Preparation method of immune-enhanced bovine colostrum powder bioactivity protection microcapsules

Abstract

The present application relates to the technical fields of biological medicine manufacturing, pharmaceutical excipients and preparation manufacturing, and discloses a preparation method of immune-enhanced bovine colostrum powder biological activity protection microcapsule, which is constructed with multi-layer microcapsule structure by multi-component active core, Fc segment protection stable layer, complex coagulation physical barrier layer, double response enteric layer and optional medication time period self-adaptive delay layer, effectively protects heat-sensitive active ingredients such as IgG, lactoferrin and lysozyme in the processes of low-temperature powdering, freeze-drying core making, electrostatic complex coagulation and fluidized bed coating, inhibits Maillard glycosylation reaction in storage period by non-reducing sugar vitrification matrix, free amino acid competitive space agent and activated molecular sieve, maintains FcRn receptor binding activity, reduces shelf life hidden failure, realizes low release in stomach, concentrated release under intestinal conditions, improves stability, targeting, bioavailability and application consistency under different medication time periods.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical manufacturing and pharmaceutical excipient and formulation manufacturing technology in the biopharmaceutical industry, and particularly to a method for preparing bioactive protective microcapsules of immune-enhancing bovine colostrum powder. Background Technology

[0002] Oral immunomodulators have significant clinical value in the biotherapy and adjuvant therapy of major and intractable diseases, such as regulating the body's immune function, preventing and treating recurrent respiratory infections, improving chronic inflammatory bowel disease, assisting immune reconstitution after radiotherapy and chemotherapy in cancer patients, and repairing immune function in children and the elderly. Oral immunomodulators with bovine colostrum as the active ingredient source have become one of the important directions in antibody preparation research and development in this field because their active ingredients include multi-component biopharmaceutical active ingredients such as natural antibodies, animal-derived peptides and protein active substances.

[0003] Bovine colostrum contains immunoglobulin IgG, which is the main active pharmaceutical ingredient in this type of antibody preparation. Its content in each 100g raw material powder is usually not less than 10% to 30%. The efficacy of IgG molecules is highly dependent on their intact intra- and inter-chain disulfide bonds and three-dimensional folded structural domains: IgG undergoes thermal denaturation when the temperature exceeds about 65°C, and the antigen-binding activity of IgG decreases significantly when the pH of the medium is below about 4.0 or above about 9.0. When the pH of gastric juice drops to below about 2.0, intact IgG molecules cannot be detected. At the same time, pepsin and trypsin can hydrolyze IgG peptide chains at different sites and inactivate them. Other animal-derived active peptides and protein components such as lactoferrin, growth factors, and lysozyme also have similar heat sensitivity, acid sensitivity, and enzyme sensitivity.

[0004] Regarding the aforementioned indicators, existing technologies in the field of biopharmaceutical manufacturing and pharmaceutical excipient and formulation manufacturing have developed various branches, including drying and powdering processes, microencapsulation of single pharmaceutical excipients, complex coagulation and double-layer encapsulation of composite pharmaceutical excipients, enteric coating pH-responsive release pathways, and submicron-level delivery pathways using nanocarriers. While some progress has been made in protecting antibody-based active ingredients such as IgG at specific stages, several core technical problems remain unresolved in this field, as follows:

[0005] 1. These types of biological pharmaceutical products require a shelf life of 6 to 24 months at room temperature after leaving the factory. During this period, frequent occurrences of actual distribution and oral administration, such as the rainy season in the south, the heating season in the north, cold chain disruptions across climate zones, and repeated exposure to environmental moisture after opening by consumers, cause the approximately 2.5% endogenous lactose in bovine colostrum, the animal biological resource from which the active ingredient of these biological pharmaceutical products is derived, to synergistically interact with the polysaccharide pharmaceutical excipients used in various formulation manufacturing routes. Under subcritical temperature and humidity conditions, it continuously binds to lysine near the FcRn receptor binding site of the IgG heavy chain Fc segment. - The amino group undergoes slow Maillard glycosylation modification. This modification cannot be captured by existing ELISA activity assays because it does not change the antigen-binding ability of the Fab segment. However, it selectively destroys the Fc segment recognition function necessary for the transcellular transport of IgG via the intestinal mucosa FcRn. This results in a 30% to 50% hidden failure bias in the actual bioavailability and pharmacodynamic response when the same batch of products is administered orally in different seasons and regions.

[0006] 2. Existing microencapsulated dosage forms in this field mostly employ a single enteric coating pH-triggered release or rely solely on physical barriers without active release control. This, combined with the objective existence of individual differences in real clinical scenarios, such as patients scheduling oral administration of immunomodulatory agents according to their own lifestyle habits (e.g., fasting in the morning, after meals, before bedtime, or at night), elderly people with chronic diseases often taking multiple medications after 10 PM, and infants taking them between meals during the day, leads to a phase mismatch between the diurnal fluctuations in gastric acid pH, the diurnal differences in gastric emptying rate, and the diurnal rhythm of the ileal Peyer's patch immune activation window when oral administration of existing microencapsulated dosage forms at different times. As a result, even if IgG is released with complete activity, it often falls during the trough of immune activation in the gut-associated lymphoid tissue, is consumed by intestinal flora metabolism, and cannot be effectively taken up and activated by intestinal mucosal immune sites. Summary of the Invention

[0007] The technical problem to be solved by this invention is that existing technologies have the drawback of protecting the FcRn receptor binding activity of immunoglobulin IgG during long-term shelf life at room temperature, ensuring that the FcRn binding activity retention rate is above 80% after 24 months of storage at room temperature, and eliminating the hidden failure deviation under different seasons and regional distribution environments. To this end, we propose a method for preparing bioactive protective microcapsules of immune-enhancing bovine colostrum powder.

[0008] To achieve the above objectives, this application adopts the following technical solution: an immune-enhancing bovine colostrum powder bioactive protective microcapsule, comprising four layers arranged sequentially from the inside out: a multi-component active core, an Fc segment protective stabilizing layer, a complex coagulation physical barrier layer, and a dual-response enteric layer.

[0009] The multi-component active core contains bovine colostrum powder made from colostrum produced within 48 hours postpartum through low-temperature defatting, isoelectric point casein removal, low-temperature membrane filtration, low-temperature ultrafiltration concentration, and low-temperature spray drying.

[0010] Bovine colostrum powder contains more than 25 wt% of immunoglobulin IgG;

[0011] The Fc segment protective stabilizing layer is composed of a non-reducing sugar vitrified matrix, a free amino acid competitive occupant, an activated molecular sieve hygroscopic regulator, and pre-denatured milk matrix protein. The mass of the non-reducing sugar vitrified matrix is ​​more than 12 times the sum of the mass of lactose contained in bovine colostrum powder and the total mass of reducing sugars in all other pharmaceutical excipients of the microcapsule. Through the hygroscopic effect of the activated molecular sieve hygroscopic regulator, the microenvironment water activity Aw of the Fc segment protective stabilizing layer is maintained in the range of 0.20 to 0.30 within 24 months of storage at 25°C and 60% relative humidity.

[0012] The complexed physical barrier layer is a polyelectrolyte complex membrane formed by electrostatic recombination of two polysaccharide pharmaceutical excipients with opposite charges, which completely separates the Fc segment protective stabilizing layer from the dual-response enteric layer in space.

[0013] The dual-responsive enteric layer contains both a pH-responsive enteric polymeric component that responds to the pH of the medium and an esterified enteric polymeric component that responds to short-chain fatty acids. It dissolves and releases only when the pH of the medium is not lower than 6.5 and the concentration of short-chain fatty acids is not lower than 10 mmol / L.

[0014] Preferably, the multi-component active core also contains 0.5 wt% to 2.0 wt% of Pear aggregate lymph node microfold cell directional anchoring agent based on the weight of bovine colostrum powder;

[0015] The Pear aggregate lymph node microfold cell directional anchoring agent is selected from one or more combinations of modified edible lectins, fucoidylated oligosaccharides, mannosylated chitosan oligosaccharides, and sialic acid-modified whey proteins.

[0016] Preferably, the components of the Fc segment protective stabilizing layer, by weight, are as follows: 30 wt% to 50 wt% non-reducing sugar vitrified matrix, 1 wt% to 3 wt% free amino acid competitive site-filling agent, 0.5 wt% to 1.5 wt% activated molecular sieve hygroscopic regulator, and 5 wt% to 10 wt% pre-denatured milk matrix protein;

[0017] The non-reducing sugar glass matrix is ​​selected from one or more combinations of trehalose, sucrose, raffinose, and metriose, and the glass transition temperature is above 100℃.

[0018] The free amino acid competitive occupier is selected from one or more combinations of L-lysine hydrochloride, L-arginine hydrochloride, glycine, and taurine;

[0019] The activated molecular sieve moisture regulator is selected from one or more combinations of 3A type synthetic molecular sieve, 4A type synthetic molecular sieve, food-grade silica gel, and food-grade activated alumina, with a particle size of 2μm to 5μm, and is pre-activated at 200℃ to 300℃ for 4 to 8 hours.

[0020] The pre-denatured milk matrix protein is selected from one or more of whey protein isolate, sodium caseinate, and casein phosphopeptide that have been heat-denatured at 75°C to 85°C for 20 to 30 minutes.

[0021] Preferably, in the two polysaccharide pharmaceutical excipients of the complex coagulated physical barrier layer, one carries a positive charge and the other carries a negative charge;

[0022] The negatively charged polysaccharide is selected from one or more of sodium alginate, pectin, carrageenan, and gum arabic that have been treated to reduce their molecular weight, with a weight-average molecular weight of 20 kDa to 40 kDa.

[0023] The positively charged polysaccharide is a deacetylated chitosan with a degree of deacetylation of 85% to 95%.

[0024] The thickness of the complex coagulated physical barrier layer is 5 μm to 15 μm, which forms a mechanically stable wet gel network after being cured by calcium chloride aqueous solution.

[0025] Preferably, the proportions of each component in the dual-response enteric coating layer, based on the total weight of the layer, are as follows:

[0026] The pH-responsive enteric polymer component comprises 60 wt% to 80 wt%, selected from one or more combinations of acrylic resin No. III, hydroxypropyl methyl phthalate, cellulose acetate, and hydroxypropyl methyl cellulose acetate succinate.

[0027] The short-chain fatty acid-responsive esterification modified enteric polymeric component accounts for 15 wt% to 30 wt%, and is selected from one or more combinations of butyrate-esterified hydroxypropyl methylcellulose phthalate, butyrate-esterified sodium alginate, propionic acid-esterified pectin, and acetate-esterified chitosan, with a degree of esterification substitution of 0.3 to 0.8.

[0028] This layer also contains 5 wt% to 10 wt% triethyl citrate plasticizer.

[0029] Preferably, the outer side of the dual-response enteric coating layer also includes an adaptive delay layer for medication administration;

[0030] The medication time-adaptive delay layer is a sustained-release membrane composed of one or more of the following: zein, gluten, soybean protein alcohol aqueous dispersion, and rice gluten, with a membrane thickness of 10 μm to 30 μm.

[0031] Based on the total weight of the microcapsules, the adaptive delay layer for medication administration accounts for 3 wt% to 5 wt%.

[0032] Preferably, the overall particle size of the microcapsules is 120 μm to 200 μm, and the particle size is expressed as the D50 value measured by a laser diffraction particle size analyzer;

[0033] The IgG-FcRn receptor binding activity of the microcapsules was retained at over 80% after 24 months of storage at 25°C and 60% relative humidity. This retention rate was determined by the surface plasmon resonance competitive binding method.

[0034] A method for preparing bioactive protective microcapsules of immune-enhancing bovine colostrum powder includes the following six steps;

[0035] Step 1: Collect colostrum from cows within 48 hours postpartum. Perform centrifugation defatting, casein removal at pH 4.6 isoelectric point, low-temperature membrane filtration with a pore size of 0.45μm, and low-temperature ultrafiltration concentration with a molecular weight cutoff of 10kDa at 4±1℃. Spray dry the ultrafiltration concentrate at an inlet air temperature of 100℃ to 115℃ and an outlet air temperature of 60℃ to 70℃ to obtain bovine colostrum powder with an IgG content of more than 25% by weight.

[0036] Step 2: Disperse the bovine colostrum powder prepared in Step 1, optional 0.5wt% to 2.0wt% of Pear aggregate lymph node microfold cell directional anchoring agent and Fc segment protective stabilizing layer components in purified water in proportion, filter through 0.22μm sterile filter and place in a vacuum freeze dryer.

[0037] The freeze-drying process is completed by pre-freezing at -40℃ to -45℃ for 4 to 6 hours, first drying at -25℃ to -30℃ for 20 to 28 hours, and second drying at 20℃ to 25℃ for 6 to 10 hours. The powder is then pulverized through an 80-mesh sieve in an environment with a relative humidity of less than 10% to obtain the core-inner stable layer composite powder.

[0038] Step 3: Disperse the composite powder prepared in Step 2 in a 1.0 wt% to 2.0 wt% low molecular weight sodium alginate aqueous solution under stirring at 300 rpm, adjust the pH to 5.3 to 5.7, add a 0.8 wt% to 1.2 wt% deacetylated chitosan acetate aqueous solution, adjust the pH to 4.3 to 4.7, adjust the pH of the system to 4.8 to 5.2 to allow the two polysaccharides to electrostatically recombine, the reaction temperature is 8℃ to 12℃, stir for 50 minutes to 70 minutes, add a 0.04 mol / L to 0.06 mol / L calcium chloride aqueous solution to solidify the alginate network for 25 minutes to 35 minutes, filter, and redissolve the wet particles in an aqueous solution containing 2 wt% to 4 wt% trehalose, and complete the secondary freeze-drying at −40℃ to −50℃ to obtain the three-layer structure intermediate particles;

[0039] Step 4: Place the intermediate microparticles prepared in Step 3 in a fluidized bed with an inlet temperature of 28°C to 32°C, an outlet temperature of 23°C to 27°C, and an atomization pressure of 0.25 MPa to 0.35 MPa. First, coat the intermediate microparticles with a pH-responsive enteric polymer aqueous dispersion until the weight gain is 12% to 15%. Then, coat them a second time with a short-chain fatty acid-responsive esterified modified enteric polymer aqueous dispersion until the weight gain is 5% to 8%. Finally, spray them with a triethyl citrate plasticizer solution and cure them at 30°C for 15 to 25 minutes to obtain a four-layer structure microcapsule.

[0040] Step 5: When it is necessary to prepare microcapsules containing an adaptive delay layer for drug administration, the four-layer microcapsules obtained in Step 4 are coated a third time with a 2.5wt% to 3.5wt% aqueous solution of zein alcohol in a fluidized bed, and the film thickness is controlled at 10μm to 30μm to obtain five-layer microcapsules.

[0041] Step 6: The prepared microcapsules are aged for 48 hours in a clean environment with relative humidity below 15% and temperature below 25℃. Three-stage release tests are performed, and IgG-FcRn receptor binding activity is tested using the surface plasmon resonance competitive binding method. Qualified batches are used as oral immunomodulatory agents.

[0042] Preferably, before step 2, the hygroscopic regulator of the activated molecular sieve is pre-activated by maintaining it at 200°C to 300°C for 4 to 8 hours, and then cooling it to room temperature in a dry nitrogen environment before putting it into use immediately. The hygroscopic capacity is more than 90% of the theoretical hygroscopic capacity of this type of molecular sieve.

[0043] In step 2, the pre-denaturation conditions for the pre-denatured milk matrix protein are as follows: prepare a 5wt% to 10wt% aqueous solution of whey protein isolate or sodium caseinate, and maintain it at 75°C to 85°C for 20 to 30 minutes. The exposed sulfhydryl content of the resulting pre-denatured milk protein is 60% to 80% of the original sulfhydryl content of the protein.

[0044] During the electrostatic recombination reaction in step 3, the zeta potential change of the system is monitored in real time, and the absolute value of the zeta potential during the recombination film formation stage is controlled within 5mV; the termination of the calcium chloride curing reaction in this step is determined by the rate of change of the system conductivity being less than 1μS·cm⁻¹ / min.

[0045] This method is carried out in an environment with a GMP cleanliness level of D or above. All water-based operations use purified water of the injection grade and do not use glutaraldehyde, formaldehyde, organic crosslinking agents, or organic solvents as processing aids.

[0046] The IgG-FcRn receptor binding activity retention rates of the finished products obtained by this method were above 90% and above 80% after 3 months of accelerated testing at 40℃ and 75% relative humidity and 24 months of long-term testing at 25℃ and 60% relative humidity, respectively.

[0047] Application of an immune-enhancing bovine colostrum powder bioactive protective microcapsule in the preparation of an oral immunomodulatory biotherapy for recurrent respiratory infections, chronic inflammatory bowel disease, intestinal barrier dysfunction, immune reconstitution after radiotherapy and chemotherapy for tumors, and clinical immunological diseases related to immunodeficiency in children and the elderly;

[0048] The dosage form of the oral immunomodulatory biological therapy drug is selected from one of the following: granules, capsules, oral suspensions, or orally disintegrating tablets;

[0049] For use in children aged 3 to 12 years with recurrent respiratory infections, the daily dose based on bovine colostrum powder weight is 50 mg / kg body weight to 150 mg / kg body weight, 1 to 3 times daily.

[0050] In applications targeting immunocompromised individuals aged 65 and above, the microcapsules used contain an adaptive time-delay layer for drug administration.

[0051] In the application of adjuvant therapy for immune reconstitution after radiotherapy and chemotherapy for tumors, the interval between the drug and radiotherapy and chemotherapy should be more than 2 hours.

[0052] The technical effects and advantages of this invention are as follows:

[0053] In this invention, a multi-layered microcapsule structure comprising a multi-component active core, an Fc segment protective stabilizing layer, a complex coagulation physical barrier layer, a dual-response enteric coating layer, and an adaptive delay layer for selectable dosing times, can maximize the protection of heat-sensitive bioactive components such as IgG, lactoferrin, and lysozyme during low-temperature powdering, freeze-drying core formation, electrostatic coagulation, and fluidized bed coating. Specifically, the non-reducing sugar vitrification matrix, the competitive occupant of free amino acids, and the activated molecular sieve collectively inhibit Maillard glycosylation induced by lactose and reducing sugars during long-term storage, maintaining the Fc segment FcRn. Receptor binding activity reduces latent failures during shelf life at room temperature and under cross-climate distribution conditions. The complex coagulation physical barrier layer further isolates the internal stable layer from the outer enteric coating material, improving the integrity of the microcapsule structure and storage stability. The dual-response enteric coating layer enables the microcapsules to release at low levels in gastric acid and low short-chain fatty acid environments, while releasing them in a concentrated manner when the intestinal pH rises and the short-chain fatty acid reaches the threshold, thereby improving intestinal targeting and bioavailability. The optional delay layer can also adapt to different medication times such as morning, after meals, before bedtime, and at night, improving the matching of the release window with the rhythm of intestinal mucosal immune activation. Attached Figure Description

[0054] The disclosure of this invention is illustrated with reference to the accompanying drawings. It should be understood that the drawings are for illustrative purposes only and are not intended to limit the scope of protection of this invention. In the drawings, the same reference numerals are used to refer to the same parts:

[0055] Figure 1 The following are SEM images of the present invention: (A) Overall morphology of microcapsules; (B) Surface morphology of single particles; (C) Wrinkled morphology of single particles; (D) Dispersed morphology of low-magnification particles; (E) Surface morphology of coated particles; (F) Edge morphology of coated particles. Detailed Implementation

[0056] It is readily understood that, based on the technical solution of this invention, those skilled in the art can propose various interchangeable structural methods and implementations without altering the essential spirit of the invention. Therefore, the following detailed embodiments and accompanying drawings are merely illustrative examples of the technical solution of this invention and should not be considered as the entirety of the invention or as limitations or restrictions on the technical solution of this invention.

[0057] The present invention will be further described in detail below through several specific embodiments and comparative examples, but the scope of protection of the present invention is not limited to these embodiments.

[0058] To facilitate understanding of the process details of the present invention by those skilled in the art and related researchers, a brief explanation of several prior art principles will be given before describing the specific operation of each embodiment.

[0059] Regarding low-temperature spray drying: Spray drying is a mature technology in the pharmaceutical and food industries for rapidly converting liquid raw materials into powder. Its basic principle is to spray liquid materials through atomizing nozzles to form fine droplets, which evaporate instantly upon contact with hot air entering the tower. The dried powder is then collected by a cyclone separator. The inlet air temperature used in conventional spray drying is usually 140°C to 160°C, but this poses a risk of instantaneous thermal denaturation for heat-sensitive proteins such as IgG. Therefore, this invention adopts a low-temperature modified spray drying process with an inlet air temperature of 100°C to 115°C. After reducing the inlet air temperature, the atomization pressure, feed rate, and residence time in the tower need to be optimized accordingly to ensure that the material moisture content is qualified. Finally, the powder moisture content is controlled below 4%.

[0060] About vacuum freeze-drying: Vacuum freeze-drying (also known as lyophilization) is a drying technology that first freezes the material at a low temperature, and then removes moisture by directly sublimating the ice into water vapor under a low vacuum environment. Its advantage is that the material remains at a low temperature throughout the sublimation drying process, minimizing damage to heat-sensitive active ingredients. It is commonly used in the formulation of biological agents. Freeze-drying typically consists of three stages: pre-freezing, primary drying (sublimation drying), and secondary drying (desorption drying). The temperature, vacuum level, and time for each stage need to be rationally designed based on the eutectic point and glass transition temperature of the material.

[0061] Regarding complex condensation: Complex condensation is a microencapsulation method that mixes two water-soluble polymers with opposite charges (polysaccharide-polysaccharide or polysaccharide-protein) under specific pH and ionic strength conditions, allowing them to combine through electrostatic attraction to form an insoluble polyelectrolyte complex (i.e., complex condensed phase), thereby forming a dense film on the surface of the core material. The advantages of this method are that the reaction takes place in an aqueous phase, no organic solvents are used, the film is dense, and the mechanical strength is high. Furthermore, the film structure can be precisely controlled by adjusting the pH, ionic strength, and polymer concentration ratio. The complex condensation reaction window is sensitive to parameters, and a stable parameter monitoring system needs to be established for industrial scale-up.

[0062] About fluidized bed coating: A fluidized bed (also known as a boiling bed) uses a high-speed airflow blown in from the bottom to suspend solid particles and create a boiling state, so that the particles are fully dispersed and uniformly contacted by the coating droplets sprayed from above, thereby forming a uniform coating layer by layer on the particle surface. The advantages of fluidized bed coating are high coating uniformity, continuous production, and the ability to stack multiple layers of coating. It is the industrial standard equipment for enteric coating and sustained-release coating.

[0063] Example 1

[0064] This embodiment fully replicates the entire preparation process of the four-layer structure microcapsules described in this invention.

[0065] Step 1: Collection of bovine colostrum and preparation of raw material powder

[0066] The raw material used in this embodiment is colostrum from Holstein cows within 36 hours postpartum, with a total collection of 100L. The selection of the time window for the raw material directly affects the IgG content. In colostrum within 24 hours postpartum, the IgG concentration can account for more than 40% of the total protein content. After 24 hours, this proportion drops rapidly to about 4%, and after 48 to 72 hours, it approaches the level of normal milk. Therefore, this invention strictly selects colostrum within 48 hours postpartum (within 36 hours in this embodiment) as the raw material to ensure a naturally high IgG content. After collection, the raw material is immediately refrigerated to below 4°C and sent to the processing workshop to avoid microbial proliferation and raw material degradation.

[0067] The raw bovine colostrum was centrifuged at 10,000 × g for 30 minutes at 4°C using a vertical centrifuge. The milk fat was collected in the upper layer and then removed to obtain skimmed colostrum. The purpose of skimming is twofold:

[0068] Firstly, it prevents the fat from oxidizing and becoming rancid during subsequent processing and microencapsulation storage, thus affecting product quality;

[0069] Secondly, the presence of fat can interfere with the operation of subsequent ultrafiltration membrane modules (easily clogging membrane pores), so it must be removed in the early stages of the process. The degreasing process is strictly controlled at 4±1℃ to avoid the loosening of IgG conformation caused by temperature rise.

[0070] Thirdly, isoelectric point treatment is used to remove casein. Casein is the most abundant protein in cow's milk (accounting for about 80% of the total milk protein). Its isoelectric point is pH 4.6. At this pH value, precipitation occurs because the net surface charge is zero. In this embodiment, dilute hydrochloric acid is used to slowly adjust the pH of the skimmed colostrum to 4.6 (operated at 4°C). After standing for 30 minutes to allow the casein to precipitate completely, the precipitate is removed by centrifugation. The purpose of removing casein is to prepare for subsequent ultrafiltration concentration. If casein is retained in the liquid phase, it will significantly increase the burden on the ultrafiltration membrane and affect the recovery rate of IgG. After isoelectric point treatment to remove casein, the resulting whey phase is rich in whey protein active substances such as IgG, lactoferrin, growth factors, and lysozyme.

[0071] Fourthly, low-temperature membrane filtration and ultrafiltration concentration are employed. The resulting whey phase is first filtered through a 0.45 μm low-temperature membrane (below 4°C) to remove residual casein flocculents and cell debris, yielding a clear whey. This whey is then concentrated to 1 / 5 of its original volume using an ultrafiltration membrane module with a molecular weight cutoff of 10 kDa (below 4°C, with the pressure difference across the membrane controlled below 0.15 MPa to avoid damage to IgG from high-pressure shear). The 10 kDa molecular weight cutoff was chosen based on the following considerations: the molecular weight of IgG is approximately 150 kDa, lactoferrin is approximately 80 kDa, EGF is approximately 6 kDa, and lysozyme is approximately 14 kDa. The 10 kDa molecular weight cutoff can retain all the target active substances while removing non-target components such as small molecule sugars, salts, and water-soluble vitamins, thereby significantly increasing the relative content of the target active substances in the subsequent powder.

[0072] Fifthly, low-temperature spray drying is used for powder production. The ultrafiltration concentrate is fed into a spray drying tower with an inlet air temperature of 110°C, an outlet air temperature of 65°C, and an atomization pressure of 0.20 MPa. The feed rate is calculated based on the tower volume. The inlet air temperature of 110°C is significantly lower than the conventional spray drying temperature of 140°C to 160°C. This parameter selection is one of the key differences between this invention and most existing bovine colostrum powder production processes. IgG begins to undergo thermal denaturation above 60°C. Instantaneous heating at a conventional inlet air temperature of 140°C can lead to a loss of approximately 10% to 15% of IgG activity. This invention uses a low-temperature inlet air temperature of 110°C, which reduces the loss of IgG activity to below 5%. After spray drying, 8.5 kg of bovine colostrum powder is collected by a cyclone separator. The IgG content is determined by ELISA to be 28.6% by weight, which meets the requirement of IgG content of more than 25% as specified in claim 1.

[0073] Step 2: Co-lyophilization of multi-component active core and Fc segment protective stabilizing layer for core fabrication

[0074] The aim is to directly form a protective microenvironment around IgG molecules by integrating the core material and the inner stabilizing layer through freeze-drying. This microenvironment consists of a vitrified matrix of non-reducing sugars, free amino acids, activated molecular sieves, and pre-denatured milk proteins, thus isolating Maillard reaction substrates from the source.

[0075] The first step is component preparation. Take 100g of bovine colostrum powder prepared in step 1, 42g of trehalose as a non-reducing sugar vitrification matrix, 2g of L-lysine hydrochloride as a competitive site-filling agent for free amino acids, 1g of type 3A synthetic molecular sieve as a hygroscopic regulator for activating the molecular sieve, and 8g of pre-denatured whey protein isolate as a pre-denatured milk matrix protein. The type 3A synthetic molecular sieve needs to be pre-activated before use: heat the molecular sieve in a muffle furnace at 250℃ for 6 hours to remove the pre-adsorbed environmental moisture and impurity gases in the molecular sieve channels, so that it has the maximum moisture absorption capacity when put into use. After activation, immediately transfer it to a sealed container filled with dry nitrogen and cool it to room temperature to avoid absorbing environmental moisture and becoming ineffective. The pre-denaturation treatment of whey protein isolate is as follows: prepare an 8% by weight aqueous solution of whey protein isolate, keep it in an 80°C water bath for 25 minutes and then immediately cool it to below 25°C. The purpose is to unfold some of the secondary structure of whey protein and expose an appropriate amount of hydrophobic groups, so that it can better form a compatible matrix with IgG in the subsequent freeze-drying process.

[0076] The second step is ingredient preparation and mixing. Add 153g of the five components to 800mL of purified water and stir at a low speed of 100 rpm at 4℃ until completely dissolved. The stirring time is approximately 30 minutes. The purpose of low-speed stirring is to avoid IgG interface denaturation caused by high-speed shearing.

[0077] The third step is sterile filtration. The obtained solution is filtered through a 0.22μm sterile filter membrane to obtain a sterile solution. The purpose of this step is to avoid microbial contamination. The subsequent freeze-drying process is a low-temperature operation that lasts for more than 30 hours. If microorganisms are present in the solution initially, they may multiply during the short window period before cooling in the pre-freezing stage. The 0.22μm filter membrane can retain most bacteria and is the standard specification for sterile filtration in the pharmaceutical industry.

[0078] Fourth is vacuum freeze drying. The sterilized liquid is divided into freeze drying trays, and the thickness of the liquid is controlled to be less than 10mm to facilitate uniform freeze drying. Then it is placed into a vacuum freeze dryer.

[0079] Operate according to the following three-stage procedure: In the pre-freezing stage, reduce the material temperature from 20℃ to −42℃ at a rate of 0.7℃ / min and maintain it for 5 hours to completely freeze the material into a solid containing ice crystals;

[0080] In the first drying (sublimation drying) stage, the vacuum pump is turned on to reduce the pressure inside the chamber to 20Pa, and the material temperature is maintained at −28℃ for 24 hours, so that the ice crystals directly sublimate into water vapor in a low vacuum environment and are captured by the cold trap.

[0081] In the secondary drying (desorption drying) stage, the material temperature is slowly raised to 23°C, and the pressure inside the chamber is further reduced to 10Pa and maintained for 8 hours to remove residual adsorbed water from the material. After freeze drying, the moisture content of the material should be controlled below 2%, presenting as a fluffy and porous sponge-like cake.

[0082] The temperature and time parameters of the three-stage process are set reasonably based on the eutectic point (approximately -35°C) and glass transition temperature (approximately -25°C) of the material. Pre-freezing must be done below the eutectic point to ensure complete freezing. The first drying temperature must be lower than the glass transition temperature to avoid collapse. The second drying temperature can be appropriately increased to accelerate desorption.

[0083] The fifth step is dry powder processing. Immediately after freeze-drying, the sponge-like cake is transferred to a drying room with a relative humidity of less than 10% for pulverization (using a turbine pulverizer). It is then passed through an 80-mesh sieve (approximately 180 μm in diameter) to obtain powder with uniform particle size. The resulting powder is 145g of core-inner stable layer composite powder, which is white to slightly yellow fine granular powder with a moisture content of approximately 1.5%.

[0084] Step 3: Wet embedding of the complex coagulated physical barrier layer

[0085] This step involves forming an intermediate film layer on the surface of the composite powder prepared in step 2 through a classic polysaccharide-polysaccharide electrostatic recombination reaction. Its function is to completely separate the enteric layer to be coated in step 4 from the stable inner layer formed in step 2 in space, thereby preventing the migration of L1 layer components to L3 layer during long-term storage and the resulting interfacial interactions.

[0086] The first step involves the preparation of a negatively charged polysaccharide solution. 7.5 g of sodium alginate, after undergoing low molecular weight treatment, was added to 500 mL of purified water and stirred at 4°C for 4 hours until completely dissolved, yielding a 1.5 wt% sodium alginate aqueous solution. The pH of this solution was adjusted to 5.5 using dilute hydrochloric acid. The selection of low molecular weight sodium alginate was based on the following considerations: Conventional sodium alginate has a weight-average molecular weight between 200 and 500 kDa, resulting in excessively high viscosity in its aqueous solution, which is detrimental to uniform coating and causes significant shear stress on the core material. The viscosity of sodium alginate after low molecular weight treatment is significantly reduced, and its Ca2+ content is also lower. 2+ Cross-linked networks can still form well.

[0087] The second step is the preparation of a positively charged polysaccharide solution. Take 4g of deacetylated chitosan with a degree of deacetylation of 90%, add purified water containing 1.5wt% glacial acetic acid to 400mL, stir at room temperature for 2 hours until completely dissolved, to obtain a 1.0wt% deacetylated chitosan acetic acid aqueous solution; adjust the pH to 4.5 with dilute sodium hydroxide.

[0088] The third step involves the dispersion and re-coagulation reaction of the core material. 100g of the composite powder prepared in step 2 is dispersed in the above-mentioned sodium alginate aqueous solution under stirring at 300 rpm. The dispersion is carried out at 4°C for 10 minutes to ensure that the composite powder particles are uniformly wetted and coated by the sodium alginate solution. Then, the deacetylated chitosan solution is slowly added dropwise to the system at a rate of 5 mL / min. After the addition is completed, the pH of the system is finely adjusted to 5.0 with dilute hydrochloric acid or dilute sodium hydroxide. This pH value is the window pH value for the optimal electrostatic re-coagulation reaction between sodium alginate (pKa of about 3.5, which is completely ionized negatively charged at pH 5.0) and chitosan (which is completely ionized positively charged at pH 5.0). The re-coagulation reaction temperature is strictly controlled at 10°C and the reaction time is 60 minutes. During this period, stirring is continued at 300 rpm to ensure that the re-coagulated film layer is uniformly deposited on the surface of the composite powder particles.

[0089] Fourth is Ca 2+ After the solidification and re-coagulation reactions are complete, slowly add 100 mL of 0.05 mol / L calcium chloride aqueous solution and continue stirring for 30 minutes. The role of calcium chloride is to react the carboxylate ions of sodium alginate with Ca2+. 2+ An ionic crosslinking reaction occurs, solidifying the complexed membrane into a wet gel-like film with higher mechanical strength. A reaction time of 30 minutes is sufficient for the solidification reaction to proceed completely. The endpoint can be determined by monitoring the change in the system's conductivity. When the rate of change in conductivity is less than 1 μS·cm... -1 Curing is considered complete when the curing time reaches 1 / minute.

[0090] The fifth step is separation and secondary freeze-drying. After solidification, the particles are filtered through a sintered metal filter plate with a pore size of 10 μm. The resulting wet particles are then quickly rinsed once with purified water (to remove residual Ca²⁺ and unreacted polysaccharides from the surface), and immediately re-dissolved in 200 mL of an aqueous solution containing 3% wt% trehalose (trehalose serves as an outer freeze-drying protectant). The resulting suspension is dispensed into freeze-drying pans, and the secondary freeze-drying is completed according to the following procedure: pre-freezing at −45℃ for 4 hours, primary drying at −30℃ / 15Pa for 20 hours, and secondary drying at 25℃ / 8Pa for 8 hours. The intermediate particles obtained after freeze-drying are white to light yellow fine particles with a particle size of approximately 80 to 120 μm and a mass of approximately 128 g.

[0091] Step 4: Fluidized Bed Dual-Material Gradient Coating with Dual-Response Enteric Coating

[0092] This step involves introducing pH-responsive components and short-chain fatty acid-responsive components through a fluidized bed secondary coating process, thereby constructing the unique dual-responsive enteric coating layer of this invention.

[0093] The first step is material preparation and equipment debugging before coating. Take 100g of the intermediate microparticles prepared in step 3 and load them into a fluidized bed. The process parameters of the fluidized bed are pre-adjusted to the following state: inlet temperature 30℃, outlet temperature 25℃, atomization pressure 0.30MPa, spray nozzle located above the fluidized chamber, and air inlet speed adjusted to ensure that the intermediate microparticles are fully suspended in the fluidized chamber but not carried to the exhaust port. The set temperature parameters are significantly lower than those of conventional enteric coating in order to avoid affecting the activity of heat-sensitive IgG, but the coating time needs to be extended accordingly.

[0094] The second step is the primary coating of the pH-responsive component. An appropriate amount of acrylic resin III is taken and diluted with purified water to a solid content of 20% by weight as the primary coating solution. The coating solution is delivered to the fluidized bed spray nozzle at a rate of 4 g / min using a peristaltic pump. The weight increase of the intermediate microparticles is monitored simultaneously. Spraying is stopped when the weight increase reaches 13.5%, and fluidization is maintained for 30 minutes to allow the coating layer to fully solidify. The purpose of primary coating is to give the microcapsules the responsiveness to the pH value of the medium. Acrylic resin III dissolves at a pH value higher than 6.0. Therefore, the microcapsules after primary coating have the preliminary functions of gastric acid protection and enteric release.

[0095] The third step is the secondary coating of short-chain fatty acid responsive components. An appropriate amount of butyrate-esterified hydroxypropyl methylcellulose (SCFA) is prepared into an aqueous dispersion with a solid content of 15% by weight using an ethanol-water mixture. The secondary coating solution is continuously delivered to the fluidized bed spray nozzle at a rate of 3 g / min using a peristaltic pump, while monitoring the weight increase. Spraying is stopped when the weight increase reaches 6.5%, and fluidization is maintained for another 30 minutes to allow the secondary coating layer to fully solidify. The purpose of the secondary coating is to give the microcapsules additional responsiveness to the concentration of short-chain fatty acids. When the esterification bonds are hydrolyzed under the action of enzymes or microbial metabolism, the original hydroxypropyl methylcellulose phthalate is released, causing the entire coating layer to further disintegrate. Since the hydrolysis rate of the butyrate esterification bonds is related to the butyric acid concentration in the medium, this layer forms a threshold response to the SCFA concentration.

[0096] The fourth step is plasticizer treatment and curing. After spraying with triethyl citrate aqueous solution, the temperature is adjusted to 30℃ and maintained for 20 minutes, so that the polymer chains in the coating layer are rearranged under the action of plasticizer to form a dense and uniform continuous film structure. After curing, fluidization is stopped, and 122g of the prepared four-layer structure microcapsules are taken out. The overall particle size distribution is 145μm to 175μm, and they are white to light yellow fine particles.

[0097] Step 6: Finished product drying and release

[0098] The microcapsules prepared in step 4 were placed in a cleanroom with a cleanliness level of D, a relative humidity of 12%, and a temperature of 22°C for another 48 hours to complete the curing process, and three-stage release tests were performed:

[0099] The cumulative release rate of the first phase was measured at 3.2% after 2 hours in simulated gastric fluid at pH 1.2 (required to be less than 5%).

[0100] The second phase, measured under simulated intestinal fluid conditions at pH 6.8 with 5 mmol / L short-chain fatty acids, showed a cumulative release rate of 12.5% ​​over 30 minutes (required to be less than 20%).

[0101] The third stage, under simulated intestinal fluid at pH 6.8 and 10 to 15 mmol / L short-chain fatty acids, showed a cumulative release rate of 86.3% after 30 minutes (required to be greater than 80%). The IgG-FcRn receptor binding activity, determined by the surface plasmon resonance competitive binding method, was 97.5% immediately after preparation.

[0102] Example 2: Five-layer microcapsules containing an adaptive delay layer for medication administration

[0103] Based on Example 1, this embodiment adds an adaptive delay layer for medication timing to the outermost layer. The resulting microcapsules are suitable for people with significant individual differences in medication timing (such as elderly patients with chronic diseases, night shift workers, etc.).

[0104] Steps 1 to 4 are exactly the same as in Example 1.

[0105] Step 5: Secondary suspension coating of the medication administration time-adaptive delay layer

[0106] This step introduces a sustained-release membrane layer composed of protein excipients through a third fluidized bed coating. The working principle of this layer is based on the difference in dissolution kinetics of protein excipients under different ambient temperature gradients: When taking the medication during the day, the temperature difference between body temperature (37℃) and ambient temperature (20 to 25℃) is large, and the hydration rate of the membrane layer from the outside to the inside is faster, resulting in relatively rapid dissolution. When taking the medication at night, due to reduced activity, decreased heat dissipation from the skin, and a higher room temperature, coupled with a significantly reduced gastric emptying rate at night, the microcapsules remain in the stomach for a longer period, and the hydration and dissolution time of the membrane layer is delayed. Ultimately, the IgG release period is automatically postponed from nighttime to the GALT activation window period of the following day.

[0107] The first step is the preparation of the coating solution. Take 3g of zein and add it to 97mL of 70% ethanol aqueous solution. Stir at room temperature for 1 hour until completely dissolved to obtain a 3.0% by weight zein alcohol-water solution. The choice of zein is based on its unique hydrophobic-hydrophilic amphoteric structure: zein is rich in hydrophobic amino acids (leucine, alanine, proline), which can only be dissolved in alcohol-water mixtures but not in pure water. Therefore, the dissolution of its membrane in aqueous intestinal fluid requires a process of swelling in the alcohol phase followed by disintegration in the aqueous phase, forming a natural sustained-release behavior.

[0108] The second step is the third fluidized bed coating. 100g of the microcapsules obtained in step 4 of Example 1 are placed in a fluidized bed. The inlet temperature is adjusted to 30°C, the outlet temperature to 25°C, and the atomization pressure to 0.25MPa. The above-mentioned zein alcohol-water solution is sprayed at a rate of 2g / min using a peristaltic pump. Spraying is stopped when the weight increases to 4%. Fluidization is maintained for 40 minutes to allow the ethanol in the membrane to fully evaporate and the zein to solidify and form a continuous and dense membrane structure. The key control parameter in this step is the coating temperature. If the temperature is too high, the ethanol will evaporate too quickly, resulting in discontinuity of the membrane and cracks. If the temperature is too low, the ethanol will not evaporate completely, resulting in membrane adhesion.

[0109] The third step is curing and quality release. The obtained microcapsules are cured in a clean room for another 48 hours before the final quality release test is performed. The obtained five-layer structure microcapsules weigh 104g, with an overall particle size distribution of 165μm to 195μm, and are white to light yellow fine particles.

[0110] Example 3: Application in children with recurrent respiratory infections

[0111] Using the four-layer microcapsules obtained in Example 1 as the active ingredient, an oral hard capsule dosage form was prepared.

[0112] Excipient formulation and mixing: Microcapsules 50: microcrystalline cellulose 30: lactose 15: croscarmellose sodium 4: magnesium stearate 1 by weight. Microcrystalline cellulose acts as a filler to provide volume and flowability of the capsule contents. Lactose acts as a flow aid and diluent to improve the compressibility of the contents. Croscarmellose sodium acts as a disintegrant to ensure rapid disintegration of the capsules in the stomach and release of the microcapsules. This rapid disintegration only releases the microcapsule body; the microcapsules themselves are still protected by their four-layer structure and do not release IgG. Magnesium stearate acts as a lubricant to prevent material adhesion during the capsule filling process. The mixture is then mixed in a double cone mixer at 30 rpm for 15 minutes to ensure uniform material distribution.

[0113] Capsule filling: The obtained mixture is filled into No. 0 hard capsule shells using a fully automatic capsule filling machine. Each capsule contains 0.5g of the mixture, corresponding to 250mg of microcapsules and approximately 140mg of bovine colostrum powder. Pharmaceutical gelatin or plant-derived HPMC capsule shells can be used for the capsule shells.

[0114] The clinical application protocol is shown in Table 1: For children aged 3 to 12 years with recurrent respiratory infections, the daily dosage is 100 mg / kg body weight based on the weight of bovine colostrum powder, twice daily, after breakfast and after dinner, for 12 consecutive weeks. A randomized controlled trial of 120 cases showed that the experimental group (80 cases taking the product of this invention) experienced a 42.3% decrease in the number of respiratory infections within 6 months compared to pre-admission levels, while the control group (40 cases taking commercially available similar products) saw a 12.6% decrease. Serum IgG levels in the experimental group increased by 27.8% compared to pre-admission levels, while those in the control group increased by 9.4%. These differences in clinical indicators demonstrate that this invention has superior efficacy to existing technologies for this indication.

[0115]

[0116] Table 1

[0117] Example 4: Application in immunocompromised individuals aged 65 and above

[0118] Using the five-layer structure microcapsules containing an adaptive delay layer for medication administration obtained in Example 2 as the active ingredient, an orally disintegrating tablet form was prepared.

[0119] Excipient formulation and mixing: The excipients are mixed in the following weight ratios: 60 microcapsules, 20 mannitol, 10 microcrystalline cellulose, 5 crospovidone, 2 aspartame, 2 peppermint flavoring, and 1 magnesium stearate. Mannitol, as a signature excipient of orally disintegrating tablets, provides a good taste and rapid disintegration performance. Microcrystalline cellulose improves compressibility. Crospovidone, as a super disintegrant, ensures complete disintegration of the tablet within 60 seconds of ingestion. Aspartame and peppermint flavoring are used to improve taste and enhance compliance in elderly patients. Magnesium stearate is used as a lubricant. After mixing, the tablets are directly compressed to form 0.5g / tablet orally disintegrating tablets. Each tablet contains 300mg of microcapsules and approximately 160mg of bovine colostrum powder.

[0120] The clinical application protocol is shown in Table 2: For immunocompromised elderly individuals aged 65 and above, medication can be taken at any time of day according to their own lifestyle habits (including fasting in the morning, after meals, before bedtime, and nighttime administration). The daily dosage is 80 mg / kg body weight based on the weight of bovine colostrum powder, once daily for 16 consecutive weeks. A randomized controlled trial of 90 cases was conducted: In the experimental group (60 cases taking the product of this invention), the serum total IgG level increased by 26.5% compared to before enrollment, regardless of the medication time. In the control group (30 cases taking commercially available similar products), the level increased by 7.8%, and the medication time had a significant impact on efficacy. The morning administration group showed an 11.2% increase, while the nighttime administration group only showed a 4.5% increase. The consistent difference in efficacy between the experimental and control groups at different medication times strongly demonstrates the practical application value of the adaptive time-delay layer of this invention in the elderly population.

[0121]

[0122] Table 2

[0123] Example 5: Extended Example of Integrated Protection of Multi-Component Active Ingredients

[0124] To verify the ability of this invention to integrate and protect multi-component animal-derived active ingredients, this embodiment adds exogenous lactoferrin and exogenous lysozyme in step 2 of embodiment 1, while the remaining steps are the same as in embodiment 1.

[0125] Specific operation: In the ingredient preparation and mixing dissolution step 2, in addition to the original 100g bovine colostrum powder, 42g trehalose, 2g L-lysine hydrochloride, 1g 3A molecular sieve, and 8g pre-denatured whey protein isolate, 3g exogenous lactoferrin (3% by weight of bovine colostrum powder) and 1g exogenous lysozyme (1% by weight of bovine colostrum powder) are added. The addition of exogenous lactoferrin and exogenous lysozyme not only expands the active component spectrum of the microcapsules (in addition to the active substances contained in bovine colostrum itself, it further strengthens the two types of functional proteins, iron-binding glycoprotein and antimicrobial enzyme protein), but also puts higher requirements on the multi-component compatibility of the Fc segment protective stabilizing layer of the present invention. Subsequent steps 3 to 6 are completely the same as in Example 1.

[0126] The multi-component activity assay data of the prepared microcapsule products are shown in Table 3: the immediate activity retention rates after preparation were 96.5% for IgG, 95.8% for lactoferrin, and 97.2% for lysozyme; after 24 months of storage at 25℃ / RH60%, the activity retention rates were 82.4% for IgG, 85.6% for lactoferrin, and 88.2% for lysozyme. The long-term retention rates of the three components were all above 80%, proving that the present invention is effective for multi-component animal-derived active ingredients, and no decrease in the stability of any component was observed due to the increase in the number of components.

[0127]

[0128] Table 3

[0129] Example 6: Critical Effect Experiment of Non-Reducing Sugar to Reducing Sugar Mass Ratio

[0130] To verify the critical threshold that the mass of the non-reducing sugar vitrified matrix is ​​more than 12 times the sum of the mass of endogenous lactose from bovine colostrum powder and the total mass of reducing sugars in all other pharmaceutical excipients of the microcapsule, all other conditions in Example 1 were kept unchanged, except for the amount of trehalose added to the Fc segment protective stabilizing layer, so that the mass ratio of non-reducing sugar to reducing sugar was 3, 5, 8, 10, 11, 12, 13, 15, 18, and 20, respectively, and corresponding microcapsule samples were prepared for storage stability testing.

[0131] Table 4. Key performance data of microcapsules at different non-reducing sugar / reducing sugar mass ratios (FcRn binding activity retention rate, %)

[0132]

[0133] Table 4

[0134] As shown in Table 4, the FcRn binding activity retention rate exhibited a significant non-linear jump in the range of mass ratio increasing from 11 to 12. Taking the data from the 3-month accelerated test at 40℃ / 75% RH as an example: it jumped from 78.4% for sample 11 to 91.6% for sample 12, a single-step increase of 13.2 percentage points. However, after the ratio exceeded 12, it continued to increase slowly and linearly by only 1.9 percentage points (91.6% to 93.5%) across the eight ratio gradients from sample 12 to sample 20. The data from 12 seasonal cycles showed a similar pattern: 60.4% for sample 11, jumping to 76.8% for sample 12, a single-step increase of 16.4 percentage points; and only 2.4 percentage points across the eight ratio gradients from sample 12 to sample 20.

[0135] The mechanism of the above-mentioned critical effect is as follows: when the total amount of non-reducing sugar vitrified matrix reaches a certain threshold, the vitrified amorphous matrix formed around the IgG molecule can just achieve complete physical isolation, preventing endogenous lactose and external reducing sugar excipients from penetrating to the IgG lysine site. This threshold is represented by a mass ratio of 12 in experimental data. When the mass ratio is below 12, the vitrified matrix does not completely cover the IgG (there is an exposed window), and reducing sugar can still undergo the Maillard reaction with IgG. When the mass ratio reaches 12 or above, the vitrified matrix forms a complete coverage, and the Maillard reaction is effectively inhibited.

[0136] To further support the limitation that the hygroscopic effect of the activated molecular sieve hygroscopic regulator keeps Aw in the range of 0.20 to 0.30, an additional test was conducted on the retention rate of FcRn binding activity of IgG samples after storage for 3 months under different Aw and temperature conditions.

[0137] Table 5 shows the retention rate (%) of FcRn binding activity of IgG samples after 3 months of storage under different Aw and temperature conditions.

[0138]

[0139] Table 5

[0140] Table 5 shows the following patterns: When Aw is below 0.20, the IgG conformation undergoes slight denaturation due to the loss of the hydration layer, and the FcRn binding activity decreases. When Aw is in the range of 0.20 to 0.30, the IgG conformation is stable and the Maillard reaction rate is significantly inhibited, with the optimal retention rate of FcRn binding activity. When Aw is above 0.30, the increased mobility of water molecules significantly accelerates the Maillard reaction rate, and the FcRn binding activity decreases significantly. This data clearly demonstrates that Aw of 0.20 to 0.30 is the Maillard reaction inhibition window.

[0141] Example 7: Application in patients with chronic inflammatory bowel disease

[0142] Using the five-layer structure microcapsules containing an adaptive delay layer for medication administration obtained in Example 2 as the active raw material, an enteric-coated granule dosage form was prepared.

[0143] Excipient formulation and mixing: Microcapsules 50g: microcrystalline cellulose 25g: lactose powder 15g: croscarmellose sodium 5g: aspartame 2g: orange flavor 2g: magnesium stearate 1g by weight. After mixing, granulate using a high-speed stirring granulator, and package into double-layered aluminum foil bags, each containing 500mg of microcapsules and approximately 265mg of bovine colostrum powder. Compared to hard capsules and orally disintegrating tablets, granules offer higher precision in release point, making them more suitable for chronic inflammatory bowel disease, an indication requiring high dosage form release precision.

[0144] The clinical application protocol is shown in Table 6: For adults with chronic inflammatory bowel disease (including those in remission of mild to moderate ulcerative colitis and those in stable Crohn's disease), the daily dose is 60 mg / kg body weight based on the weight of bovine colostrum powder, twice daily (after breakfast and dinner), taken with warm water, for 12 consecutive weeks. A randomized controlled trial of 60 cases showed the following: In the experimental group (40 cases taking the product of this invention), the level of the inflammatory factor IL-6 decreased by 36.8% compared to before enrollment, CRP decreased by 33.5%, and the Mayo score of the intestinal mucosa decreased by 1.8 points (out of 4). In the control group (20 cases taking commercially available similar products), IL-6 decreased by 11.2%, CRP decreased by 8.7%, and the Mayo score decreased by 0.5 points. Clinical indicators show that this invention has significant anti-inflammatory and mucosal repair effects in this indication.

[0145]

[0146] Table 6

[0147] Example 8: Application in individuals with intestinal barrier dysfunction

[0148] Using the four-layer microcapsules obtained in Example 1 as the active raw material, a dry suspension granule dosage form for oral suspension was prepared.

[0149] Excipient formulation and mixing: Microcapsules 50: lactose powder 25: xanthan gum 8: sodium carboxymethyl cellulose 5: aspartame 2: sorbitol 5: strawberry flavor 4: magnesium stearate 1 by weight. Xanthan gum and sodium carboxymethyl cellulose act as suspending agents to ensure uniform dispersion of microcapsules in water. Sorbitol acts as a sweetener and humectant to improve taste and stability. After mixing, the mixture is packaged into composite material bags, each containing 2.0g of microcapsules and approximately 560mg of bovine colostrum powder. When taking, add 50mL of warm water, shake to form a suspension, and take immediately.

[0150] The clinical application protocol is shown in Table 7: For individuals with intestinal barrier dysfunction (including those with stress-induced intestinal barrier damage, chronic diarrhea-related barrier function decline, and those requiring perioperative intestinal barrier protection), the daily dose is 70 mg / kg body weight based on bovine colostrum powder, twice daily, 30 minutes before breakfast and 30 minutes before dinner, for 8 consecutive weeks. Clinical data from 50 cases were observed: In the experimental group, serum zonulin levels decreased by 31.6% compared to pre-enrollment levels, the lactulose / mannitol ratio (an intestinal permeability indicator) decreased by 42.5%, diamine oxidase (DAO) levels decreased by 27.8%, and the subjective abdominal distension symptom score decreased by 45.2%. All these indicators collectively demonstrate the significant effect of this invention on intestinal barrier function repair. In particular, the significant decrease in zonulin and the lactulose / mannitol ratio directly reflects the repair of intestinal tight junction protein function and the improvement of intestinal permeability.

[0151]

[0152] Table 7

[0153] Example 9: Application of adjuvant therapy for immune reconstitution after tumor radiotherapy and chemotherapy

[0154] Using the four-layer microcapsules obtained in Example 1 as the active ingredient, enteric-coated hard capsules were prepared.

[0155] Excipient formulation and mixing: Microcapsules 55: microcrystalline cellulose 25: lactose 15: cross-linked sodium carboxymethyl cellulose 4: magnesium stearate 1 by weight. After mixing, the mixture is filled into No. 1 enteric-coated hard capsule shells using a fully automatic capsule filling machine. Each capsule contains 0.6g of the mixture, corresponding to 300mg of microcapsules and approximately 168mg of bovine colostrum powder. The enteric-coated hard capsule shell used in this embodiment further enhances the protection of IgG activity (i.e., in addition to the enteric coating layer of the microcapsules, the capsule shell itself also has enteric properties), forming a dual enteric protection mechanism, which is particularly suitable for tumor patients with abnormal gastric acid secretion.

[0156] The clinical application protocol is shown in Table 8: For patients undergoing adjuvant therapy for immune reconstitution after radiotherapy and chemotherapy (solid tumor patients should start taking the medication within 4 weeks after radiotherapy and chemotherapy, including patients with common solid tumors such as breast cancer, colon cancer, lung cancer, and cervical cancer), the daily dose is 60 mg / kg body weight based on bovine colostrum powder, twice daily. The timing of administration is as follows: the interval between administration and radiotherapy / chemotherapy drugs should be more than 2 hours (to avoid interactions caused by simultaneous presence of chemotherapy drugs in the gastrointestinal tract). This treatment is continued for 16 weeks. Clinical data from 40 cases were observed.

[0157] The percentage of CD3+ / CD4+ T cells in the experimental group increased by 22.4% compared to before enrollment;

[0158] The CD4+ / CD8+ T cell ratio has returned to near the normal reference range.

[0159] Natural killer (NK) cell activity increased by 26.8%;

[0160] The incidence of treatment-related infections decreased by 38.5% compared to the historical control group;

[0161] Patient treatment adherence scores improved by 31.5%;

[0162]

[0163] Table 8

[0164] Various immunological indicators jointly demonstrate the significant adjuvant therapeutic effect of this invention on immune reconstitution after tumor radiotherapy and chemotherapy. In particular, the recovery of CD3+ / CD4+ T cell and NK cell activity directly reflects the restoration of cellular immune function.

[0165] Detailed data on the release behavior of dual-response enteric coatings under different pH and SCFA concentration combinations.

[0166] To further support the limitation that the dual-response enteric coating only dissolves and releases under conditions where the medium pH is not lower than 6.5 and the short-chain fatty acid concentration is not lower than 10 mmol / L, the four-layer microcapsules obtained in Example 1 were used as samples to conduct release tests under different combinations of pH and SCFA concentrations.

[0167] Table 9. Cumulative release rate (%) of dual-response enteric coating at different pH and SCFA concentrations over 30 minutes.

[0168]

[0169] Table 9

[0170] As shown in Table 9, the dual-response enteric coating of this invention maintains a cumulative release rate of less than 60% over 30 minutes under pH conditions ranging from 1.2 (artificial gastric juice) to 6.0, regardless of SCFA concentration. Particularly at pH 1.2, even with an SCFA concentration as high as 15 mmol / L, the cumulative release rate is only 4.8%, demonstrating that pH is a hard constraint in the dual response. At pH 6.5 and above, SCFA concentration significantly affects the release behavior: when SCFA concentration is below 10 mmol / L, the cumulative release rate over 30 minutes is only 42.5% at most; when SCFA concentration reaches 10 mmol / L or above, the cumulative release rate rapidly jumps to over 82.5%, demonstrating that the SCFA concentration threshold response is another soft constraint in the dual response. This data clearly shows that effective release of the microcapsules only occurs when both pH ≥ 6.5 and SCFA ≥ 10 mmol / L are simultaneously met.

[0171] GALT activation window alignment data at different medication times

[0172] To support the requirement that the proportion of IgG released within the Peyer's Spatch immune activation window in the ileum to the total drug-loaded dose of the microcapsules be more than 70% when the drug is taken at different times, a SHIME artificial gastrointestinal biomimetic model modified with Peyer's Spatch organoids was used to compare the five-layer structure microcapsules (including the drug administration time adaptive delay layer) obtained in Example 2 with the prior art microcapsules prepared in Comparative Example 1.

[0173] Table 10 Comparison of GALT activation window alignment (%) between the microcapsules of the present invention (Example 2) and Comparative Example 1 at different medication periods

[0174]

[0175] Table 10

[0176] As shown in Table 10, the release rate of the microcapsules of this invention within the GALT activation window was consistently above 70% during all eight typical medication administration periods, reaching a maximum of 85.2% and a minimum of 71.8% (under the condition of morning medication administration in the elderly). The overall performance was uniform and stable. In contrast, the data for Comparative Example 1 showed dramatic fluctuations. The alignment between the morning (07:00) fasting and post-breakfast conditions was acceptable (38% to 42%), while the alignment under the morning, bedtime, and nighttime medication administration conditions in the elderly was extremely poor (only 8.6% to 18.5%). This fluctuation revealed the significant differences in performance compared to Comparative Example 1. The existing technology represented by this invention is extremely sensitive to differences in the timing of medication administration, and the timing of medication administration for clinical patients is often difficult to strictly control. The medication administration stability of the microcapsules of this invention comes from the synergistic effect of the dual-response enteric layer (L3) and the medication administration time adaptive delay layer (L4). The L3 layer ensures that the release is precisely locked in the Peyer's spatch enrichment area, and the L4 layer utilizes the differences in dissolution kinetics of protein-like sustained-release membranes under different temperature gradients to adaptively delay the release time, so that the IgG release time under nighttime medication conditions is automatically postponed to the GALT activation window period of the next day.

[0177] Time evolution data of FcRn binding activity retention rate under long-term storage conditions

[0178] To support the requirement that the IgG-FcRn binding activity retention rates were above 90% and above 80% after 3 months of accelerated testing at 40℃ / 75% and long-term testing at 25℃ / 60% for 24 months, respectively, the four-layer microcapsules obtained in Example 1 were subjected to system storage stability testing.

[0179] Table 11 shows the time evolution (%) of FcRn binding activity retention rate of the microcapsules prepared in Example 1 under different storage conditions.

[0180]

[0181] Table 11

[0182] As shown in Table 11, the microcapsules prepared in this invention retain 82.5% of FcRn binding activity under long-term test conditions of 25℃ / RH60% for 24 months, which meets the requirement of a retention rate of more than 80% after long-term test. Under accelerated test conditions of 40℃ / RH75% for 3 months, the retention rate is 91.6%, which meets the requirement of a retention rate of more than 90% after accelerated test.

[0183] Comparative Example 1: Reproduction and Implementation of Existing Technology

[0184] To objectively compare the performance advantages of the present invention with those of the closest prior art, a comparative replication was performed according to the existing technology process path, using the same batch of bovine colostrum as in Example 1 as the raw material.

[0185] Specific procedures: Take the same batch of bovine colostrum as in Example 1, and perform Bifidobacterium fermentation and immunization treatment on the bovine colostrum according to the method (inoculate Bifidobacterium at a 5% inoculum amount into the defatted immunized bovine colostrum sterilized by ultra-high pressure, and culture at 37°C for 18 hours). Add freeze-drying protectant (8% skim milk powder + 5% sucrose + 2% monosodium glutamate) and then perform vacuum freeze-drying to obtain composite freeze-dried powder. The core (microcrystalline cellulose ball core) and the composite freeze-dried powder are granulated into particles by air suspension granulation at a mass ratio of 1:8. The particles are then coated with 30% enteric-coated Opadry coating solution in an air suspension granulator (coating weight gain of 15%) to obtain microcapsules of Comparative Example 1, with an overall particle size of approximately 200 to 240 μm.

[0186] Comparative Example 2: Reproduction and Implementation of Existing Technology

[0187] Following the existing technical process, the same batch of bovine colostrum as in Example 1 was used as the raw material.

[0188] Specific operation: Take 100g of bovine colostrum powder from the same batch as in Example 1, mix 30g of trehalose and 20g of casein in 500mL of purified water, and stir until completely dissolved; spray dry the resulting liquid at an inlet air temperature of 130℃ and an outlet air temperature of 70℃ to obtain the powder described in Comparative Example 2 (without subsequent coating treatment), with an overall particle size of about 3 to 5μm.

[0189] Comparative Example 3: Reproduction and Implementation of Existing Technology

[0190] According to existing food science processes, purified IgG extracted from the same batch of bovine colostrum as in Example 1 was used as the core material.

[0191] Specific operation: Mix maltodextrin and gum arabic at a mass ratio of 1:2 to prepare a 5% by weight aqueous solution, add purified IgG (add at 5% by weight of wall material), add soybean oil at an oil-water ratio of 3:4 and perform high-speed shear emulsification (10,000 rpm, 10 minutes), spray dry the emulsion at an inlet air temperature of 140℃ and an outlet air temperature of 80℃ to obtain the IgG microcapsules described in Comparative Example 3, with an overall particle size of about 1 to 5 μm.

[0192] Comparison of overall performance between Comparative Examples 1 to 3 and Examples 1 and 2

[0193] Table 12 compares the overall performance of the present invention with that of three prior art technologies.

[0194] Microcapsule basic parameters Overall particle size D50 (μm) 160 180 220 4 3 number of microcapsule layers 4 5 2 0 1 Whether to use organic crosslinking agents no no no no no Activity indicators Total IgG activity retention rate (after preparation) 96.8% 96.5% 88.5% 91.2% 81.2% FcRn binding activity (after preparation) 97.5% 97.8% 92.4% 94.1% 89.5% FcRn binding activity (accelerated in 3 months at 40℃ / RH75%) 91.6% 92.3% 54.8% 62.5% 51.3% FcRn binding activity (25℃ / RH60% long-term for 24 months) 82.5% 84.2% 47.6% 56.8% 44.2% FcRn binding activity (after 12 seasonal cycles) 76.8% 79.5% 28.5% 38.2% 25.6% Release rate pH 1.2 artificial gastric fluid 2-hour cumulative release rate 3.2% 2.8% 8.5% Fully released 65.4% Cumulative release rate at pH 6.8 + SCFA 5mM for 30 min 12.5% 11.8% 86.5% Fully released Fully released Cumulative release rate at pH 6.8 + SCFA 10mM for 30 min 86.3% 88.5% 92.6% — — GALT activates window alignment GALT alignment when taken on an empty stomach at 7:00 AM 76.5% 78.2% 38.4% — — GALT was taken at 13:00 after lunch to ensure alignment. 85.2% 87.5% 50.3% — — GALT alignment when taken 22 hours before bedtime 73.2% 76.8% 14.5% — — Clinical efficacy (recurrent respiratory infections in children) The rate of decrease in the number of infection outbreaks within 6 months 42.3% 43.6% 12.6% 8.4% 10.2%

[0195] Table 12

[0196] As can be seen from the comprehensive data in Table 12, the microcapsules prepared in this invention, compared with Example 2, showed significant and unexpected synergistic effects in all test dimensions of Comparative Examples 1, 2, and 3. In particular, in terms of long-term retention of FcRn binding activity, the 82.5% of this invention was much higher than that of Comparative Example 1 (47.6%, a difference of 34.9 percentage points), Comparative Example 2 (56.8%, a difference of 25.7 percentage points), and Comparative Example 3 (44.2%, a difference of 38.3 percentage points). In terms of GALT alignment when taking the medication at 22 hours before bedtime, the 73.2% of this invention was much higher than that of Comparative Example 1 (14.5%, a difference of 58.7 percentage points). In terms of clinical efficacy, the 42.3% reduction rate of infection incidence in children of this invention was much higher than that of the 8.4% to 12.6% of the comparative examples. None of the above data can be obtained by simply adding up the data of any comparative example.

[0197] The technical scope of this invention is not limited to the content described above. Those skilled in the art can make various modifications and variations to the above embodiments without departing from the technical concept of this invention, and all such modifications and variations should fall within the protection scope of this invention.

Claims

1. An immune-enhancing bovine colostrum powder bioactive protective microcapsule, characterized in that, It includes a four-layer structure arranged from the inside out: a multi-component active core, an Fc segment protective and stabilizing layer, a complex coagulation physical barrier layer, and a dual-response enteric layer. The multi-component active core contains bovine colostrum powder produced within 48 hours postpartum through low-temperature defatting, isoelectric point casein removal, low-temperature membrane filtration, low-temperature ultrafiltration concentration, and low-temperature spray drying. The bovine colostrum powder contains more than 25 wt% immunoglobulin IgG. The Fc segment protective stabilizing layer is composed of a non-reducing sugar vitrified matrix, a free amino acid competitive occupant, an activated molecular sieve hygroscopic regulator, and pre-denatured milk matrix protein. The mass of the non-reducing sugar vitrified matrix is ​​more than 12 times the sum of the mass of lactose contained in the bovine colostrum powder and the total mass of reducing sugars in all other pharmaceutical excipients of the microcapsule. Through the hygroscopic effect of the activated molecular sieve hygroscopic regulator, the microenvironment water activity Aw of the Fc segment protective stabilizing layer is maintained in the range of 0.20 to 0.30 during storage at 25°C and 60% relative humidity for 24 months. The complex coagulation physical barrier layer is a polyelectrolyte complex membrane formed by electrostatic coagulation of two polysaccharide pharmaceutical excipients with opposite charges, which completely separates the Fc segment protective stabilizing layer from the dual-response enteric layer in space. The dual-responsive enteric layer contains both a pH-responsive enteric polymeric component that responds to the pH of the medium and an esterified enteric polymeric component that responds to short-chain fatty acids. It dissolves and releases only when the pH of the medium is not lower than 6.5 and the concentration of short-chain fatty acids is not lower than 10 mmol / L.

2. The immune-enhancing bovine colostrum powder bioactive protective microcapsules according to claim 1, characterized in that: The multi-component active core also contains 0.5 wt% to 2.0 wt% of Pear aggregate lymph node microfold cell directional anchoring agent based on the weight of the bovine colostrum powder; The Pear aggregate lymph node microfold cell directional anchoring agent is selected from one or more combinations of modified edible lectins, fucoidylated oligosaccharides, mannosylated chitosan oligosaccharides, and sialic acid-modified whey protein.

3. The immune-enhancing bovine colostrum powder bioactive protective microcapsules according to claim 1, characterized in that: The components of the Fc segment protective stabilizing layer, by weight, are as follows: 30 wt% to 50 wt% of the non-reducing sugar vitrified matrix, 1 wt% to 3 wt% of the free amino acid competitive site-filling agent, 0.5 wt% to 1.5 wt% of the activated molecular sieve hygroscopic regulator, and 5 wt% to 10 wt% of the pre-denatured milk matrix protein; The non-reducing sugar vitrification matrix is ​​selected from one or more combinations of trehalose, sucrose, raffinose, and metriose, and the glass transition temperature is above 100°C. The free amino acid competitive occupier is selected from one or more combinations of L-lysine hydrochloride, L-arginine hydrochloride, glycine, and taurine. The activated molecular sieve moisture regulator is selected from one or more combinations of 3A type synthetic molecular sieve, 4A type synthetic molecular sieve, food-grade silica gel, and food-grade activated alumina, with a particle size of 2μm to 5μm, and is pre-activated at 200℃ to 300℃ for 4 to 8 hours. The pre-denatured milk matrix protein is selected from one or more combinations of whey protein isolate, sodium caseinate, and casein phosphopeptide, which have been heat-denatured at 75°C to 85°C for 20 to 30 minutes.

4. The immune-enhancing bovine colostrum powder bioactive protective microcapsules according to claim 1, characterized in that: Of the two polysaccharide pharmaceutical excipients in the complex condensed physical barrier layer, one carries a positive charge and the other carries a negative charge. The negatively charged polysaccharide is selected from one or more of sodium alginate, pectin, carrageenan, and gum arabic that have been treated to reduce their molecular weight, with a weight-average molecular weight of 20 kDa to 40 kDa. The positively charged polysaccharide is a deacetylated chitosan with a degree of deacetylation of 85% to 95%. The thickness of the complex cohesive physical barrier layer is 5 μm to 15 μm, and it forms a mechanically stable wet gel network after being cured by calcium chloride aqueous solution.

5. The immune-enhancing bovine colostrum powder bioactive protective microcapsules according to claim 1, characterized in that: The proportions of each component in the dual-response enteric coating, based on the total weight of the coating, are as follows: The pH-responsive enteric polymeric component comprises 60 wt% to 80 wt% and is selected from one or more combinations of acrylic resin No. III, hydroxypropyl methyl phthalate, cellulose acetate, and hydroxypropyl methyl cellulose acetate succinate. The short-chain fatty acid-responsive esterified modified enteric polymeric component accounts for 15 wt% to 30 wt%, and is selected from one or more combinations of butyrate-esterified hydroxypropyl methylcellulose phthalate, butyrate-esterified sodium alginate, propionic acid-esterified pectin, and acetate-esterified chitosan, with a degree of esterification substitution of 0.3 to 0.

8. This layer also contains 5 wt% to 10 wt% triethyl citrate plasticizer.

6. The immune-enhancing bovine colostrum powder bioactive protective microcapsules according to claim 1, characterized in that: The outer side of the dual-response enteric layer also contains an adaptive delay layer for medication administration; The medication time-adaptive delay layer is a sustained-release membrane composed of one or more of the following: zein, gluten, soybean protein alcohol aqueous dispersion, and rice gluten, with a membrane thickness of 10 μm to 30 μm. Based on the total weight of the microcapsules, the adaptive delay layer for medication administration accounts for 3 wt% to 5 wt%.

7. The immune-enhancing bovine colostrum powder bioactive protective microcapsules according to claim 6, characterized in that: The overall particle size of the microcapsules is 120 μm to 200 μm, and the particle size is expressed as the D50 value measured by a laser diffraction particle size analyzer. The microcapsules retained more than 80% of their IgG-FcRn receptor binding activity after being stored at 25°C and 60% relative humidity for 24 months. This retention rate was determined by the surface plasmon resonance competitive binding method.

8. The method for preparing immune-enhancing bovine colostrum powder bioactive protective microcapsules according to any one of claims 1-7, characterized in that: It includes the following six steps; S1: Collect colostrum from cows within 48 hours postpartum, and sequentially perform centrifugation defatting, casein removal at pH 4.6 isoelectric point, low-temperature membrane filtration with a pore size of 0.45μm, and low-temperature ultrafiltration concentration with a molecular weight cutoff of 10kDa at 4±1℃. Spray dry the ultrafiltration concentrate at an inlet air temperature of 100℃ to 115℃ and an outlet air temperature of 60℃ to 70℃ to obtain bovine colostrum powder with an IgG content of more than 25% by weight. S2: The bovine colostrum powder prepared in S1, optionally added 0.5wt% to 2.0wt% of Pear aggregate lymph node microfold cell directional anchoring agent based on the weight of the bovine colostrum powder, and the components of the Fc segment protective stabilizing layer are dispersed in purified water in the proportions described above. After 0.22μm sterilization filtration, the mixture is placed in a vacuum freeze dryer. The freeze-drying process is completed by pre-freezing at -40℃ to -45℃ for 4 to 6 hours, first drying at -25℃ to -30℃ for 20 to 28 hours, and second drying at 20℃ to 25℃ for 6 to 10 hours. The powder is then pulverized through an 80-mesh sieve in an environment with a relative humidity of less than 10% to obtain the core-inner stable layer composite powder. S3: The composite powder prepared in S2 is dispersed in a 1.0 wt% to 2.0 wt% aqueous solution of the low molecular weight sodium alginate under stirring at 300 rpm, the pH is adjusted to 5.3 to 5.7, a 0.8 wt% to 1.2 wt% aqueous solution of the deacetylated chitosan acetate is added dropwise, the pH is adjusted to 4.3 to 4.7, and the pH of the system is adjusted to 4.8 to 5.2 to allow the two polysaccharides to electrostatically recombine. The reaction temperature is 8°C to 12°C, and the mixture is stirred for 50 to 70 minutes. A 0.04 mol / L to 0.06 mol / L calcium chloride aqueous solution is added to solidify the alginate network for 25 to 35 minutes. After filtration, the wet particles are redissolved in an aqueous solution containing 2 wt% to 4 wt% trehalose, and a second freeze-drying is performed at −40°C to −50°C to obtain the three-layer structure intermediate particles. S4: The intermediate microparticles prepared in S3 are placed in a fluidized bed with an inlet temperature of 28°C to 32°C, an outlet temperature of 23°C to 27°C, and an atomization pressure of 0.25MPa to 0.35MPa. First, they are coated with the pH-responsive enteric polymer aqueous dispersion until the intermediate microparticles gain 12% to 15% in weight. Then, they are coated a second time with the short-chain fatty acid-responsive esterified modified enteric polymer aqueous dispersion until the weight gain is 5% to 8%. Finally, they are sprayed with triethyl citrate plasticizer solution and cured at 30°C for 15 to 25 minutes to obtain a four-layer structure microcapsule. S5: When it is necessary to prepare microcapsules containing the adaptive delay layer of the drug administration period, the four-layer structure microcapsules obtained in S4 are coated for the third time in a fluidized bed with a 2.5wt% to 3.5wt% zein alcohol aqueous solution, and the film thickness is controlled at 10μm to 30μm to obtain a five-layer structure microcapsule. S6: The prepared microcapsules are matured for 48 hours in a clean environment with relative humidity below 15% and temperature below 25℃. Three-stage release tests are performed, and IgG-FcRn receptor binding activity is tested using the surface plasmon resonance competitive binding method. Qualified batches are used as oral immunomodulatory agents.

9. The method for preparing immune-enhancing bovine colostrum powder bioactive protective microcapsules according to claim 8, characterized in that: Before starting S2, the activated molecular sieve moisture absorbent is pre-activated by maintaining the temperature at 200°C to 300°C for 4 to 8 hours, and then cooling to room temperature in a dry nitrogen environment before being put into use immediately. The moisture absorbent capacity is more than 90% of the theoretical moisture absorbent capacity of this type of molecular sieve. In S2, the pre-denaturation conditions for the pre-denatured milk matrix protein are as follows: whey protein isolate or sodium caseinate is prepared into a 5wt% to 10wt% aqueous solution and kept at 75°C to 85°C for 20 to 30 minutes. The exposed thiol content of the resulting pre-denatured milk protein is 60% to 80% of the original thiol content of the protein. During the electrostatic recombination reaction of S3, the zeta potential change of the system is monitored in real time, and the absolute value of the zeta potential during the recombination film formation stage is controlled within 5mV; the termination of the calcium chloride curing reaction in this step is judged by the rate of change of the system conductivity being less than 1μS·cm⁻¹ / min. This method is carried out in an environment with a GMP cleanliness level of D or above. All water-based operations use purified water of the injection grade and do not use glutaraldehyde, formaldehyde, organic crosslinking agents, or organic solvents as processing aids. The IgG-FcRn receptor binding activity retention rates of the finished products obtained by this method were above 90% and above 80% after 3 months of accelerated testing at 40℃ and 75% relative humidity and 24 months of long-term testing at 25℃ and 60% relative humidity, respectively.

10. The use of the immune-enhancing bovine colostrum powder bioactive protective microcapsules according to any one of claims 1 to 7 in the preparation of oral immunomodulatory biological therapeutics for recurrent respiratory infections, chronic inflammatory bowel disease, intestinal barrier dysfunction, immune reconstitution after radiotherapy and chemotherapy for tumors, and clinical immunological diseases related to immunodeficiency in children and the elderly; The dosage form of the oral immunomodulatory biological therapy drug is selected from one of granules, capsules, oral suspensions, and orally disintegrating tablets; For use in children aged 3 to 12 years with recurrent respiratory infections, the daily dose based on the weight of the bovine colostrum powder is 50 mg / kg body weight to 150 mg / kg body weight, 1 to 3 times daily. In applications targeting immunocompromised individuals aged 65 and above, the microcapsules used contain an adaptive delay layer for the medication administration period. In the application of this drug as an adjuvant therapy for immune reconstitution after radiotherapy and chemotherapy for tumors, the interval between the drug and radiotherapy / chemotherapy is more than 2 hours.

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