of the encapsulated functional factors. Furthermore, pure WPI particles lack intelligent responsiveness to the digestive environment and cannot effectively resist pepsin hydrolysis, causing premature release of the encapsulated material in the stomach, failing to achieve intestinal-targeted delivery, and hindering the utilization and highlighting of WPI's advantages. To overcome the shortcomings of single protein carriers, researchers often introduce polysaccharides for modification or compounding.

[0004] Hyaluronic acid (HA) is a linear macromolecular anionic mucopolysaccharide with unique advantages: it is an endogenous substance in the human body, making it extremely safe; its molecular structure is stable and not easily degraded in gastric and intestinal juices, effectively resisting the attack of pepsin and the effects of low pH environments, making it an ideal "protective shell" material; simultaneously, its good bioadhesion may prolong its retention time in the intestines. Traditional techniques for constructing protein-polysaccharide complexes, such as Maillard reactions, chemical cross-linking, and electrostatic compounding, each have their drawbacks. Maillard reactions require prolonged heating, which may destroy heat-sensitive active ingredients; chemical cross-linking agents pose a risk of toxic residues; and electrostatic compounding is highly dependent on the pH environment of the system. Conventional protein-polysaccharide complex encapsulation or layer-by-layer encapsulation of mogrosides are unsatisfactory in terms of encapsulation, sustained release, and functional performance. There are also few reports on the use of protein-protein / proteopolysaccharide complexes for encapsulating mogrosides in existing technologies.

[0005] In the current context, there is an urgent need for a novel delivery system that can comprehensively address the problem of low bioavailability of mogrosides. Finding a method for preparing proteins, polysaccharides, and ideal delivery systems that are highly compatible with mogrosides is a challenge faced by technical workers in this field. Summary of the Invention

[0006] Purpose of the invention:

[0007] The purpose of this invention is to develop a nanodelivery system that can effectively protect mogrosides, achieve targeted release into the intestine, and significantly enhance their health benefits.

[0008] Another objective of this invention is to provide a method for preparing the above-mentioned mogroside delivery system, wherein the method is carried out entirely in an aqueous phase, the process is mild and does not use any chemical cross-linking, nor does it require the assistance of other materials or reagents.

[0009] The present invention also aims to use the delivery system to prepare foods that help control body fat, maintain healthy blood sugar levels, maintain healthy blood lipid levels, and help regulate gut microbiota; or to use the delivery system to prepare drugs with anti-obesity, hypoglycemic, and anti-inflammatory properties.

[0010] Technical issues:

[0011] To address the problems of low bioavailability, poor stability in gastric acid environment, and insufficient intestinal targeting in existing mogroside delivery technologies, and to improve the encapsulation efficiency and physiological activity of mogrosides, we seek preparation methods for proteins, polysaccharides, and corresponding delivery systems that are highly compatible with mogroside encapsulation.

[0012] Technical solution:

[0013] A method for preparing a mogroside delivery system, comprising whey protein isolate, hyaluronic acid, and mogroside through self-assembly, wherein the delivery system is a nanoparticle with a defined core-shell structure, the core being whey protein isolate encapsulating mogroside, and the shell being hyaluronic acid; the nanoparticle has a particle size of 400-500 nm and a zeta potential of -10 mV to -15 mV.

[0014] The preparation method of the mogroside delivery system includes the following steps:

[0015] (1) Dissociation of whey protein isolate: Dissolve whey protein isolate in deionized water to prepare a protein solution of 8-12 mg / mL, add urea to a final concentration of 18-22 mM, adjust the pH to 6.8-7.2, and incubate at 2-8℃ for 10-14 hours to obtain the dissociated protein solution;

[0016] (2) Hyaluronic acid modification and mogroside loading: Add hyaluronic acid to the dissociated protein solution obtained in step (1) to a final concentration of 0.08-0.12 mg / mL, mix well, add mogroside to a final concentration of 3-5 mg / mL, and continue to incubate at 2-8℃ for 10-14 hours to obtain a mixed solution;

[0017] (3) Dialysis recombination: The mixed solution obtained in step (2) is placed in a dialysis bag with a molecular weight cutoff of 500-1000 Da and dialyzed for 40-56 hours to remove urea;

[0018] (4) Purification: After removing urea, the sample was transferred to a dialysis bag with a molecular weight cutoff of 8000-14000 Da and dialyzed for 20-28 hours to remove free mogrosides and obtain a nanoparticle dispersion.

[0019] (5) Freeze-drying: The nanoparticle dispersion is freeze-dried to obtain nanoparticle powder.

[0020] Preferably, the urea concentration in step (1) is 20 mM and the protein concentration is 10 mg / mL.

[0021] Preferably, the concentration of hyaluronic acid in step (2) is 0.1 mg / mL and the concentration of mogroside is 4 mg / mL.

[0022] Preferably, the dialysis process in steps (3) and (4) is carried out at 4°C, and the dialysis solution is replaced every 8 hours.

[0023] Preferably, in step (5), the nanoparticle dispersion is pre-filtered through a 0.45μm microporous membrane before freeze-drying.

[0024] Preferably, the delivery system is used to prepare foods that help control body fat, maintain healthy blood sugar levels, maintain healthy blood lipid levels, and help regulate gut microbiota; and to prepare pharmaceuticals with anti-obesity, hypoglycemic, and anti-inflammatory effects.

[0025] Preferably, the delivery system is suitable for sugar substitutes for diabetic patients, low-calorie functional foods for weight management, health foods for regulating blood lipid metabolism, and prebiotic foods for improving gut microbiota.

[0026] Preferably, the food is a solid beverage, nutrition bar, or liquid nutritional preparation.

[0027] Preferably, the content of mogroside V in the mogroside is not less than 80%, the purity of the whey protein isolate is not less than 90%, and the molecular weight of the hyaluronic acid is 100,000-150,000 Daltons.

[0028] Compared with existing technologies, the beneficial effects are:

[0029] (1) Breaking away from the conventional thinking of directly embedding or layer-by-layer embedding multiple wall materials after mixing / composite, a mogroside delivery system with excellent delivery performance was prepared by combining whey protein isolate, hyaluronic acid and mogroside in a mixed system, with the assistance of urea-induced self-assembly technology. The selected whey protein isolate has excellent amphiphilicity and embedding ability, forming a nanoparticle core and loading mogroside; hyaluronic acid, with its anti-digestion and mucosal adhesion, is mainly responsible for constructing the nanoparticle shell and achieving intestinal targeted delivery; mogroside, as the core functional factor, is mainly responsible for playing a role in regulating the bioactivity of glucose and lipid metabolism. The synergistic effect of the three realizes the optimization of the whole process from effective embedding to precise delivery to functional performance. The whey protein isolate, composed of multiple protein components such as β-lactoglobulin, α-lactalbumin, and bovine serum albumin, dissociates under urea induction, and the various protein structures unfold, with a large number of hydrophobic groups exposed on the molecular surface and relatively uniform overall distribution. The binding mechanism with mogroside is diversified and the compatibility is significantly improved, the interaction force is enhanced and the stability is increased. During protein recombination, the addition of hyaluronic acid and precise control of its concentration allow it to be tightly adsorbed onto the surface of the WPI-MG complex through electrostatic interactions, gradually forming a "HA-encapsulated" structure. This structure also forms a network support, inhibiting complex aggregation and making the complex more uniformly dispersed in aqueous solution. Ultimately, it spontaneously forms well-structured, dense, uniform, and stable "core-shell" nanoparticles.

[0030] (2) The innovative application of urea-induced self-assembly technology to the core-shell structure construction of WPI and HA. Compared with traditional chemical cross-linking methods and complex processes, urea-induced self-assembly technology provides a green, mild, and controllable method. This method avoids high temperatures and chemical cross-linking agents / organic solvents, operates under mild conditions, maximizes the efficacy of active ingredients, and is carried out entirely in the aqueous phase, significantly reducing energy consumption and generating no harmful waste. Due to the simplified process steps, reduced equipment investment, and significantly lower production costs, it provides technical support for large-scale production.

[0031] (3) This study overcomes the technical bottlenecks of low encapsulation efficiency, poor gastric stability, low bioavailability, and poor sustained-release effect of mogrosides, achieving efficient encapsulation, precise delivery, high bioavailability, and physiological efficacy. The encapsulation rate of mogrosides by nanoparticles increased from less than 45% in physical mixing to over 75%, an increase of more than 70%. The release rate in simulated gastric juice after 2 hours was less than 10%, only 3.95%, while continuous release was achieved in intestinal juice, with a cumulative release rate of 65%-70% (69.99%). This effectively solves the key problems of gastric acid hydrolysis inactivation and poor intestinal absorption of mogrosides, realizing the value enhancement of mogrosides from "ordinary raw material" to "highly effective functional factor". Animal experiments show that H0.1-WU-M nanoparticles can significantly enhance the in vivo efficacy of mogrosides. The nanoparticle treatment group of this application showed significant improvement in weight control, blood glucose regulation, and blood lipid regulation. Through the construction of the core-shell structure, the stability of the nanoparticles was significantly improved, and the storage period was extended by about 50%. Meanwhile, the solubility of mogrosides after encapsulation is increased by approximately 120%, and the bioavailability is increased from less than 10% to over 85%. It can be widely used in functional foods, special dietary foods, and pharmaceuticals, demonstrating significant market competitiveness and industrial development potential. Attached Figure Description

[0032] Figure 1 TEM images of H-WU nanowall materials and H-WU-M nanoparticles

[0033] Figure 2 DSC curves of H-WU nanowall material (A) and H-WU-M nanoparticles (B)

[0034] Figure 3 The effects of simulated gastrointestinal digestion on nanoparticle size (A) and zeta potential (B)

[0035] Figure 4 Release curve of mogrosides under simulated gastrointestinal digestion conditions

[0036] Figure 5 The Influence of Simulated Gastrointestinal Digestion on the Microstructure of Nanoparticles

[0037] Figure 6 Effects of mogrosides and their nanoparticles on mouse body weight

[0038] Figure 7 Effects of mogrosides and their nanoparticles on body weight and Lee's index

[0039] Figure 8 Effects of mogrosides and their nanoparticles on fasting blood glucose in mice

[0040] Figure 9 Effects of mogrosides and their nanoparticles on oral glucose tolerance test (OGTTB) in mice

[0041] Figure 10 Graph showing the changing trends of mogroside encapsulation rate and drug loading of different wall materials Figure 1 (AE: H-WU nanowall material modified with HA concentrations of 0.1, 0.5, 1, 1.5, and 2 mg / mL; FJ: H-WU-M nanoparticles modified with HA concentrations of 0.1, 0.5, 1, 1.5, and 2 mg / mL) Detailed Implementation

[0042] Main raw materials and reagents:

[0043] Mogrosides (Mogroside V ≥ 80%) Hunan Huacheng Bioresources Co., Ltd.

[0044] Whey protein isolate (94% purity) - Fonterra, New Zealand

[0045] Hyaluronic acid (food grade, molecular weight 110,000) Shandong Focus Freda Biotechnology Co., Ltd.

[0046] Urea Tianjin Tianli Chemical Reagent Co., Ltd.

[0047] Example 1:

[0048] Preparation of the delivery system (H0.1-WU-M or HW-MOG):

[0049] H0.1-WU-M: H (hyaluronic acid) 0.1 (concentration) -W (whey protein isolate) U (urea) -M (monk fruit glycoside)

[0050] (1) Protein solution preparation and urea-induced dissociation: Accurately weigh 1.0 g of whey protein isolate (WPI) and dissolve it in 100 mL of deionized water to prepare a 10 mg / mL protein solution. Add 1 M urea stock solution to make the final concentration 20 mM. Adjust the pH to 7.0 with 0.1 M NaOH solution. Place the mixture in a 4℃ refrigerator and incubate for 12 hours to complete protein dissociation.

[0051] (2) Polysaccharide modification and active ingredient loading: Add 1.0 mL of 10 mg / mL hyaluronic acid (HA) solution to the dissociated protein solution obtained in step (1) to make the final concentration 0.1 mg / mL, vortex mix, then add 400 mg of mogroside (mogroside V content ≥80%) to make the final concentration 4 mg / mL, and continue to incubate at 4℃ for 12 hours;

[0052] (3) Dialysis recombination: The mixture obtained in step (2) is transferred to a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed with deionized water at 4°C for 48 hours, during which the dialysate is replaced every 8 hours.

[0053] (4) Purification: The dialysate was then transferred to a dialysis bag with a molecular weight cutoff of 8000 Da and dialyzed for 24 hours to remove unencapsulated free mogrosides and obtain a nanoparticle dispersion.

[0054] (5) Post-processing and product acquisition: The nanoparticle dispersion was filtered through a 0.45 μm microporous membrane and then freeze-dried to obtain a light yellow nanoparticle powder. Characterization showed that the average particle size of the nanoparticles was 472.3 nm, the PDI was 0.21, the Zeta potential was -13.37 mV, and the encapsulation rate of mogrosides reached 76.55%.

[0055] By adjusting the concentration of hyaluronic acid, a series of self-assembled nanoparticles such as H0.5-WU-M, H1-WU-M, H1.5-WU-M, and H2-WU-M can also be obtained.

[0056] Example 2: Preparation of HW (HA-WPI or H0.1-WU) wall material: (prepared according to Example 1, except that no mogrosides are added).

[0057] (1) Protein solution preparation and urea-induced dissociation: Accurately weigh 1.0 g of whey protein isolate (WPI) and dissolve it in 100 mL of deionized water to prepare a 10 mg / mL protein solution. Add 1 M urea stock solution to make the final concentration 20 mM. Adjust the pH to 7.0 with 0.1 M NaOH solution. Place the mixture in a 4℃ refrigerator and incubate for 12 h to complete protein dissociation.

[0058] (2) Polysaccharide modification: Add 1.0 mL of 10 mg / mL hyaluronic acid (HA) solution to the dissociated protein solution obtained in step (1) to make the final concentration 0.1 mg / mL, vortex to mix, and continue to incubate at 4°C for 12 hours;

[0059] (3) Dialysis recombination: The mixture obtained in step (2) is transferred to a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed with deionized water at 4°C for 48 hours, with the dialysate being replaced every 8 hours to obtain a dispersion.

[0060] (4) Post-processing and product acquisition: The dispersion was filtered through a 0.45 μm microporous membrane and then freeze-dried to obtain hyaluronic acid-modified whey protein isolate self-assembled nanowall materials. H0.5-WU, H1-WU, H1.5-WU, and H2-WU self-assembled nanowall materials were obtained by adjusting the concentration of hyaluronic acid.

[0061] Example 3: Preparation of WU20-M intervention group (W-MOG) nanoparticles: prepared according to Example 1, except that hyaluronic acid was not added.

[0062] (1) Protein solution preparation and urea-induced dissociation: Accurately weigh 1.0 g of whey protein isolate (WPI) and dissolve it in 100 mL of deionized water to prepare a 10 mg / mL protein solution. Add 1 M urea stock solution to make the final concentration 20 mM. Adjust the pH to 7.0 with 0.1 M NaOH solution. Place the mixture in a 4℃ refrigerator and incubate for 12 h to complete protein dissociation.

[0063] (2) Loading of active ingredients: Add about 400 mg of mogroside (mogroside V content ≥80%) to the dissociated protein solution obtained in step (1) to make a final concentration of 4 mg / mL, and continue to incubate at 4℃ for 12 hours;

[0064] (3) Dialysis recombination: The mixture obtained in step (2) is transferred to a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed with deionized water at 4°C for 48 hours, during which the dialysate is replaced every 8 hours.

[0065] (4) Purification: The dialysate was then transferred to a dialysis bag with a molecular weight cutoff of 8000 Da and dialyzed for 24 hours to remove unencapsulated free mogrosides and obtain a nanoparticle dispersion.

[0066] (5) Post-processing and product acquisition: The nanoparticle dispersion was filtered through a 0.45μm microporous membrane and then freeze-dried to obtain WU20-M intervention group (W-MOG) nanoparticle powder.

[0067] Example 4:

[0068] Preparation of the delivery system (H0.1-WU-M or HW-MOG):

[0069] H0.1-WU-M: H (hyaluronic acid) 0.1 (concentration) -W (whey protein isolate) U (urea) -M (monk fruit glycoside)

[0070] (1) Protein solution preparation and urea-induced dissociation: Accurately weigh 1.0 g of whey protein isolate (WPI) and dissolve it in 100 mL of deionized water to prepare a 10 mg / mL protein solution. Add 1 M urea stock solution to make the final concentration 20 mM. Adjust the pH to 7.0 with 0.1 M NaOH solution. Place the mixture in a 4℃ refrigerator and incubate for 12 hours to complete protein dissociation.

[0071] (2) Polysaccharide modification and active ingredient loading: Add 1.0 mL of 10 mg / mL hyaluronic acid (HA) solution to the dissociated protein solution obtained in step (1) to make the final concentration 0.1 mg / mL, vortex mix, then add 400 mg of mogroside (mogroside V content ≥80%) to make the final concentration 4 mg / mL, and continue to incubate at 4℃ for 12 hours;

[0072] (3) Dialysis recombination: The mixture obtained in step (2) is transferred to a dialysis bag with a molecular weight cutoff of 900 Da and dialyzed with deionized water at 4°C for 48 hours, during which the dialysate is replaced every 8 hours.

[0073] (4) Purification: The dialysate was then transferred to a dialysis bag with a molecular weight cutoff of 10,000 Da and dialyzed for 24 hours to remove unencapsulated free mogrosides and obtain a nanoparticle dispersion.

[0074] (5) Post-processing and product acquisition: The nanoparticle dispersion was filtered through a 0.45 μm microporous membrane and then freeze-dried to obtain a light yellow nanoparticle powder.

[0075] Example 5:

[0076] Preparation of the delivery system (H0.1-WU-M or HW-MOG):

[0077] H0.1-WU-M: H (hyaluronic acid) 0.1 (concentration) -W (whey protein isolate) U (urea) -M (monk fruit glycoside)

[0078] (1) Protein solution preparation and urea-induced dissociation: Accurately weigh 1.0 g of whey protein isolate (WPI) and dissolve it in 100 mL of deionized water to prepare a 10 mg / mL protein solution. Add 1 M urea stock solution to make the final concentration 20 mM. Adjust the pH to 7.0 with 0.1 M NaOH solution. Place the mixture in a 4℃ refrigerator and incubate for 12 hours to complete protein dissociation.

[0079] (2) Polysaccharide modification and active ingredient loading: Add 1.0 mL of 10 mg / mL hyaluronic acid (HA) solution to the dissociated protein solution obtained in step (1) to make the final concentration 0.1 mg / mL, vortex mix, then add 400 mg of mogroside (mogroside V content ≥80%) to make the final concentration 4 mg / mL, and continue to incubate at 4℃ for 12 hours;

[0080] (3) Dialysis recombination: The mixture obtained in step (2) is transferred to a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed with deionized water at 4°C for 48 hours, during which the dialysate is replaced every 8 hours.

[0081] (4) Purification: The dialysate was then transferred to a dialysis bag with a molecular weight cutoff of 12000 Da and dialyzed for 24 hours to remove unencapsulated free mogrosides and obtain a nanoparticle dispersion.

[0082] (5) Post-processing and product acquisition: The nanoparticle dispersion was filtered through a 0.45 μm microporous membrane and then freeze-dried to obtain a light yellow nanoparticle powder.

[0083] Experimental Example 1: Core-shell microstructure analysis:

[0084] The microstructural changes of mogroside before and after encapsulation were observed by TEM at different HA concentrations. The results are shown in [Figure 1]. Figure 1 The HA-modified particle images show spherical particles with a dark core encased in a light gray outer layer. The dark portion represents protein, and the light gray outer layer corresponds to HA, indicating that H-WU and H-WU-M have a core-shell structure. The particles gradually increase in size with increasing HA concentration. No mogrosides (the gray color of WU-M) were observed in the H-WU-M particles. Compared to H-WU, the particles are larger, indicating that mogrosides and H-WU form a tightly packed spherical structure.

[0085] Experimental Example 2: Thermal Stability Analysis

[0086] The effect of different HA concentrations on the thermal stability of mogrosides before and after encapsulation on nanoparticles was detected using DSC (preferred method: 4 mg of powder sample was weighed and placed in an aluminum crucible and sealed. An empty crucible was used as a control. The temperature was increased from 30 °C to 200 °C at a rate of 10 °C / min, and nitrogen was purged at a rate of 50 mL / min to obtain DSC spectra). Results are shown below. Figure 2 The thermal denaturation temperature of the HA-modified nanomaterials increased with increasing HA concentration, rising from 140℃ for H0.1-WU to 181.67℃ for H2-WU. After encapsulation with mogrosides, the thermal denaturation temperature of the nanoparticles was significantly higher than that of the nanomaterials with the same HA concentration (see...). Figure 2(A and B in the figure) indicates that the addition of mogroside can improve the thermal stability of nanoparticles.

[0087] Experiment Example 3: In vitro simulated gastrointestinal digestion evaluation of the delivery system:

[0088] (1) Gastric digestion stage: Take 25 mL of nanoparticle dispersion, add 25 mL of 0.1 M HCl solution and mix well. Adjust the pH to 2.0 and add 10 mg of pepsin. Digest for 60 minutes at 37℃ and 100 rpm shaking.

[0089] (2) Intestinal digestion stage: After gastric digestion is completed, adjust the pH to 7.0, add 20 mg of trypsin, and continue digestion for 120 minutes under the same conditions;

[0090] (3) Sample collection and analysis: Samples were taken at 0, 30, 60, 90, 120 and 180 minutes to determine the release rate of mogrosides; the average particle size and zeta potential of WU20-M and H0.1-WU-M nanoparticles in each stage of simulated in vitro gastrointestinal digestion were determined to explore the gastrointestinal stability of nanoparticles during digestion; TEM was used to study the changes in the microstructure of nanoparticles after simulated gastrointestinal digestion.

[0091] See release characteristics Figure 3 In the gastric environment, the release rate of nanoparticles was only 3.95% within 2 hours, effectively avoiding hydrolysis by gastric acid. Upon entering the intestinal environment, continuous release was achieved under the action of trypsin, with a cumulative release rate of 69.99% after 120 minutes. During the SGF (simulated gastric juice) digestion stage, mogrosides in WU20-M nanoparticles were released rapidly, while those in H0.1-WU-M nanoparticles were released slowly, with cumulative release amounts of 10.06% and 3.95%, respectively. During the SIF (simulated intestinal juice) digestion stage, both types of nanoparticles exhibited continuous release. At 120 min, the release rate of mogrosides from H0.1-WU-M was higher than that from WU20-M. At the end of SIF digestion, the mogrosides released from WU20-M and H0.1-WU-M reached 65.36% and 69.99%, respectively. Data show that H0.1-WU-M nanoparticles can achieve controlled release in a simulated gastrointestinal tract, and H0.1-WU-M exhibits certain resistance to digestion during SGF digestion. The nanoparticles prepared in this invention demonstrate excellent delivery characteristics in a simulated gastrointestinal environment.

[0092] Particle size test results are shown below Figure 4In the diagram, the particle sizes of WU20-M and H0.1-WU-M before digestion were approximately 360.8 nm and 472.9 nm, respectively. After simulated gastric juice digestion, the particle size of WU20-M nanoparticles significantly increased to approximately 580.3 nm (p<0.05), while the particle size of H0.1-WU-M did not increase significantly. This may be because the low pH environment of gastric juice leads to the loss of surface charge of protein particles, reducing electrostatic repulsion and promoting flocculation between particles. Simultaneously, pepsin hydrolyzes proteins, causing them to re-aggregate, resulting in particle enlargement. HA, being an anionic polysaccharide, coats the particle surface, preventing protein aggregation in the low pH environment and also inhibiting pepsin hydrolysis. After entering the intestinal digestion stage, the average particle sizes of WU20-M and H0.1-WU-M nanoparticles significantly increased to approximately 628.9 nm and 762.7 nm, respectively (p < 0.05). This is likely due to the strong hydrolytic ability of trypsin, which rapidly breaks down protein particles into smaller peptides. This may also lead to protein re-aggregation, resulting in larger particles. This indicates that H0.1-WU-M nanoparticles are more stable in gastric digestive fluids.

[0093] The zeta potential detection results are shown in […]. Figure 4 In the B group, the Zeta potentials of WU20-M and H0.1-WU-M before digestion were approximately -9.76 mV and -14.32 MV, respectively. After simulated gastric digestion, the Zeta potentials of the nanoparticles significantly decreased to approximately -3.93 mV and -8.06 mV (p<0.05), which is attributed to the loss of surface charge due to the low pH environment. Upon entering the intestinal digestion stage, the Zeta potential of the nanoparticles significantly increased and was higher than the pre-digestion Zeta potential (p<0.05). This may be because trypsin breaks down the protein into small peptides, and the intestinal fluid pH is 7.0, higher than the isoelectric point of the protein, enhancing the electrostatic repulsion between particles and maintaining particle stability.

[0094] Results of microstructure changes are shown in Figure 5 Before digestion, mogrosides were observed to be small particles; after digestion, the particles enlarged, possibly due to the binding of mogrosides to proteases. The microstructure of WU20-M before and after digestion showed that the protein particle structure gradually became looser and the particles enlarged. This is because the protein was hydrolyzed into small peptides and then re-aggregated to form larger particles. The microstructure of H0.1-WU-M before and after digestion showed no significant change in particle structure after gastric digestion, but the particles enlarged and became looser after intestinal digestion. This further demonstrates that H0.1-WU-M has anti-digestive properties during gastric digestion, allowing for better delivery of mogrosides to the intestines.

[0095] Hyaluronic acid modification significantly enhances the anti-digestion properties of nanoparticles. The intact core-shell structure effectively resists pepsin hydrolysis, protecting mogrosides and ensuring their stability in the gastric environment while enabling controlled release in the intestinal environment. In contrast, samples without hyaluronic acid modification or urea treatment exhibit significant leakage in the stomach, failing to achieve targeted delivery.

[0096] Comparative test

[0097] Animal model establishment and gavage: Seventy 6-week-old SPF-grade male C57BL / 6 mice weighing 19-20g were selected (purchased from Liaoning Changsheng Biotechnology Co., Ltd.). Feed preparation: basal diet (10% fat energy supply) XT93M; high-fat diet (60% fat energy supply) XTHF60, both purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd.

[0098] Animals were housed at 22±1℃ and 55±10% humidity in a 12-hour light / dark cycle. All animals had free access to purified water and food for 12 weeks. After one week of acclimatization, animals were randomly divided into 7 experimental groups (grouping shown in Table 1), with 10 animals in each group. From week 2 to week 12, except for the control group, all animals had free access to a high-fat diet, with a gavage dose of 400 mg / kg / day.

[0099] In vivo bioefficacy evaluation: Body weight, Lee's index, fat, blood lipids, fasting blood glucose, and oral glucose tolerance test (OGTT) were measured in each group of mice.

[0100] Indicator Testing:

[0101] The weight test results show (see) Figure 6 At the end of week 12, the body weight of the mice treated with the intervention was significantly reduced (p<0.05). The body weight of mice in the WPI and HW groups decreased by 5.92% and 3.36%, respectively, while that in the MOG group decreased by 13.90%, the W-MOG group by 18.66%, and the HW-MOG group by 28.22%.

[0102] Lee's Index of Body Mass (see Figure 7 Lee's Index is one of the key indicators for assessing the degree of obesity in the body.

[0103] Table 1 Grouping of experimental mice

[0104] feed Gavage Control group (CON) Basic feed purified water High-fat group (HFD) High-fat feed purified water WPI Wall Material Control Group (WPI) High-fat feed 1000 mg / kg / day HA-WPI wall material control group (HW) High-fat feed 1010 mg / kg / d Monk fruit glycoside intervention group (MOG) High-fat feed 400 mg / kg / d WU20-M intervention group (W-MOG) High-fat feed 1400 mg / kg / d H0.1-WU-M intervention group (HW-MOG) High-fat feed 1410 mg / kg / d

[0105] After grouping the mice, they underwent different feeding interventions for 12 weeks, with mouse weight measured weekly. Mice were fasted for 12 hours prior to the experiment but allowed free water. At the end of the experiment, mice were fasted for 12 hours but allowed free water, and their weight was measured again. Lee's index was calculated using the following formula:

[0106]

[0107] Where m: body weight; L: body length.

[0108] There was no significant difference in obesity levels between the WPI, HW, and MOG groups and the HFD group. Mice administered WU20-M (W-MOG) and H0.1-WU-M (HW-MOG) nanoparticles by gavage showed a significant decrease in obesity levels (p<0.05), with the HW-MOG group exhibiting the best anti-obesity effect, reducing obesity to 3.25 (p<0.05). Figure 7 The results showed that the anti-obesity effect of mogrosides was significantly enhanced after encapsulation, with HW-MOG showing the best effect, and the weight of mice tended to stabilize at week 10.

[0109] Fat analysis results (see Table 2) showed that the fat indices of mice in the HFD, WPI, HW, and MOG groups were significantly higher than those in the CON group, while the fat indices of mice in the W-MOG and HW-MOG groups were significantly lower (p<0.05), indicating that the nanoparticles inhibited fat accumulation in mice. After high-fat diet induction and nano-wall material intervention, the liver indices of the HFD, WPI, and HW groups significantly increased to 4.66-4.73% (p<0.05), and the liver color became lighter. The liver in the HFD group became significantly yellow, due to excessive fat accumulation in hepatocytes. After intervention with mogroside alone and with mogroside-encapsulated particles, the liver indices significantly decreased. The liver indices of the HW-MOG group (3.62%) were closest to the CON group level (3.76%), and the liver color was bright red. There were no significant differences in the organ indices of the spleen, kidney, pancreas, and thymus among the groups, indicating that the nano-wall material and nanoparticles had no toxic effects on the organs. The results showed that W-MOG and HW-MOG could significantly enhance the regulation of fat accumulation in mice, with HW-MOG exhibiting the strongest regulatory capacity.

[0110] Lipid analysis showed that the levels of TG, TC, and LDL-C in mice in the HFD, WPI, HW, and MOG groups were significantly higher than those in the CON group (p<0.05). After nanoparticle intervention, the levels of TG, TC, and LDL-C significantly decreased, with the HW-MOG group showing the lowest levels at 1.18 mmol / L, 3.95 mmol / L, and 1.29 mmol / L, respectively, showing no significant difference from the CON group (p>0.05). In the HFD, WPI, and HW groups, HDL-C levels significantly decreased after high-fat diet induction and nanoparticle intervention (p<0.05), but significantly increased after mogroside and nanoparticle intervention (p<0.05), and were significantly higher than those in the CON group. Since HDL-C helps transport cholesterol from the blood to the liver, thereby regulating lipid levels, the results indicate that mogroside has a regulatory effect on lipid levels. LEP is a protein hormone secreted by adipocytes that can regulate energy balance by controlling food intake and increasing energy expenditure. GLP-1 is a hormone secreted by the gut that plays an anti-obesity role, primarily acting on the hypothalamus to inhibit the expression of appetite-related genes. Compared with the CON group, mice in the HFD, WPI, HW, and MOG groups showed significantly increased LEP levels and significantly decreased GLP-1 levels. After nanoparticle intervention, LEP levels decreased significantly, while GLP-1 levels increased significantly (p<0.05), showing no significant difference in LEP and GLP-1 levels compared to the CON group. These results indicate that nanoparticles can effectively regulate blood lipid levels and LEP and GLP-1 levels, thereby achieving a regulatory effect on obesity.

[0111] Fasting blood glucose test results (see) Figure 8 Fasting blood glucose levels in mice were monitored for 12 weeks. Compared with the CON group, the blood glucose level in the HFD group increased significantly from week 6 (p<0.05), the blood glucose level in the WPI and HW groups increased significantly from week 10 (p<0.05), and the blood glucose level in the MOG, W-MOG and HW-MOG groups did not change significantly (p>0.05). This indicates that mogrosides and their nanoparticles can inhibit the increase in blood glucose caused by a high-fat diet.

[0112] Results of the oral glucose tolerance test (OGTT) were as follows (see Figure 9The oral glucose tolerance test (OGTT) of mice was monitored after 12 weeks. After gavage administration of glucose, blood glucose levels in mice showed an increasing trend, reaching a peak at 30 minutes, followed by a gradual decrease. Compared with the CON group, the blood glucose levels of HFD, WPI, HW, and MOG mice were significantly higher (p<0.05), indicating that glucose tolerance was reduced in high-fat mice, mice treated with nanomaterials, and mice treated with mogroside alone. After nanoparticle intervention, the blood glucose levels of the W-MOG and HW-MOG groups were significantly lower than those of the HFD group (p<0.05). The blood glucose level of HW-MOG at 120 minutes was not significantly different from that of the CON group, indicating the best inhibitory effect on blood glucose increase. These results indicate that a high-fat diet leads to impaired glucose tolerance in mice, and ingestion of encapsulated mogroside can effectively improve glucose tolerance abnormalities induced by a high-fat diet in obese mice.

[0113]

[0114] Figure 10 The results showed the effect of different wall materials on the encapsulation efficiency (EE) and loading capacity (LC) of mogrosides (adjusted according to Example 1). The whey protein isolate and hyaluronic acid used in this application were significantly better than soy protein isolate (SPI) and sodium alginate (SA). The encapsulation efficiency of this application was 76.55%.

[0115] The above embodiments of the present invention are mainly used to help understand the technical solutions of the present invention, but do not constitute a limitation of the present invention. Any improvements or equivalent substitutions made based on the technical solutions of the present invention, as long as they do not depart from the spirit and scope of the technical solutions of the present invention, should be covered within the protection scope of the present invention.

Claims

1. A method for preparing a mogroside delivery system, characterized in that, Includes the following steps: (1) Dissociation of whey protein isolate: Dissolve whey protein isolate in deionized water to prepare a protein solution of 8-12 mg / mL, add urea to a final concentration of 18-22 mM, adjust the pH to 6.8-7.2, and incubate at 2-8℃ for 10-14 hours to obtain the dissociated protein solution; (2) Hyaluronic acid modification and mogroside loading: Add hyaluronic acid to the dissociated protein solution obtained in step (1) to a final concentration of 0.08-0.12 mg / mL, mix well, add mogroside to a final concentration of 3-5 mg / mL, and continue to incubate at 2-8℃ for 10-14 hours to obtain a mixed solution; (3) Dialysis recombination: The mixed solution obtained in step (2) is placed in a dialysis bag with a molecular weight cutoff of 500-1000 Da and dialyzed for 40-56 hours to remove urea; (4) Purification: After removing urea, the sample was transferred to a dialysis bag with a molecular weight cutoff of 8000-14000 Da and dialyzed for 20-28 hours to remove free mogrosides and obtain a nanoparticle dispersion. (5) Freeze-drying: The nanoparticle dispersion obtained in step (4) is freeze-dried to obtain nanoparticle powder.

2. The mogroside delivery system prepared by the method according to claim 1.

3. The mogroside delivery system according to claim 2, characterized in that, The nanoparticles have a particle size of 400-500 nm and a zeta potential of -10 mV to -15 mV.

4. A functional food, characterized in that, It includes the mogroside delivery system as described in any one of claims 2-3.

5. The functional food according to claim 4, characterized in that, (1) Dissociation of whey protein isolate: Dissolve whey protein isolate in deionized water to prepare a protein solution of 10 mg / mL, add urea to a final concentration of 20 mM, adjust the pH to 7.0, and incubate at 4°C for 12 hours to obtain a dissociated protein solution; (2) Hyaluronic acid modification and mogroside loading: Add hyaluronic acid to the dissociated protein solution obtained in step (1) to a final concentration of 0.1 mg / mL, mix well, add mogroside to a final concentration of 4 mg / mL, and continue incubation at 4°C. (2) Incubate for 12 hours to obtain a mixed solution; (3) Dialysis recombination: Place the mixed solution obtained in step (2) into a dialysis bag with a molecular weight cutoff of 500-1000 Da and dialyze for 48 hours to remove urea; (4) Purification: After removing urea, transfer it to a dialysis bag with a molecular weight cutoff of 8000-14000 Da and dialyze for 24 hours to remove free mogrosides and obtain a nanoparticle dispersion; (5) Freeze-drying: Freeze-dry the nanoparticle dispersion obtained in step (4) to obtain nanoparticle powder.

6. The functional food according to claim 5, characterized in that, The food is a solid beverage, nutrition bar, or liquid nutritional preparation.

7. The application of a combination system of whey protein isolate, hyaluronic acid, and mogrosides in the construction of core-shell nanoparticle powders with intestinal targeted release function, characterized in that... The nanoparticle powder is the nanoparticle powder according to any one of claims 1-5, which can achieve a release rate of less than 10% in simulated gastric fluid after 2 hours, a cumulative release rate of 65%-70% in simulated intestinal fluid, and an encapsulation rate of mogrosides of not less than 75% for the nanoparticles.

8. The method for preparing a mogroside delivery system according to claim 1, characterized in that, The mogroside is mogroside V.

9. The method for preparing a mogroside delivery system according to claim 8, characterized in that, The entire reaction takes place in the aqueous phase.

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