Dintine 48k gel type sports nutritious food capable of promoting dissolution and process

Abstract

The invention relates to the technical field of health-care food and pharmaceutical preparations, and discloses a dissolution-promoting Echinostatin 48k gel type sports nutritional food and a process thereof, the content of the food is a multiphase suspension composition, and the multiphase suspension composition comprises 6.0-10.0 parts of a dispersion phase, 0.5-10.0 parts of an emulsifier, 0.5-10.0 parts of an emulsifier, and 0.5-10.0 parts of a stabilizer. 18.0 to 22.0 parts of red yeast rice powder; 60.0 to 68.0 parts of a base oil; 4.0 to 6.0 parts of a phospholipid; and 1.5 to 3.0 parts of beeswax; wherein the dispersion phase is enzyme activity double-layer composite microspheres subjected to surface lipophilic modification. The biological enzyme is entrapped in a polymer matrix to form a glassy-state compound core, conformational movement of protein is limited from the molecular level, denaturation and inactivation of the protein under the influence of temperature and humidity are effectively prevented, the endogenous deep locking technology for enzyme activity endows the product with excellent long-term storage stability, and the product has good application prospects. Therefore, the effective shelf life of the product is obviously prolonged, and the lasting and reliable effects of the terminal product are ensured.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of health food and pharmaceutical preparations, in particular to a solubility-promoted echinatin 48k gel-type sports nutrition food and process. BACKGROUND

[0002] Bio-enzyme preparations such as natto kinase and earthworm kinase, and traditional fermentation products such as red yeast rice, have been widely concerned in the health field due to their potential in assisting the regulation of physiological functions and promoting metabolism. Combining these active ingredients with different physicochemical properties and mechanisms of action to develop a complex preparation in order to achieve synergistic effects is an important direction of current product development, especially in soft capsule dosage forms.

[0003] However, the implementation of such combinations faces huge technical challenges. First, the chemical nature of bio-enzymes such as natto kinase and earthworm kinase is protein, and its three-dimensional structure is the basis of its activity, but it also exhibits inherent instability. Changes in temperature and humidity during the production, storage and transportation of the product can easily cause denaturation and inactivation, leading to rapid decay of the product's efficacy during the shelf life. In addition, when such enzyme preparations are taken orally, their activity will be rapidly destroyed in the strong acid and digestive enzyme environment of the stomach without effective protection, making it difficult to reach the intestine and play a role, thereby greatly reducing the bioavailability.

[0004] Further, there are insurmountable obstacles at the formulation process level. When the protected enzyme active microparticles, usually with a hydrophilic surface, are mixed with lipophilic ingredients such as red yeast rice in an oil-based matrix to prepare the soft capsule content, serious technical problems arise. Due to the large interfacial tension between the solid microparticles and the oil phase matrix, they are naturally incompatible, which easily leads to microparticle aggregation and rapid settling. This unstable suspension system makes the content distribution of the final product extremely uneven, seriously affecting the accuracy of the product dosage and the reliability of the quality. At the same time, traditional mixing methods such as high shear or heating, which are used to manufacture uniform suspensions, in turn destroy the delicate microstructure constructed to protect the enzyme activity, rendering the previous protection measures ineffective.

[0005] Therefore, there is still a lack of a comprehensive technical solution in the prior art that can simultaneously solve the intrinsic stability of enzymes, in vivo release and physical stability of the preparation. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application provides a solubility-promoted echinatin 48k gel-type sports nutrition food and process, which solves the problems of enzyme inactivation, physical instability of solid-liquid two-phase state, and inability to achieve intestinal targeted release when high-activity bio-enzymes are compounded with lipid matrices to produce soft capsules.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions: a dissolution-promoting 48k gel-type sports nutrition food, the content of which is a multi-phase suspension composition, comprising, by weight: 6.0 to 10.0 parts of a dispersed phase; 18.0 to 22.0 parts of red yeast rice flour; 60.0 to 68.0 parts of base oil; 4.0 to 6.0 parts of phospholipids; and 1.5 to 3.0 parts beeswax; Wherein, the dispersed phase is an enzyme-active double-layer composite microsphere with surface lipophilic modification, and the microsphere comprises: an enzyme-polymer matrix glassy complex core comprising nattokinase and / or lumbrokinase; A double-layer composite shell is coated on the outside of the core, wherein the double-layer composite shell is composed of an inner layer of pH-sensitive enteric material and an outer layer of intestinal mucosal bioadhesive material.

[0008] Preferably, the enzyme-polymer matrix glassy complex core further comprises a water-soluble polymer excipient selected from trehalose, pullulan or a combination thereof.

[0009] Preferably, the pH-sensitive enteric material of the inner layer is acrylic resin, and the intestinal mucosal bioadhesive material of the outer layer is chitosan or its derivatives.

[0010] Preferably, the surface lipophilic modification is achieved by coating a layer of polyglycerol ricinoleate on the surface of the double-layer composite microsphere.

[0011] Preferably, the matrix oil is linseed oil, and the red yeast rice powder is derived from a strain.

[0012] A process for preparing a dissolution-promoting 48k gel-type sports nutrition food comprises the following steps: S1: Preparation of dispersed phase: An enzyme comprising nattokinase and / or lumbrokinase is spray-dried with a water-soluble polymer excipient to form an "enzyme-polymer matrix glassy complex" core; the core is then coated with a pH-sensitive enteric material and an intestinal mucosal bioadhesive material in sequence using fluidized bed technology to form double-layer composite microspheres; and the double-layer composite microspheres are subjected to surface lipophilic modification. S2: Prepare the continuous phase: Disperse red yeast rice powder in a lipid system containing matrix oil and excipients to form a functional lipid suspension; S3: Final mixing and encapsulation: The surface lipophilically modified microspheres prepared in step S1 are gently dispersed in the lipid suspension prepared in step S2 in a suspended manner, and the final mixture is encapsulated in soft capsules.

[0013] Preferably, the parameters of the spray drying process in step S1 are: air inlet temperature 120-150°C, air outlet temperature 70-90°C.

[0014] Preferably, the fluidized bed coating process in step S1 comprises: firstly performing inner layer coating using an acrylic resin solution, and then performing outer layer coating using a chitosan solution.

[0015] Preferably, the surface lipophilic modification treatment in step S1 refers to spraying the double-layer composite microspheres with polyglycerol ricinoleate.

[0016] The final mixing process in step S3 is performed at a temperature of 35-45° C. and a slow stirring speed of less than 30 RPM.

[0017] The present invention provides a dissolution-promoting 48k gel-type sports nutrition food and a process thereof. It has the following beneficial effects: 1. By encapsulating the enzyme within a polymer matrix to form a glassy complex core, this invention restricts the protein's conformational motion at the molecular level, effectively preventing its denaturation and inactivation under the influence of temperature and humidity. This endogenous, deep-locking technology for enzyme activity imparts excellent long-term storage stability to the product, significantly extending its effective shelf life and ensuring the end product's long-lasting and reliable efficacy.

[0018] 2. This invention successfully constructs a precise targeted release system. Utilizing a double-layer composite shell structure, the inner pH-sensitive material ensures the microspheres can safely pass through the highly acidic environment of the stomach, preventing premature loss of core enzyme activity. The outer bioadhesive material prolongs the retention time of the formulation in the intestine, achieving efficient and concentrated release of the active ingredient in the intestine, thereby significantly improving its bioavailability.

[0019] 3. This invention solves the physical incompatibility problem of the solid-liquid phase in a multiphase system. By subjecting the microspheres to a critical surface lipophilic modification treatment, coupled with a specific low-temperature, slow-speed mixing process, the hydrophilic microspheres are stably and evenly suspended in the lipid continuous phase, effectively preventing product aggregation, sedimentation, and stratification during standing or storage. This not only ensures the uniformity of the final soft capsule content and dosage accuracy, but also improves the overall quality and appearance of the product.

[0020] 4、The present application realizes the synergistic effect of different mechanism active ingredients. By combining the high-activity enzyme preparation microspheres as the dispersed phase with the functional monascus substrate as the continuous phase, the two components can play their respective thrombolytic and auxiliary regulation functions, form a functional complement, and produce a result far superior to a single component. The compound formula design of the present application provides an innovative solution to improve overall efficacy.

[0021] 5、The present application provides a complete preparation process that can effectively protect the activity of biological macromolecules, especially solving the technical bottleneck of successfully applying high-activity and high-environmental sensitivity enzyme preparations to lipid-based soft capsules. The present application forms a protective chain from enzyme endogenous stabilization, multi-layer microcapsule structure protection, to the final mild mixing process, ensuring that the precise micro design can be completely preserved in macro production, laying a solid technical foundation for developing more functional composite soft capsule products. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The present application is a process flow diagram. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present application will be described below in conjunction with the drawings of the present application specification. Obviously, the described embodiments are only part of the embodiments of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the present application.

[0024] Please refer to the accompanying Figure 1 The present application provides a solubility-promoting spiculisin 48k gel-type sports nutrition food and process, including the following steps: Embodiment 1: The experimental steps of this embodiment are as follows: Enzyme core preparation: weigh 20g of natto kinase and 10g of earthworm kinase, and mix with 80g of trehalose and 40g of pullulan. Dissolve the mixed powder in purified water and make up to 1L. Stir at 8℃ and 200RPM for 45 minutes. Pump the solution into a spray dryer, set the inlet air temperature to 140℃ and the outlet air temperature to 85℃, and dry to obtain a glassy composite core.

[0025] Preparation of double-layer microspheres: The core powder described above was placed in a fluidized bed. First, an 8% ethanolic solution of acrylic resin No. II was used as the inner coating solution at 35°C until the core weight reached 12% of its dry weight. Subsequently, a 1.5% acidic aqueous solution of chitosan was used as the outer coating solution. Coating was continued until the core weight reached 5% of its total weight after the inner coating. Finally, the microspheres were dried at 45°C for 45 minutes.

[0026] Surface modification: The prepared double-layer microspheres were placed in a V-type mixer and sprayed with 1.0% polyglycerol ricinoleate (PGPR) by weight of the total microspheres at a speed of 30 RPM, and the mixing was continued for 20 minutes.

[0027] Prepare the lipid continuous phase: By weight, combine 65 parts flaxseed oil, 20 parts red yeast rice powder, 5 parts soybean lecithin, and 2 parts beeswax. In a mixing tank, heat 650g of flaxseed oil to 45°C, add 20g of beeswax and 50g of soybean lecithin, and stir to dissolve. Then, add 200g of red yeast rice powder and high-speed shear at 4000 RPM for 8 minutes to form a uniform suspension.

[0028] Final mixing and encapsulation: Maintain the lipid suspension at 40°C and slowly stir at 20 RPM. Slowly add the modified enzyme microspheres prepared in Step 3 (approximately 80 g, to meet the 8-part ratio). After continuous stirring for 30 minutes, compress the contents into 500 mg softgel capsules and dry them under standard conditions for 36 hours to obtain the finished product.

[0029] Example 2: The experimental steps of this embodiment are as follows: Enzyme core preparation: Weigh 15g of nattokinase and 10g of lumbrokinase and mix with 50g of trehalose (enzyme to excipient weight ratio of 1:2). Dissolve the mixed powder in purified water and dilute to 1L. Stir at 100 RPM at 10°C for 60 minutes. Pump the solution into a spray dryer with an inlet temperature of 120°C and an outlet temperature of 70°C for drying to obtain a glassy composite core.

[0030] Preparation of double-layer microspheres: The core powder described above was placed in a fluidized bed. First, a 5% ethanolic solution of acrylic resin No. II was used as the inner coating solution at 30°C until the core weight reached 10% of its dry weight. Subsequently, a 1.0% acidic chitosan aqueous solution was used as the outer coating solution. Coating was continued until the core weight reached 3.0% of its total weight after the inner coating. Finally, the microspheres were dried at 40°C for 60 minutes.

[0031] Surface modification: The prepared double-layer microspheres were placed in a V-type mixer and sprayed with PGPR (0.5% by weight of the total weight of the microspheres) at a speed of 20 RPM for 30 minutes.

[0032] Prepare the lipid continuous phase: By weight, combine 68 parts flaxseed oil, 18 parts red yeast rice powder, 4 parts soybean lecithin, and 1.5 parts beeswax. In a mixing tank, heat 680g of flaxseed oil to 40°C, add 15g of beeswax and 40g of soybean lecithin, and stir to dissolve. Then, add 180g of red yeast rice powder and high-speed shear at 3000 RPM for 10 minutes to form a uniform suspension.

[0033] Final mixing and encapsulation: Maintain the lipid suspension at 35°C and slowly stir at 15 RPM. Slowly add the modified enzyme microspheres prepared in Step 3 (approximately 60 g, to meet the 6-part ratio). After stirring for 40 minutes, compress the contents into 500 mg softgel capsules and dry them under standard conditions for 48 hours to obtain the finished product.

[0034] Example 3: The experimental steps of this embodiment are as follows: Enzyme core preparation: Weigh 25g of nattokinase and 10g of lumbrokinase and mix with 135g of trehalose and 40g of pullulan (enzyme to excipient weight ratio of 1:5). Dissolve the mixed powder in purified water and dilute to 1L. Stir at 300 RPM at 4°C for 30 minutes. Pump the solution into a spray dryer with an inlet temperature of 150°C and an outlet temperature of 90°C for drying to obtain a glassy composite core.

[0035] Preparation of double-layer microspheres: The core powder was placed in a fluidized bed. First, a 10% ethanolic solution of acrylic resin II was used as the inner coating solution at 40°C until the core weight reached 15% of its dry weight. Subsequently, a 2.0% acidic chitosan aqueous solution was used as the outer coating solution. Coating was continued until the core weight reached 8.0% of its total weight after the inner coating. Finally, the microspheres were dried at 50°C for 30 minutes.

[0036] Surface modification: The prepared double-layer microspheres were placed in a V-type mixer and sprayed with PGPR (2.0% by weight of the total weight of the microspheres) at a speed of 40 RPM for 15 minutes.

[0037] Prepare the lipid continuous phase: By weight, combine 60 parts flaxseed oil, 22 parts red yeast rice powder, 6 parts soybean lecithin, and 3 parts beeswax. In a mixing tank, heat 600g of flaxseed oil to 50°C, add 30g of beeswax and 60g of soybean lecithin, and stir to dissolve. Then, add 220g of red yeast rice powder and high-speed shear at 5000 RPM for 5 minutes to form a uniform suspension.

[0038] Final mixing and encapsulation: The lipid suspension was maintained at 45°C, with slow stirring at 25 RPM, and the modified enzyme microspheres prepared in Step 3 (about 100 g, to meet the ratio of 10 parts) were slowly added. After 20 minutes of continuous stirring, the contents were pressed into soft capsules at a specification of 500 mg per capsule, and dried under standard conditions for 24 hours to obtain the finished product.

[0039] Comparative Example 1: Compared with Example 1, the difference is that the microsphere preparation of Steps 1, 2, and 3 is not performed, and the untreated natto kinase powder and earthworm kinase powder are directly added to the lipid continuous phase in Step 4 for mixing. The rest are the same.

[0040] Comparative Example 2: Compared with Example 1, the difference is that only the enzyme core is coated with an inner layer of enteric coating in Step 2, without an outer layer of bioadhesion coating. The rest are the same.

[0041] Comparative Example 3: Compared with Example 1, the difference is that the surface lipophilic modification treatment of the composite microspheres in Step 3 is omitted, and the double-layer composite microspheres prepared in Step 2 are directly used in the final mixing of Step 5. The rest are the same.

[0042] Comparative Example 4: Compared with Example 1, the difference is that the composition does not contain the dispersed phase, i.e., no enzyme active microspheres prepared in Steps 1, 2, and 3 are added, and only the lipid matrix soft capsules containing red yeast rice powder are prepared. The rest are the same.

[0043] Comparative Example 5: Compared with Example 1, the difference is that in the final content composition ratio, the weight fraction of the dispersed phase (enzyme microspheres) is reduced from 8 parts to 4 parts, and the weight fraction of flaxseed oil is increased to 69 parts accordingly, to maintain the total weight fraction unchanged. The rest are the same.

[0044] Comparative Example 6: Compared with Example 1, the difference is that in the final mixing process of Step 5, the mixing temperature is increased to 70°C, and high-speed shearing (4000 RPM) is used instead of slow stirring. The rest are the same.

[0045] Test Example 1: Physical stability and uniformity test of product content Purpose of the experiment: The purpose of this experiment is to verify the key role of the "surface lipophilic modification" process (Comparative Comparative Example 3) and the "low temperature slow mixing" process (Comparative Comparative Example 6) in maintaining the physical stability of the product content and ensuring the uniformity of the final product content through comparative observation and quantitative analysis.

[0046] In the preparation process of Example 1, Example 2, Example 3, Comparative Example 3, and Comparative Example 6, the final content suspension without soft capsule encapsulation, and the finished soft capsule product after encapsulation are obtained.

[0047] The experiment included the following steps: Suspension stability test: Immediately after the final mixing step of Example 1-3, Comparative Example 3 and Comparative Example 6 was completed, 50 mL of the uniform content suspension was withdrawn from each without being encapsulated.

[0048] The five samples were each filled into a 100 mL graduated sealed glass measuring cylinder of the same specification, and sealed with a lid.

[0049] The measuring cylinders were placed vertically on a bench at room temperature (25°C ± 2°C) without vibration.

[0050] After standing for 24 hours, the state of the contents in each measuring cylinder was carefully observed by visual inspection, and recorded in detail. The observation points included: whether there was a visible separation of the oil phase and the solid phase (microspheres); whether the top oil phase became clear; whether there was a clear white microsphere precipitate layer at the bottom, and the volume of the precipitate layer was recorded (if any).

[0051] Content uniformity test: From the final product soft capsules of Example 1-3, Comparative Example 3 and Comparative Example 6, 10 capsules were randomly selected from each.

[0052] The weight of the contents of each soft capsule was accurately weighed and recorded.

[0053] The contents of each capsule were transferred to separate centrifuge tubes, and an appropriate amount of 0.5% Tween-80 in phosphate buffer (PBS, pH 7.4) was used to demulsify and vortex for 5 minutes to ensure that the enzyme in the contents was fully extracted into the buffer.

[0054] The extract was centrifuged at 4000g for 10 minutes, and the supernatant was taken.

[0055] The fibrin plate method was used to determine the activity titer of nattokinase in each supernatant (unit: FU / capsule).

[0056] According to the measured activity data of the 10 capsules, the average activity value, standard deviation (SD) of each group was calculated, and finally the relative standard deviation (RSD%) was calculated. The calculation formula is: RSD = (standard deviation / average value) x 100%.

[0057] Experimental results: Table 1 Physical stability and content uniformity test results of each group of samples

[0058] The above test results clearly show that the technical scheme adopted by the present application can significantly improve the physical stability and content uniformity of the product contents. The results of Examples 1, 2 and 3 show that the contents can maintain a uniform suspension state for a long time after standing, and the content uniformity of the active ingredient in the final product is extremely high (RSD is far less than 5%). This is due to a core innovation of the present application: the key surface lipophilic modification of the enzyme active microspheres. The modification process coats the originally hydrophilic microsphere surface with a layer of lipophilic molecular film, greatly reducing the interfacial tension between it and the oil phase matrix, so that it can be stably suspended in the continuous phase like an oil droplet, thereby fundamentally solving the physical incompatibility problem between the solid-liquid two phases.

[0059] In contrast, the results of Comparative Example 3 are quite different. After omitting the surface lipophilic modification step, the unmodified microspheres, due to their hydrophilic surface and natural repulsion with the hydrophobic oil phase, quickly agglomerate and settle after mixing, resulting in a serious oil-solid separation phenomenon. This macroscopic inhomogeneity directly leads to a large difference in the content of active ingredients in each capsule during encapsulation, and its RSD value of 17.58% indicates that the product quality is completely uncontrollable. This result inversely proves that the surface lipophilic modification is an indispensable key step for building the stable multi-phase system described in the present application.

[0060] The results of Comparative Example 6 reveal the importance of another innovative point of the preparation process of the present application. Although the microspheres in Comparative Example 6 are surface-modified, the stability and uniformity of the final product are still very poor due to the use of high-temperature and high-shear mixing methods. The mechanism is that the intense physical conditions (high temperature and high shear) are enough to destroy the precise lipophilic modification film on the surface of the microspheres, and even the overall structure of the microspheres may be destroyed. Once the modification layer is destroyed, the microspheres will again expose their hydrophilic surface, thereby again triggering the agglomeration and sedimentation problems similar to those of Comparative Example 3. This fully demonstrates that the low-temperature, slow-speed mixing process adopted by the present application is necessary to protect the integrity of the microsphere structure and ensure that its function is fully retained in the final product.

[0061] Test Example 2: In vitro simulation of gastrointestinal release behavior test Purpose of the experiment: The purpose of this experiment is to verify the key role of the "pH-sensitive bioadhesive double-layer composite shell" designed in the present application in protecting the core enzyme active ingredient (nattokinase) to safely pass through the gastric acid environment and achieve targeted and efficient release in the intestinal environment by simulating the environment of the human stomach and intestine.

[0062] Experimental materials: Samples: final product soft capsules of Example 1, Example 2, Example 3, Comparative Example 1, and Comparative Example 2.

[0063] Reagents: simulated gastric fluid (take 2.0 g of sodium chloride, 7.0 mL of dilute hydrochloric acid, add appropriate amount of pepsin, dilute to 1000 mL with purified water, and adjust the pH to 1.2); simulated intestinal fluid (take 6.8 g of potassium dihydrogen phosphate, add appropriate amount of trypsin, adjust the pH to 6.8 with 0.1 mol / L sodium hydroxide solution, and dilute to 1000 mL with purified water).

[0064] The experiment includes the following steps: The dissolution test method specified in the Chinese Pharmacopoeia was used. The dissolution medium temperature was set at 37°C ± 0.5°C and the basket speed was set at 50 RPM.

[0065] Six soft capsules were taken from each of the five groups of Examples 1-3, Comparative Example 1 and Comparative Example 2, and placed in six hanging baskets respectively.

[0066] Intragastric Release Phase: Simultaneously lower all six baskets into a dissolution vessel containing 900 mL of simulated gastric fluid and immediately start the timer. Run for exactly 120 minutes. At the end of 120 minutes, precisely draw 5 mL of solution from each dissolution vessel and replace with fresh simulated gastric fluid of the same temperature and volume.

[0067] Intestinal release stage: After 120 minutes, all the hanging baskets were immediately taken out of the simulated gastric fluid and quickly immersed in another set of dissolution cups containing 900 mL of simulated intestinal fluid, and the operation continued under the same conditions.

[0068] At 30, 60, and 120 minutes of operation in the simulated intestinal fluid, 5 mL of solution was accurately drawn from each dissolution vessel and replenished with fresh simulated intestinal fluid of the same temperature and volume.

[0069] All sample solutions were filtered through a 0.45 μm filter membrane, and the activity concentration of nattokinase in the filtrate was determined using the fibrin plate method.

[0070] The cumulative release rate (%) at each time point was calculated based on the measured concentration and sampling volume.

[0071] Experimental results: Table 2 Cumulative release rate of each group of samples in the simulated gastrointestinal tract in vitro (%)

[0072] The results of this in vitro simulated release experiment strongly confirm the effectiveness and advanced nature of the microsphere delivery system designed by this invention. The samples of Examples 1, 2, and 3 all exhibited ideal release profiles: after incubation for 2 hours in the highly acidic environment of simulated gastric fluid, the release rate of active enzyme was extremely low (less than 5%). However, after transfer to simulated intestinal fluid, it was released rapidly and completely, with cumulative release rates exceeding 90% within 2 hours. This perfectly demonstrates the mechanism of the "pH-sensitive shell" in the inner layer of the microspheres: the enteric material is structurally stable at low pH values, acting like a solid "chemical shield" that protects the enzyme core from being destroyed by gastric acid; upon entering the intestinal environment with a higher pH value, the material rapidly dissolves, achieving a fixed, targeted release of the active ingredient.

[0073] The results of Comparative Example 1 confirm the necessity of the protective structure of the present invention from the opposite side. When the unprotected enzyme powder was directly mixed into the oil phase, it was "released" in large quantities (85.6%) in the simulated gastric fluid. However, this was not an effective release, but a devastating exposure. The biological enzyme will quickly denature and inactivate in such an acidic environment, resulting in its subsequent almost no activity in the intestinal environment. This result clearly shows that without the effective isolation and protection of the microcapsule structure described in the present invention, the core biological enzyme would have basically lost its effectiveness before reaching the intestine, and the product would therefore lose its core efficacy.

[0074] The comparison between Comparative Example 2 and the embodiment further highlights the ingenuity of the "double-layer composite shell" design of the present invention. Comparative Example 2 only has an inner enteric coating. Although it also successfully protects the enzyme from passing through the gastric acid environment, its release rate in simulated intestinal fluid is significantly slower than that of the embodiment, and about 30% of the active substance has not been released after 2 hours. The mechanistic difference lies in that the "outer bioadhesive shell" in the embodiment not only plays a role in prolonging the retention time in vivo, but also its hydrophilic and swelling properties promote the rapid disintegration of the entire shell structure during the in vitro dissolution process. Without this outer layer, the dissolution rate of the enteric layer alone is slow, resulting in a delayed and incomplete release of the active ingredient. The double-layer shell design of the present invention ensures precise targeting while also achieving efficient and rapid drug release, thereby maximizing bioavailability.

[0075] Test Example 3: Accelerated stability test (enzyme activity retention) Purpose of the experiment This experiment aims to evaluate the stability of the product of the present invention under harsh temperature and humidity conditions during long-term storage, so as to verify the decisive role of the "enzyme-polymer matrix glassy complex core" technology and the overall microcapsule structure adopted in the present invention in protecting the core enzyme activity from environmental influences and ensuring the stability of the product's efficacy during the shelf life.

[0076] Experimental materials and equipment: Samples: The final finished soft capsules of Example 1, Example 2, Example 3, Comparative Example 1, and Comparative Example 6 were all sealed and packaged using the same aluminum-plastic blister plate.

[0077] Equipment: constant temperature and humidity chamber, high performance liquid chromatography (or microplate reader for activity determination), vortex mixer, high-speed refrigerated centrifuge.

[0078] The experiment includes the following steps: Initial activity measurement (month 0): Ten pellets were randomly sampled from each of the five groups of final products of Examples 1-3, Comparative Example 1 and Comparative Example 6.

[0079] Referring to the content uniformity determination method in Test Example 1, the contents of 10 capsules of each sample group were subjected to demulsification, extraction and centrifugation to obtain an enzyme activity extract.

[0080] The average activity of nattokinase in each group of extracts was determined using national standard methods (such as the fibrin plate method), and the initial activity value was accurately recorded. This value was used as the 100% activity baseline.

[0081] Accelerated stability test: The remaining sealed finished products of the five groups of samples were placed together in a constant temperature and humidity chamber with set conditions of temperature 40℃±2℃ and relative humidity 75%±5%.

[0082] Start the test and begin timing.

[0083] Periodic sampling and measurement: At the end of the first, third and sixth months of the trial, 10 samples were taken out from the constant temperature and humidity chamber for each group.

[0084] Repeat the extraction and activity determination method in step 1 to determine the average enzyme activity of each group at that time point.

[0085] Data processing: The activity retention rate of nattokinase was calculated based on the average activity value measured at each time point.

[0086] The calculation formula is: Activity retention rate (%) = (average activity at the current time point / initial average activity) × 100%.

[0087] Experimental results: Table 3 Nattokinase activity retention rate of each group of samples under accelerated conditions (%)

[0088] The results of the accelerated stability test deeply reveal the breakthrough innovation of the present invention in protecting the core enzyme activity from the perspective of product shelf life. The samples of Examples 1, 2, and 3 can still maintain an enzyme activity retention rate of more than 90% under harsh conditions for up to 6 months, showing excellent stability. Its core mechanism lies in the original enzyme-polymer matrix glassy complex core technology of the present invention. Through a specific spray drying process, the enzyme molecules are instantly embedded in the amorphous glassy matrix formed by trehalose and pullulan. This high-viscosity molecular cage fundamentally limits the movement and conformational changes of the enzyme protein segments, effectively inhibits thermal denaturation, degradation, and aggregation that lead to loss of activity, and achieves endogenous and deep locking of enzyme activity.

[0089] In contrast, the results of Comparative Example 1 showed a cliff-like activity decay, with activity almost completely lost after 6 months. This fully exposes the inherent flaws of traditional preparation methods. In Comparative Example 1, the enzyme powder, which was not specially protected, had a protein structure in a free state of motion under a continuous warm environment, making it extremely prone to irreversible folding errors and denaturation, leading to the destruction of the active center. Even when encapsulated in an oil phase, this inherent instability at the molecular level could not be prevented. This comparative result irrefutably proves that without the glass-stabilized core described in this invention, the product would have no meaningful shelf life and commercial value.

[0090] The results of Comparative Example 6 cleverly reveal the necessity of the preparation process of the present invention. Although this comparative example uses the core components of the present invention, its activity retention rate is much lower than that of the embodiment, indicating that its protective structure has been significantly damaged. The mechanism is that the mixing process of high temperature and high shear force is destructive to the already formed precise microsphere structure. The severe physical force is enough to destroy the integrity of the microspheres and may even cause the glassy core to undergo a phase transition, thereby breaking the "lock" on the enzyme molecules and exposing them to an unstable environment again.

[0091] Test Example 4: In vitro thrombolytic activity test Purpose of the experiment: The purpose of this experiment is to intuitively evaluate and compare the thrombolytic ability of products with different formulas through an in vitro simulation of a thrombosis model, thereby verifying the synergistic effect of the enzymatic active component (dispersed phase) and the red yeast rice matrix (continuous phase) in the present invention, and proving the scientific nature and superiority of the component ratio described in the present invention.

[0092] Experimental materials and equipment: Samples: the final finished soft capsules of Example 1, Example 2, Example 3, Comparative Example 4, and Comparative Example 5.

[0093] Reagents: fibrinogen, agarose, thrombin, phosphate buffered saline (PBS, pH 7.4).

[0094] Equipment: Petri dish (90 mm diameter), constant temperature incubator, water bath, hole punch (5 mm diameter), pipette, vernier caliper.

[0095] The experiment includes the following steps: Preparation of fibrin slabs; Accurately weigh 1.5 g of agarose, dissolve it in 100 mL of PBS buffer, and heat and boil until completely dissolved to prepare a 1.5% agarose gel.

[0096] The agarose gel was placed in a 50°C water bath to cool and then kept warm.

[0097] Take another 10 mL of PBS buffer, add 0.2 g of fibrinogen, and stir to dissolve in a 37°C water bath.

[0098] Add the fibrinogen solution to the 50°C agarose gel and shake gently. Then add 100 units (U) of thrombin solution and mix quickly.

[0099] Immediately pour the mixture into a horizontal culture dish, 20 mL per dish, and let it stand at room temperature for 30 minutes until it completely solidifies to form an opaque fibrin gel plate.

[0100] Sample processing: From the five groups of final products of Examples 1-3, Comparative Example 4 and Comparative Example 5, 5 pellets were randomly sampled from each group.

[0101] Carefully cut open the capsule and squeeze the contents completely into a separate centrifuge tube.

[0102] Add 2 mL of PBS buffer to each tube and vortex for 5 minutes to mix thoroughly to prepare a sample suspension to be tested.

[0103] Thrombolytic activity assay: Use a 5 mm diameter sterile puncher to make even holes in the prepared fibrin plate.

[0104] Use a pipette to accurately draw 20 μL of the sample suspension in each group and add it to the corresponding wells.

[0105] Place the plate with the sample in a constant temperature incubator at 37°C and incubate for 18 hours.

[0106] After incubation, remove the plate and use a vernier caliper to measure the diameter of the transparent thrombolytic zone formed around each well (accurate to 0.1 mm). Measure three parallel wells for each sample group and calculate the average value.

[0107] Experimental results: Table 4 In vitro thrombolytic activity test results of each group of samples

[0108] The results of the in vitro thrombolytic activity test visually and powerfully prove the functional synergistic effect of the composition of the present application. Examples 1, 2, and 3 all exhibit strong thrombolytic ability, forming a clear thrombolytic ring much larger than the control group. The mechanism of this remarkable effect lies in the fact that the present application has creatively constructed a dispersed-continuous phase synergistic system: the enzyme active microspheres composed of natto kinase and acaenase (dispersed phase) as the direct thrombolytic pioneer, can efficiently hydrolyze fibrin and directly dissolve thrombus; while the lipid matrix containing red yeast rice powder (continuous phase) provides auxiliary support. This scientific functional zoning and synergistic cooperation makes the overall thrombolytic effect of the final product far exceed the simple addition of each component.

[0109] The results of Comparative Example 4 are the key to understanding the synergistic effect of the present application. After completely removing the dispersed phase containing enzyme active microspheres, its thrombolytic ring diameter decreases sharply to almost negligible 5.2 mm. This shows that although red yeast has certain physiological activity, its direct and rapid thrombolytic ability is very weak. This result clearly defines the functional role of each component and irrefutably proves that the enzyme active microspheres in the present application are the core and foundation to achieve strong thrombolytic efficacy. Without the participation of the dispersed phase, the core technical effect of the present application cannot be achieved.

[0110] The comparison between Comparative Example 5 and the examples further reveals the scientificity and innovation of the present application in component proportioning. When the amount of the dispersed phase (enzyme microspheres) is halved, the thrombolytic activity also decreases significantly, and its thrombolytic ring diameter is much smaller than that of the examples. This shows that the thrombolytic effect of the product has a clear dose-dependent relationship with the concentration of the core enzyme component. This result confirms that the component weight ratio defined in the present application is not arbitrarily set, but is an effective concentration range that can ensure the product to exert excellent efficacy. Below this range, the core efficacy of the product will be greatly discounted, thus highlighting the precision of the formula design of the present application.

[0111] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A dissolution-promoting 48k gel-type sports nutrition food, characterized in that: The content of the food is a multi-phase suspension composition, which comprises, by weight: 6.0 to 10.0 parts of a dispersed phase; 18.0 to 22.0 parts of red yeast rice flour; 60.0 to 68.0 parts of base oil; 4.0 to 6.0 parts of phospholipids; and 1.5 to 3.0 parts beeswax; Wherein, the dispersed phase is an enzyme-active double-layer composite microsphere with surface lipophilic modification, and the microsphere comprises: an enzyme-polymer matrix glassy complex core comprising nattokinase and / or lumbrokinase; A double-layer composite shell is coated on the outside of the core, wherein the double-layer composite shell is composed of an inner layer of pH-sensitive enteric material and an outer layer of intestinal mucosal bioadhesive material.

2. The dissolution-promoting jiting 48k gel-type sports nutrition food according to claim 1, characterized in that: The enzyme-polymer matrix glassy complex core further comprises a water-soluble polymer excipient selected from trehalose, pullulan or a combination thereof.

3. The dissolution-promoting jiting 48k gel-type sports nutrition food according to claim 1, characterized in that: The pH-sensitive enteric material of the inner layer is acrylic resin, and the intestinal mucosal bioadhesive material of the outer layer is chitosan or its derivatives.

4. The dissolution-promoting tricin 48k gel-type sports nutrition food according to claim 1, characterized in that: The surface lipophilic modification is achieved by coating a layer of polyglycerol ricinoleate on the surface of the double-layer composite microsphere.

5. The dissolution-promoting tricin 48k gel-type sports nutrition food according to claim 1, characterized in that: The matrix oil is linseed oil, and the red yeast rice powder is derived from a bacterial strain.

6. A process for preparing the dissolution-promoting spondin 48k gel-type sports nutrition food according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1: Preparation of dispersed phase: An enzyme comprising nattokinase and / or lumbrokinase is spray-dried with a water-soluble polymer excipient to form an "enzyme-polymer matrix glassy complex" core; the core is then coated with a pH-sensitive enteric material and an intestinal mucosal bioadhesive material in sequence using fluidized bed technology to form double-layer composite microspheres; and the double-layer composite microspheres are subjected to surface lipophilic modification. S2: Prepare the continuous phase: Disperse red yeast rice powder in a lipid system containing matrix oil and excipients to form a functional lipid suspension; S3: Final mixing and encapsulation: The surface lipophilically modified microspheres prepared in step S1 are gently dispersed in the lipid suspension prepared in step S2 in a suspended manner, and the final mixture is encapsulated in soft capsules.

7. The process for preparing the dissolution-promoting 48k gel-type sports nutrition food according to claim 1, characterized in that: The parameters of the spray drying process described in step S1 are: inlet air temperature 120-150°C, outlet air temperature 70-90°C.

8. The process for preparing the dissolution-promoting 48k gel-type sports nutrition food according to claim 1, characterized in that: The fluidized bed coating process described in step S1 includes: firstly using an acrylic resin solution to perform an inner layer coating, and then using a chitosan solution to perform an outer layer coating.

9. The process for preparing the dissolution-promoting 48k gel-type sports nutrition food according to claim 1, characterized in that: The surface lipophilic modification treatment described in step S1 refers to the surface spraying treatment of the double-layer composite microspheres using polyglycerol ricinoleate.

10. The process for preparing the dissolution-promoting 48k gel-type sports nutrition food according to claim 1, characterized in that: The final mixing process in step S3 is performed at a temperature of 35-45° C. and a slow stirring speed of less than 30 RPM.

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