Layered sustained-release bc39 protein powder compound and application thereof

Abstract

The application relates to the technical field of nutritional health food, and discloses a layered sustained-release BC39 protein powder compound and application thereof, which comprises the following steps: preparing porous starch by treating corn starch with a compound amylase, combining the porous starch with silicon dioxide to obtain a compound carrier, adsorbing part of Bacillus coagulans BC39 under negative pressure, vacuum drying, and sealing the carrier to obtain a bacteria-loaded core, spraying a bottom spraying liquid containing pectin and oligofructose and a cover liquid containing ethyl cellulose to form an inner layer, spraying a middle layer coating liquid containing hydroxypropyl cellulose, carboxymethyl starch sodium and part of the bacterial strains, spraying an outer layer coating liquid containing hydroxypropyl methyl cellulose, mannitol and the remaining bacterial strains to obtain three-layer microcapsules, and physically mixing the microcapsules with whey protein and soybean protein isolate. The application realizes multi-stage site release of probiotics in the gastrointestinal tract by using a multilayer coating structure, provides amino acid relay supply by combining double absorption rate proteins, and cooperatively improves the muscle protein synthesis efficiency.

Description

Technical Field

[0001] This invention relates to the field of nutritional and health food technology, specifically to a layered sustained-release BC39 protein powder compound and its application. Background Technology

[0002] Muscle loss and sarcopenia are health challenges faced by the elderly and frail. Conventional nutritional interventions involve supplementing with protein powders, primarily whey protein or soy protein isolate. However, with the deepening research into the human digestive system, the gut microbiota-mediated regulation of muscle metabolism has been shown to have a key impact on amino acid absorption and muscle protein synthesis. Combining probiotics with proteins has become an evolving direction for intervention methods.

[0003] Existing probiotic protein compound products have technical limitations in practical applications. Conventional powder mixing processes expose probiotics directly to the gastric environment after oral ingestion. Live bacteria are subjected to low-pH gastric juice erosion, resulting in protein denaturation and inactivation. Although some products have introduced basic microencapsulation technology, they can only establish gastric juice resistance and a free release process in the whole intestine. They cannot establish specific release concentration gradients for the different physiological microenvironments of the duodenum, jejunum, and colon. The single release behavior hinders the core strains from gaining absolute abundance dominance in the colon region and completing long-term colonization.

[0004] The efficiency of protein digestion and absorption in the human body depends on good intestinal mucosal permeability and microecological balance. In traditional compound preparations, the release sites and cycles of probiotics are detached from the protein degradation rate, failing to form a physical synergy of spatial distribution and temporal release. The transmembrane transport of free amino acids such as leucine by small intestinal villi is restricted due to the lack of synchronous promotion of corresponding intestinal probiotic metabolites, which restricts the activation of muscle protein synthesis signaling pathways and ultimately limits the physiological efficacy of compound preparations in improving muscle metabolism. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a layered sustained-release BC39 protein powder compound and its application, which solves the problems of low probiotic survival rate, uncontrollable segmented targeted release, and hindered synergistic muscle synthesis promotion effect caused by the disconnect between the probiotic release cycle and protein digestion and absorption in existing probiotic protein compound products during gastrointestinal delivery.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a layered sustained-release BC39 protein powder compound, the preparation process of which includes the following steps:

[0008] Corn starch is made into starch milk and the pH is adjusted. Porous starch is obtained by isothermal reaction with composite amylase, enzyme inactivation treatment and drying treatment. The porous starch is mixed with silica powder to obtain a lightweight porous composite carrier.

[0009] A portion of Bacillus coagulans BC39 was prepared into an inner layer bacterial solution, which was then added to the lightweight porous composite carrier and subjected to negative pressure adsorption and vacuum drying to obtain the bacterial core.

[0010] An inner layer bottom spray solution containing pectin and fructooligosaccharides, and an inner layer top spray solution containing ethyl cellulose were prepared separately. The inner layer bottom spray solution and the inner layer top spray solution were sequentially sprayed onto the surface of the bacterial core in a fluidized bed and continuously dried to obtain inner layer microparticles.

[0011] Hydroxypropyl cellulose, sodium carboxymethyl starch, and a portion of Bacillus coagulans BC39 were formulated into a middle layer coating solution; the middle layer coating solution was sprayed onto the surface of the inner layer particles in a fluidized bed and continuously dried to obtain middle layer coated particles;

[0012] Hydroxypropyl methylcellulose, mannitol, and the remaining Bacillus coagulans BC39 were formulated into an outer coating solution; the outer coating solution was sprayed onto the surface of the middle coating particles in a fluidized bed and continuously dried to obtain a three-layer sustained-release microcapsule;

[0013] The three-layered sustained-release microcapsules, whey protein, and soy protein isolate are physically mixed to obtain the layered sustained-release BC39 protein powder complex.

[0014] By employing the technical solution of this invention, and by using a porous carrier combined with a three-layer fluidized bed coating process, and mixing the prepared microcapsules with proteins at different digestion rates, multi-segment targeted release and synergistic absorption effects are achieved. The specific mechanism is as follows:

[0015] The hydroxypropyl methylcellulose in the outer coating solution hydrates in the low pH environment of gastric juice, and the polymer chains extend to form a high-viscosity gel network layer, which acts as a physical barrier to the penetration of gastric acid into the interior. As the system enters the duodenum, mannitol, as a water-soluble porogen, dissolves, and the gel network produces micropores, releasing a small portion of Bacillus coagulans BC39 contained in the outer layer. This strain is used to activate the immune response of intestinal epithelial cells in the early stage.

[0016] After the microcapsules enter the jejunum and ileum, the sodium carboxymethyl starch in the middle coating absorbs water, causing a dramatic volume expansion that destroys the remaining gel skeleton in the outer layer. Hydroxypropyl cellulose gradually dissolves in the weakly alkaline intestinal fluid, releasing Bacillus coagulans BC39 contained in the middle layer. At this time, the rapidly digestible whey protein has been degraded into free amino acids and oligopeptides in the stomach and upper small intestine. The metabolic enzymes produced by the released middle layer flora promote the increased absorption efficiency of branched-chain amino acids such as leucine by the villi of the small intestine.

[0017] As the microparticles reach the colon, the ethyl cellulose in the inner coating liquid, acting as a water-insoluble material, maintains the structural integrity of the microparticles. The pectin molecules in the inner bottom spray liquid contain an α-1,4-D-galacturonic acid polymer structure, which is broken down by polysaccharide-degrading enzymes secreted by the colon's native microbiota. The pectin skeleton disintegrates, causing the ethyl cellulose membrane to rupture and exposing the bacterial core.

[0018] The Bacillus coagulans BC39 inside the porous starch is released in large quantities here, along with co-encapsulated fructooligosaccharides providing the initial carbon source, and the strain proliferates. At the same time, the slowly digested soy protein isolate reaches the lower digestive tract, maintaining a sustained supply of amino acids and reducing muscle protein breakdown.

[0019] Preferably, the steps of obtaining porous starch through a composite amylase isothermal reaction, enzyme inactivation treatment, and drying treatment include: adjusting the pH of the starch milk to 4.8 to 5.2 using citric acid solution, and carrying out the isothermal reaction at 45 to 55°C for 12 to 24 hours; subsequently adding hydrochloric acid solution to adjust the pH to 2.0 to 2.4 and maintaining it for 10 to 20 minutes for the enzyme inactivation treatment; then neutralizing the pH to 7.0 using sodium hydroxide solution, followed by centrifugation, washing, and drying treatment; the step of mixing the porous starch with silica powder includes: putting it into a powder mixer and mixing it at a speed of 900 to 1100 r / min for 25 to 35 minutes.

[0020] By limiting the preparation parameters of porous starch, the pH and temperature are controlled within the optimal catalytic range of amylase. The amorphous region inside the starch granules preferentially undergoes hydrolysis, while the crystalline framework is preserved, forming interconnected micropores from the surface to the interior. Changing the pH to 2.0 to 2.4 denatures and inactivates the enzyme protein, terminating the hydrolysis reaction and maintaining pore size stability. Silica powder has an extremely high specific surface area and adheres to the surface of porous starch under high-speed mixing, reducing van der Waals forces and electrostatic attraction between powders, improving the fluidity of the carrier and preventing moisture absorption and agglomeration.

[0021] Preferably, the steps of performing negative pressure adsorption and vacuum drying include: performing negative pressure adsorption for 20 to 30 minutes under a vacuum of 0.04 to 0.08 MPa; and then performing vacuum drying at 35 to 40°C until the moisture content is less than 5 wt%.

[0022] By employing negative pressure adsorption and vacuum drying processes, air escapes from the system to form a pressure gradient. When normal pressure is restored or bacterial solution is added, capillary force forces droplets containing Bacillus coagulans BC39 deep into the micropores of porous starch. Vacuum drying is carried out at a low temperature of 35 to 40°C. Water molecules have a lower boiling point under low pressure and are rapidly vaporized and removed, preventing the heat-sensitive Bacillus from undergoing protein denaturation and inactivation due to high temperature. At the same time, the moisture content of less than 5 wt% keeps the spores in a dormant state, improving storage stability.

[0023] Preferably, the steps of preparing the inner layer bottom spray liquid containing pectin and fructooligosaccharides and the inner layer top coat liquid containing ethyl cellulose include: mixing pectin and fructooligosaccharides as dry powders and then slowly dispersing them in purified water, mechanically stirring at 350 to 450 r / min at 55 to 65°C for 40 to 45 minutes until completely dissolved, and then naturally cooling to 20 to 25°C to obtain the inner layer bottom spray liquid.

[0024] By employing the formulation conditions of the inner bottom spray liquid, the dry powder premixing disperses the pectin and fructooligosaccharide particles, preventing the rapid hydration of the surface when the pectin comes into direct contact with water molecules, thus preventing the formation of water-blocking gel clusters. Hot water at 55 to 65°C provides molecular kinetic energy, breaking the hydrogen bonds between polysaccharide molecules, promoting chain segment extension and complete dissolution. After cooling to room temperature, a uniform and stable polymer solution is formed, ensuring the uniform particle size of the fluidized bed atomized droplets.

[0025] Preferably, the continuous drying step includes: after spraying the inner layer base spray liquid and the inner layer top coat liquid, controlling the air inlet temperature at 35 to 45°C and continuing to dry for 10 to 15 minutes; after spraying the middle layer coating liquid, controlling the air inlet temperature at 35 to 40°C and continuing to dry for 10 to 15 minutes; and after spraying the outer layer coating liquid, controlling the air inlet temperature at 34 to 38°C and continuing to dry for 15 to 20 minutes.

[0026] By employing a decreasing inlet air temperature gradient, the drying process of fluidized bed coating exhibits a gradually decreasing heat distribution from the inside out. As the number of coating layers increases, the microcapsule volume increases, shortening the heat transfer path required for the evaporation of outer layer moisture. This reduces the outer layer inlet air temperature, slows down the solvent evaporation rate, and allows sufficient time for the hydroxypropyl methylcellulose polymer segments to rearrange and crosslink on the droplet surface, reducing film-forming stress and porosity. The temperature gradient also prevents excessive heat transfer from the outer layer to the inner layer, ensuring the survival rate of the strains in the core area.

[0027] Preferably, the step of preparing the intermediate coating solution from hydroxypropyl cellulose, sodium carboxymethyl starch, and a portion of Bacillus coagulans BC39 includes: mixing and dissolving hydroxypropyl cellulose and sodium carboxymethyl starch at a mass ratio of 5:2; the step of preparing the outer coating solution from hydroxypropyl methyl cellulose, mannitol, and the remaining Bacillus coagulans BC39 includes: mixing and dissolving hydroxypropyl methyl cellulose and mannitol at a mass ratio of 3:1.

[0028] By employing a specific coating solution mass ratio, the middle layer uses a 5:2 ratio, utilizing the skeletal support of hydroxypropyl cellulose to create an antagonistic balance with the high water absorption and swelling rate of sodium carboxymethyl starch. The swelling of sodium carboxymethyl starch generates internal tension. When the generated tension exceeds the binding force of the hydroxypropyl cellulose gel layer, the middle layer undergoes mechanical disintegration. The outer layer uses a 3:1 ratio, with mannitol molecules uniformly embedded in the hydroxypropyl methyl cellulose macromolecular network. In an aqueous environment, mannitol dissolves, leaving microporous channels that regulate the rate at which water molecules enter the internal skeleton, thus controlling the dissolution cycle of the outer layer in gastric acid.

[0029] Preferably, based on the total mass of Bacillus coagulans BC39 contained in the compound: the step of preparing a portion of Bacillus coagulans BC39 into an inner layer bacterial solution includes: preparing the inner layer bacterial solution using 50% to 60% of the total mass of Bacillus coagulans BC39; the step of preparing a middle layer coating solution using hydroxypropyl cellulose, sodium carboxymethyl starch, and a portion of Bacillus coagulans BC39 includes: preparing the middle layer coating solution using 25% to 30% of the total mass of Bacillus coagulans BC39; the step of preparing an outer layer coating solution using hydroxypropyl methyl cellulose, mannitol, and the remaining Bacillus coagulans BC39 includes: preparing the outer layer coating solution using 15% to 20% of the total mass of Bacillus coagulans BC39; and the sum of the mass percentages of Bacillus coagulans BC39 used in the inner, middle, and outer layers is 100%.

[0030] By employing a gradient distribution mechanism for gut microbiota quality allocation, an increasing gradient of bacterial concentration is established. 15% to 20% of the outer layer and 25% to 30% of the middle layer of bacteria are released freely in the small intestine. The secreted metabolites, such as short-chain fatty acids, lower the local pH of the intestine, inhibit the growth of harmful bacteria, and improve the permeability of the small intestinal mucosa. 50% to 60% of the high-concentration bacteria in the inner layer are concentrated and released in the colon, establishing an abundance dominance in the colonic region, thereby supporting intestinal colonization and producing a long-term regulatory effect.

[0031] Preferably, the steps of preparing the inner bottom spray liquid containing pectin and fructooligosaccharides and the inner top coat liquid containing ethyl cellulose further include: mixing pectin and fructooligosaccharides at a mass ratio of 4 to 5: 5 to 8; wherein the fructooligosaccharides serve as prebiotics for probiotic colonization, and the pectin, together with the ethyl cellulose in the inner top coat liquid, serves as a colon-targeting material.

[0032] By employing the synergistic constraint of pectin and fructooligosaccharides, the ratio of pectin to fructooligosaccharides ensures a balance between membrane structure stability and biodegradability. Fructooligosaccharides are difficult to hydrolyze by gastric juice and human intestinal digestive enzymes. After reaching the colon, they provide fermentation substrates for Bacillus coagulans BC39, promoting the bacterial community to enter the logarithmic growth phase through glycolysis. Pectin macromolecules are intertwined in the ethyl cellulose network and do not degrade in the stomach and small intestine where corresponding glycosidases are lacking. After reaching the colon, they undergo enzymatic chain scission, disrupting membrane compactness and triggering site-directed release.

[0033] Preferably, the step of physically mixing the three-layer sustained-release microcapsules, whey protein and soy protein isolate includes: mixing 10 to 20 parts by weight of the three-layer sustained-release microcapsules, 50 to 70 parts by weight of the whey protein and 30 to 50 parts by weight of the soy protein isolate at 25°C at a rotation speed of 300 to 500 r / min for 10 to 20 minutes.

[0034] By employing a three-component dry-mixing process, whey protein, rich in leucine, rapidly produces a peak in plasma free amino acids within 1 to 2 hours after oral administration, initiating the muscle protein synthesis signaling pathway. Soy protein isolate has a slower digestion and absorption rate, maintaining a continuous supply of plasma amino acids after meals and reducing muscle protein breakdown. Physical dry mixing at 25°C and medium speed avoids mechanical shear heating that could cause the polymer on the surface of the microcapsules to melt or break, ensuring the uniform distribution of each component in the final product system.

[0035] Secondly, the present invention also provides the application of a layered sustained-release BC39 protein powder compound in the preparation of nutritional foods or health foods for promoting muscle protein synthesis-related nutritional metabolism.

[0036] By employing the application scheme of this invention, sustained-release microencapsulated probiotics are combined with proteins of dual absorption rates. This utilizes the intestinal flora-mediated muscle metabolism regulation mechanism to produce physiological effects. After Bacillus coagulans BC39 colonizes the intestinal segment, it secretes peptidoglycan and short-chain fatty acids, downregulating the level of systemic inflammatory factors and reducing muscle protein degradation caused by inflammation. At the same time, it improves the intestinal microenvironment and promotes the transport efficiency of whey and soy protein isolate enzymatic hydrolysates by the small intestinal villi, thus doubly increasing the net muscle protein synthesis rate in the elderly or frail. This invention is suitable for promoting nutritional metabolism related to muscle protein synthesis.

[0037] In summary, the present invention has at least one of the following beneficial technical effects:

[0038] 1. This invention utilizes a fluidized bed three-layer coating structure to achieve multi-level targeted release of Bacillus coagulans BC39 in the gastrointestinal tract. The hydroxypropyl methylcellulose in the outer coating forms a gel in the acidic environment of the stomach to block liquid permeation. The sodium carboxymethyl starch in the middle layer absorbs water and swells in the weakly alkaline intestinal fluid, breaking through the outer shell skeleton. The pectin in the inner layer undergoes specific chain scission due to the polysaccharide-degrading enzymes inherent in the colon. Due to the combination of polymer materials with specific degradation logic, the microcapsules undergo segmental dissolution in the duodenum, jejunum and colon, ensuring that the internal core is protected from damage by low pH gastric juice. Furthermore, an increasing bacterial abundance gradient is established according to the physiological microenvironment of different intestinal segments, improving the colonization efficiency of live bacteria.

[0039] 2. This invention uses enzymatically hydrolyzed porous starch composite silica as a lightweight carrier, and combines negative pressure adsorption and low-temperature vacuum drying processes to improve the storage stability of the strains inside the microparticle core. The porous starch has a continuous pore network inside. Driven by the pressure gradient, the liquid strains are forced into the deep layer of the microparticle to achieve physical integration. Combined with the removal of free water in a low-temperature environment of 35 to 40°C, this invention avoids the risk of protein denaturation and inactivation of heat-sensitive strains when exposed to high temperatures, and strictly limits the moisture content of the powder to below 5 wt%, which promotes the reduction of metabolic consumption of dormant spores, thereby improving the biological activity of the finished powder during long-term storage.

[0040] 3. This invention constructs a time-dimensional amino acid relay supply and synergistic absorption mechanism by dry-mixing layered sustained-release microcapsules with whey protein and soy protein isolate. After oral ingestion, whey protein rapidly enzymatically releases leucine and initiates the muscle synthesis signaling pathway, while soy protein isolate maintains a sustained supply of subsequent amino acids due to its slower digestion rate. The simultaneous microparticle degradation process causes Bacillus coagulans BC39 to be released freely throughout the intestine. The secreted metabolic enzymes and produced short-chain fatty acids improve local intestinal permeability and promote the transmembrane absorption efficiency of the degradation products of the two proteins by intestinal villi. This forms a spatiotemporal synergistic response between microorganisms and amino acids, enhancing the intervention effect of targeting muscle synthesis. Attached Figure Description

[0041] Figure 1 This is a schematic diagram of the vertical spatial distribution of viable bacteria in each group of samples after simulated transport vibration treatment according to the present invention.

[0042] Figure 2 This is a schematic diagram showing the relative standard deviation of the viable bacterial count distribution in each group of samples of the present invention;

[0043] Figure 3 This is a schematic diagram of the cumulative release rate curves of each group of samples in a continuous in vitro simulated gastrointestinal environment.

[0044] Figure 4 This is a schematic diagram showing the distribution of the release amount of each group of samples in different in vitro simulated digestion stages.

[0045] Figure 5 This is a schematic diagram showing the cumulative concentration changes of free leucine in each group of samples in an in vitro static protein digestion model of the present invention.

[0046] Figure 6 This is a schematic diagram showing the cumulative concentration changes of total free amino acids in each group of samples in an in vitro static protein digestion model of the present invention.

[0047] Figure 7 This is a schematic diagram illustrating the co-evolution trajectory of viable bacterial proliferation and short-chain fatty acid metabolites in the in vitro simulated colon fermentation system for each group of samples of the present invention. Detailed Implementation

[0048] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0050] Heyndrickxia coagulans BC-39 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 11, 2026, with accession number GDMCC No:68241, and its specific form as a raw material is a food-grade microbial powder prepared based on Heyndrickxia coagulans BC-39.

[0051] Whey protein, CAS number 84082-51-9, is food grade, specifically whey protein isolate with a protein mass fraction of not less than 90 wt%.

[0052] Soy protein isolate, CAS number 9010-10-0, is food grade and has a protein content of not less than 90 wt%.

[0053] Pectin, CAS number 9000-69-5, is food grade, specifically high methoxylated citrus pectin, with an esterification degree of not less than 50%. Its main component is a linear polysaccharide polymer composed of D-galacturonic acid linked by α-1,4 glycosidic bonds.

[0054] Ethyl cellulose, CAS No. 9004-57-3, is a food additive grade with a degree of substitution of 2.25 to 2.58 and an ethoxy mass fraction of 44 wt% to 51 wt%.

[0055] Fructooligosaccharides, CAS number 308066-66-2, are food grade and have a degree of polymerization of 2 to 9.

[0056] The compound amylase is a food-grade powdered compound enzyme preparation composed of α-amylase and saccharifying enzyme. The CAS number of α-amylase is 9000-90-2, and the CAS number of saccharifying enzyme is 9032-08-0. The total enzyme activity is not less than 100,000 U / g, and the enzyme activity ratio of α-amylase to saccharifying enzyme is 1:3 to 1:5.

[0057] In this invention, the units of measurement for all materials and solvents are uniformly referred to as parts by weight.

[0058] Example 1:

[0059] This embodiment provides a layered sustained-release BC39 protein powder formulation, including the following steps:

[0060] 200 parts by weight of corn starch were dispersed in 800 parts by weight of purified water to prepare starch slurry, and the pH was adjusted to 5.2 using citric acid solution. 6 parts by weight of compound amylase were added to the starch slurry and reacted at 55°C for 24 hours. Then, hydrochloric acid solution was added to adjust the pH to 2.0 and held for 20 minutes to inactivate the enzyme. The pH was then neutralized to 7.0 using sodium hydroxide solution. After centrifugation, washing, and drying, porous starch was obtained. 120 parts by weight of porous starch and 12 parts by weight of silica were added to a powder mixer and mixed at 1100 r / min for 35 minutes to obtain a lightweight porous composite carrier.

[0061] Mix 5 parts by weight of pectin and 5 parts by weight of oligofructose into a dry powder until homogeneous, then slowly disperse in 190 parts by weight of purified water. Stir mechanically at 450 r / min for 40 minutes at 65°C until completely dissolved and then naturally cooled to 20°C to obtain the inner bottom spray liquid; dissolve 10 parts by weight of ethyl cellulose in 90 parts by weight of ethanol to obtain the inner top coat liquid.

[0062] 6.0 parts by weight of Bacillus coagulans BC39 were dispersed in 30 parts by weight of purified water to form an inner layer bacterial solution. Then, 100 parts by weight of a lightweight porous composite carrier were added, and the solution was subjected to negative pressure adsorption at a vacuum of 0.08 MPa for 30 minutes. Subsequently, the solution was vacuum dried at 40°C until the moisture content was less than 5 wt% to obtain the bacterial core. The bacterial core was placed in a fluidized bed coating machine, and the inlet air temperature was controlled at 45°C. The inner layer bottom spray liquid and the inner layer top spray liquid were sprayed onto the surface of the bacterial core in sequence, and the solution was dried for 15 minutes to obtain the inner layer microparticles.

[0063] Dissolve 5 parts by weight of hydroxypropyl cellulose and 2 parts by weight of sodium carboxymethyl starch in 80 parts by weight of purified water, then uniformly disperse 2.5 parts by weight of Bacillus coagulans BC39 to prepare a middle layer coating solution; control the fluidized bed inlet air temperature to 40°C, spray the middle layer coating solution onto the surface of the inner layer particles, and continue to dry for 15 minutes to obtain the middle layer coated particles.

[0064] Three parts by weight of hydroxypropyl methylcellulose and one part by weight of mannitol were dissolved in 50 parts by weight of purified water, and then 1.5 parts by weight of Bacillus coagulans BC39 were uniformly dispersed in the solution to prepare an outer coating solution. The fluidized bed inlet air temperature was controlled at 38°C, and the outer coating solution was sprayed onto the surface of the middle coating microparticles and dried for 20 minutes to obtain a three-layer sustained-release microcapsule.

[0065] 15 parts by weight of three-layer sustained-release microcapsules, 60 parts by weight of whey protein and 40 parts by weight of soy protein isolate were put into a powder mixer and mixed at 25°C and 500 r / min for 20 minutes to obtain the layered sustained-release BC39 protein powder compound.

[0066] Example 2:

[0067] This embodiment provides a layered sustained-release BC39 protein powder formulation, including the following steps:

[0068] 150 parts by weight of corn starch were dispersed in 600 parts by weight of purified water to prepare starch slurry, and the pH was adjusted to 5.0 using citric acid solution. 3 parts by weight of compound amylase were added to the starch slurry and reacted at 50°C for 18 hours. Then, hydrochloric acid solution was added to adjust the pH to 2.2 and held for 15 minutes to inactivate the enzyme. The pH was then neutralized to 7.0 using sodium hydroxide solution. After centrifugation, washing, and drying, porous starch was obtained. 75 parts by weight of porous starch and 10 parts by weight of silica were added to a powder mixer and mixed at 1000 r / min for 30 minutes to obtain a lightweight porous composite carrier.

[0069] Four parts by weight of pectin and eight parts by weight of oligofructose were mixed into a dry powder until homogeneous. Then, the mixture was slowly dispersed in 188 parts by weight of purified water and mechanically stirred at 400 r / min for 45 minutes at 60°C until completely dissolved. The mixture was then naturally cooled to 25°C to obtain the inner bottom spray liquid. Eight parts by weight of ethyl cellulose were dissolved in 72 parts by weight of ethanol to obtain the inner top coat liquid.

[0070] 12.0 parts by weight of Bacillus coagulans BC39 were dispersed in 60 parts by weight of purified water to form an inner layer bacterial solution. Then, 80 parts by weight of a lightweight porous composite carrier were added, and the solution was subjected to negative pressure adsorption at a vacuum of 0.06 MPa for 25 minutes. Subsequently, the solution was vacuum dried at 38°C until the moisture content was less than 5 wt% to obtain the bacterial core. The bacterial core was placed in a fluidized bed coating machine, and the inlet air temperature was controlled at 40°C. The inner layer bottom spray liquid and the inner layer top spray liquid were sprayed onto the surface of the bacterial core in sequence, and the solution was dried for 12 minutes to obtain the inner layer microparticles.

[0071] 7.5 parts by weight of hydroxypropyl cellulose and 3 parts by weight of sodium carboxymethyl starch were dissolved in 120 parts by weight of purified water, and then 5.0 parts by weight of Bacillus coagulans BC39 were uniformly dispersed in to prepare a middle layer coating solution. The fluidized bed inlet air temperature was controlled at 38°C, and the middle layer coating solution was sprayed onto the surface of the inner layer particles and dried for 15 minutes to obtain the middle layer coated particles.

[0072] 4.5 parts by weight of hydroxypropyl methylcellulose and 1.5 parts by weight of mannitol were dissolved in 75 parts by weight of purified water, and then 3.0 parts by weight of Bacillus coagulans BC39 were uniformly dispersed in the solution to prepare an outer coating solution. The fluidized bed inlet air temperature was controlled at 36°C, and the outer coating solution was sprayed onto the surface of the middle coating microparticles and dried for 18 minutes to obtain a three-layer sustained-release microcapsule.

[0073] 20 parts by weight of three-layer sustained-release microcapsules, 70 parts by weight of whey protein and 50 parts by weight of soy protein isolate were put into a powder mixer and mixed at 25°C and 400 r / min for 15 minutes to obtain the layered sustained-release BC39 protein powder compound.

[0074] Example 3:

[0075] This embodiment provides a layered sustained-release BC39 protein powder formulation, including the following steps:

[0076] 100 parts by weight of corn starch were dispersed in 400 parts by weight of purified water to prepare a starch slurry, and the pH was adjusted to 4.8 using citric acid solution. 1 part by weight of compound amylase was added to the starch slurry, and the mixture was reacted at a constant temperature of 45°C for 12 hours. Then, hydrochloric acid solution was added to adjust the pH to 2.4 and held for 10 minutes to inactivate the enzyme. The pH was then neutralized to 7.0 using sodium hydroxide solution. After centrifugation, washing, and drying, porous starch was obtained. 45 parts by weight of porous starch and 8 parts by weight of silica were added to a powder mixer and mixed at 900 r / min for 25 minutes to obtain a lightweight porous composite carrier.

[0077] 4.5 parts by weight of pectin and 6.5 parts by weight of oligofructose were mixed into a dry powder until homogeneous, and then slowly dispersed in 189 parts by weight of purified water. The mixture was mechanically stirred at 350 r / min for 42 minutes at 55°C until completely dissolved and then naturally cooled to 22°C to obtain the inner bottom spray liquid. 6 parts by weight of ethyl cellulose were dissolved in 54 parts by weight of ethanol to obtain the inner top coat liquid.

[0078] 5.0 parts by weight of Bacillus coagulans BC39 were dispersed in 25 parts by weight of purified water to form an inner layer bacterial solution. Then, 50 parts by weight of a lightweight porous composite carrier were added, and the solution was subjected to negative pressure adsorption at a vacuum of 0.04 MPa for 20 minutes. Subsequently, the solution was vacuum dried at 35°C until the moisture content was less than 5 wt% to obtain the bacterial core. The bacterial core was placed in a fluidized bed coating machine, and the inlet air temperature was controlled at 35°C. The inner layer bottom spray liquid and the inner layer top spray liquid were sprayed onto the surface of the bacterial core in sequence, and the solution was dried for 10 minutes to obtain the inner layer microparticles.

[0079] 2.5 parts by weight of hydroxypropyl cellulose and 1 part by weight of sodium carboxymethyl starch were dissolved in 40 parts by weight of purified water, and then 3.0 parts by weight of Bacillus coagulans BC39 were uniformly dispersed in to prepare a middle layer coating solution. The fluidized bed inlet air temperature was controlled at 35°C, and the middle layer coating solution was sprayed onto the surface of the inner layer particles and dried for 10 minutes to obtain the middle layer coated particles.

[0080] 1.5 parts by weight of hydroxypropyl methylcellulose and 0.5 parts by weight of mannitol were dissolved in 25 parts by weight of purified water, and then 2.0 parts by weight of Bacillus coagulans BC39 were uniformly dispersed in the solution to prepare an outer coating solution. The fluidized bed inlet air temperature was controlled at 34°C, and the outer coating solution was sprayed onto the surface of the middle coating microparticles and dried for 15 minutes to obtain a three-layer sustained-release microcapsule.

[0081] Ten parts by weight of three-layer sustained-release microcapsules, 50 parts by weight of whey protein and 30 parts by weight of soy protein isolate were put into a powder mixer and mixed at 300 r / min for 10 minutes at 25°C to obtain the layered sustained-release BC39 protein powder compound.

[0082] Example 4:

[0083] This embodiment provides a layered sustained-release BC39 protein powder formulation, including the following steps:

[0084] 200 parts by weight of corn starch were dispersed in 800 parts by weight of purified water to prepare starch slurry, and the pH was adjusted to 5.2 using citric acid solution. 6 parts by weight of compound amylase were added to the starch slurry and reacted at 55°C for 24 hours. Then, hydrochloric acid solution was added to adjust the pH to 2.0 and held for 20 minutes to inactivate the enzyme. The pH was then neutralized to 7.0 using sodium hydroxide solution. After centrifugation, washing, and drying, porous starch was obtained. 120 parts by weight of porous starch and 12 parts by weight of silica were added to a powder mixer and mixed at 1100 r / min for 35 minutes to obtain a lightweight porous composite carrier.

[0085] Four parts by weight of pectin and eight parts by weight of oligofructose were mixed into a dry powder until homogeneous. Then, the mixture was slowly dispersed in 188 parts by weight of purified water and mechanically stirred at 400 r / min for 45 minutes at 60°C until completely dissolved. The mixture was then naturally cooled to 25°C to obtain the inner bottom spray liquid. Nine parts by weight of ethyl cellulose were dissolved in 81 parts by weight of ethanol to obtain the inner top coat liquid.

[0086] 5.5 parts by weight of Bacillus coagulans BC39 were dispersed in 28 parts by weight of purified water to form an inner layer bacterial solution. Then, 100 parts by weight of a lightweight porous composite carrier were added, and the solution was subjected to negative pressure adsorption for 28 minutes under a vacuum of 0.07 MPa. Subsequently, the solution was vacuum dried at 39°C until the moisture content was less than 5 wt% to obtain the bacterial core. The bacterial core was placed in a fluidized bed coating machine, and the inlet air temperature was controlled at 42°C. The inner layer bottom spray liquid and the inner layer top spray liquid were sprayed onto the surface of the bacterial core in sequence, and the solution was dried for 14 minutes to obtain the inner layer microparticles.

[0087] Dissolve 5 parts by weight of hydroxypropyl cellulose and 2 parts by weight of sodium carboxymethyl starch in 80 parts by weight of purified water, then uniformly disperse 2.8 parts by weight of Bacillus coagulans BC39 to prepare a middle layer coating solution; control the fluidized bed inlet air temperature to 39°C, spray the middle layer coating solution onto the surface of the inner layer particles, and continue to dry for 12 minutes to obtain the middle layer coated particles.

[0088] Three parts by weight of hydroxypropyl methylcellulose and one part by weight of mannitol were dissolved in 50 parts by weight of purified water, and then 1.7 parts by weight of Bacillus coagulans BC39 were uniformly dispersed in the solution to prepare an outer coating solution. The fluidized bed inlet air temperature was controlled at 38°C, and the outer coating solution was sprayed onto the surface of the middle coating microparticles and dried for 18 minutes to obtain a three-layer sustained-release microcapsule.

[0089] 18 parts by weight of three-layer sustained-release microcapsules, 65 parts by weight of whey protein and 45 parts by weight of soy protein isolate were put into a powder mixer and mixed at 25°C and 450 r / min for 18 minutes to obtain the layered sustained-release BC39 protein powder compound.

[0090] Comparative Example 1:

[0091] Compared with Example 1, the difference is that the complex amylase reaction and enzyme inactivation steps of starch milk are omitted, and ordinary corn starch is directly mixed with silicon dioxide as a carrier. All other aspects are the same.

[0092] Comparative Example 2:

[0093] Compared with Example 1, the difference is that hydroxypropyl methylcellulose is used in place of pectin in the inner bottom spray solution in equal parts by weight, otherwise the same.

[0094] Comparative Example 3:

[0095] Compared with Example 1, the difference is that equal parts by weight of polyvinylpyrrolidone are used to replace hydroxypropyl cellulose and sodium carboxymethyl starch in the intermediate coating solution, while the rest are the same.

[0096] Comparative Example 4:

[0097] Compared with Example 1, the difference is that 10 parts by weight of Bacillus coagulans BC39 were added to the inner layer bacterial solution, and Bacillus coagulans BC39 was not added to the middle layer coating solution and the outer layer coating solution, while the rest were the same.

[0098] Comparative Example 5:

[0099] Compared with Example 1, the difference is that instead of adding fructooligosaccharides to the inner bottom spray liquid, fructooligosaccharides are directly added to the powder mixer and mixed with protein powder. All other aspects are the same.

[0100] Test Example 1:

[0101] 100g of each of the layered sustained-release BC39 protein powder complexes prepared in Examples 1, 2, 3, and Comparative Example 1 were extracted. Each layered sustained-release BC39 protein powder complex was allowed to pass naturally through a 10-mesh sieve and poured into a 500mL dry glass graduated cylinder. The volume when the powder surface was flush was recorded, and the ratio of mass to volume was calculated to obtain the bulk density. The glass graduated cylinder containing the powder was then fixed on a tapped density meter. The vibration frequency was set to 250 times / minute, the vibration drop distance was 3mm, and the powder volume was read after 3000 vibrations. The tapped density was calculated, and the Karl Fischer index was calculated by combining the values ​​of bulk density and tapped density. Each sample was measured in parallel 3 times and the average value was taken.

[0102] Take appropriate amounts of each component of the stratified slow-release BC39 protein powder compound and put them into cylindrical transparent acrylic test tubes with an inner diameter of 50 mm and a height of 400 mm. Control the powder filling height to 300 mm and seal the opening of the cylindrical transparent acrylic test tube with a rubber stopper.

[0103] The sealed cylindrical transparent acrylic test tube was vertically fixed on the electromagnetic simulated transport vibration table. The vibration frequency of the electromagnetic simulated transport vibration table was set to 5Hz and the vertical amplitude to 25mm. The simulated transport vibration treatment was carried out continuously for 120 minutes.

[0104] After the vibration treatment is completed, the cylindrical transparent acrylic test tube is removed. Keeping the cylindrical transparent acrylic test tube in a vertical position, the same mass of powder sample is extracted from the following areas: 10mm to 30mm below the top of the powder height inside the cylindrical transparent acrylic test tube, the middle area at half the depth of the powder bed, and 10mm to 30mm above the bottom of the test tube. These are recorded as upper layer sampling, middle layer sampling, and lower layer sampling, respectively.

[0105] Weigh 1.00 g of powder from the upper, middle, and lower layers of the sample, and add them separately to homogenization bags containing 99 mL of sterile physiological saline. Homogenize by tapping for 2 minutes to dissolve the microcapsules and release the bacteria. After serial dilution, take 0.1 mL of the diluted solution and spread it on an agar plate containing bromocresol purple. Incubate upside down at 37°C for 48 hours, count the typical characteristic colonies, calculate the viable count of Bacillus coagulans BC39 in each layer of the sample, and calculate the relative standard deviation of the viable count distribution of the upper, middle, and lower layers of each sample group.

[0106] The test results are shown in Table 1.

[0107] Table 1. Data Recording Table for Density Measurement and Vibration Segregation Measurement of Each Group of Samples:

[0108] Example 1 0.382 0.457 16.41 10.15 10.42 10.87 3.48 Example 2 0.408 0.489 16.56 19.34 20.08 21.15 4.54 Example 3 0.371 0.446 16.82 9.78 10.05 10.53 3.73 Comparative Example 1 0.635 0.821 22.66 3.12 8.86 21.43 84.09

[0109] According to Table 1 and appendix Figure 1 and attached Figure 2 The data shows that Examples 1, 2, and 3 used porous starch mixtures obtained through a constant-temperature reaction with composite amylase and enzyme inactivation treatment as structural carriers. Taking Example 1 as an example, the bulk density of Example 1 was 0.382 g / cm³. 3 The Karl Fischer index was 16.41%. After 120 minutes of vertical vibration treatment, the viable count in the upper layer of the powder from the surface to the bottom layer was 10.15 × 10⁻⁶. 8 CFU / g, viable count in the lower layer was 10.87 × 10⁻⁶. 8 The overall measurement record shows that the relative standard deviation of the viable bacteria count in Examples 1, 2 and 3 is between 3.48% and 4.54%. The measurement results reflect that the porous carrier framework reduces the absolute density of microcapsule particles, reduces the physical difference in aerodynamic properties between the microcapsules containing live bacteria and the bulk light protein powder, and maintains the spatial homogeneity of the powder mixing system under mechanical vibration.

[0110] Comparative Example 1 omitted the complex amylase reaction and enzyme inactivation treatment steps for pore formation, and used untreated ordinary corn starch to construct the inner layer. The bulk density of Comparative Example 1 was 0.635 g / cm³. 3 The Karl Fischer index was 22.66%. After 120 minutes of vertical vibration treatment, Comparative Example 1 showed differences in vertical spatial distribution, with the upper layer viable count of Comparative Example 1 measuring 3.12 × 10⁻⁶. 8 CFU / g, the viable bacterial count in the lower layer increased to 21.43 × 10⁻⁶. 8 With a CFU / g and a relative standard deviation of 84.09%, the test data confirmed that the density of solid microcapsules lacking internal pores is higher than that of conventional bulk protein powder. Under simulated vibration conditions during transportation, they migrate downwards under gravity, leading to powder segregation.

[0111] Test Example 2:

[0112] Sterile simulated gastric fluid with pH 1.2 and containing pepsin was prepared; sterile simulated intestinal fluid with pH 6.8 and containing pancreatin was prepared; and sterile simulated colonic fluid with pH 7.4 and containing specific pectinase was prepared. Layered sustained-release BC39 protein powder complexes containing fluorescein isothiocyanate as a tracer were prepared for Examples 1, 4, 2, and 3, respectively.

[0113] 10.00 g of each of the layered sustained-release BC39 protein powder complexes containing fluorescein isothiocyanate as a tracer in Examples 1, 4, 2, and 3 were weighed. Examples 1, 4, 2, and 3 were respectively added to dissolution vessels of a dissolution meter containing 900 mL of sterile simulated gastric fluid. The temperature of the dissolution meter was set to 37°C and the stirring speed was set to 50 rpm. The dissolution was run continuously for 120 minutes. At 60 minutes and 120 minutes of continuous operation, 5 mL of digestive fluid was drawn from each dissolution vessel of the dissolution meter, and 5 mL of fresh sterile simulated gastric fluid at the same temperature was added to the corresponding dissolution vessel of the dissolution meter. The drawn digestive fluid was filtered through a microporous membrane and the filtrate was collected for testing.

[0114] After 120 minutes of aseptic simulated gastric fluid treatment, the mixture in the dissolution vessel of the dissolution apparatus was transferred to a centrifuge for centrifugation. The residual microcapsule precipitate at the bottom was collected and transferred to a dissolution vessel containing 900 mL of aseptic simulated intestinal fluid. The dissolution apparatus was run at 37°C and 50 rpm for another 240 minutes, bringing the total digestion time to 360 minutes. At 240 minutes and 360 minutes of total digestion time, 5 mL of digestion fluid was extracted and 5 mL of fresh aseptic simulated intestinal fluid at the same temperature was added. The extracted digestion fluid was filtered and the filtrate was collected.

[0115] After a total digestion time of 360 minutes, the mixture in the dissolution vessel of the dissolution apparatus was centrifuged to separate and collect the residual microcapsule precipitate. The microcapsule precipitate was transferred to the dissolution vessel of the dissolution apparatus containing 900 mL of sterile simulated colon fluid. The same operating parameters were maintained and the process continued for 720 minutes. At the total digestion time of 480 minutes, 600 minutes and 720 minutes, 5 mL of digestion solution was extracted and filtered to collect the filtrate.

[0116] Using a UV-Vis fluorescence spectrophotometer, the fluorescence intensity of the filtrate collected at each time point was measured under the conditions of excitation wavelength 490 nm and emission wavelength 520 nm. The concentration of fluorescein isothiocyanate in the filtrate was converted according to the standard curve, and the cumulative release rate of Examples 1, 4, Comparative Example 2 and Comparative Example 3 at the corresponding time points was calculated.

[0117] The test results are shown in Table 2.

[0118] Table 2. Data record of cumulative release rate of each group of test samples in the in vitro simulated gastrointestinal tract:

[0119] Example 1 8.14 14.52 24.67 38.91 68.23 86.45 94.12 Example 4 10.21 16.89 27.34 41.05 71.56 89.11 95.84 Comparative Example 2 11.34 18.05 55.62 79.43 82.15 84.67 85.02 Comparative Example 3 15.67 24.89 35.41 48.72 78.38 89.24 95.45

[0120] According to Table 2 and appendix Figure 3 and attached Figure 4 The data shows that Examples 1 and 4 exhibited phased release distribution characteristics in a continuous in vitro digestion environment. Taking Example 1 as an example, at the end of the 120-minute simulated gastric fluid treatment, the cumulative release rate of Example 1 was 14.52%, reflecting that the outer layer of the three-layer coating structure has an anti-dissolution barrier effect in a strongly acidic environment. At the end of the 360-minute simulated intestinal fluid treatment, the cumulative release rate of Example 1 was 38.91%, and the core microparticles were in a slow release state. After entering the simulated colonic fluid treatment stage, the cumulative release rate of Example 1 increased to 94.12% at 720 minutes. The test results confirmed that the inner porous framework disintegrated and released under the intervention of specific pectinase, completing the spatial fixed-point delivery of the core microparticles.

[0121] Comparative Examples 2 and 3 lack a complete three-layer coating control system. Comparative Example 2 showed a cumulative release rate of 79.43% at the end of the 360-minute simulated intestinal fluid treatment, exhibiting a high release rate in the simulated intestinal fluid stage, which led to a decrease in the residual load particles entering the simulated colonic fluid stage. Comparative Example 3 showed a cumulative release rate of 24.89% at the end of the 120-minute simulated gastric fluid treatment, and as high as 48.72% at the end of the 360-minute simulated intestinal fluid treatment.

[0122] Test data indicates that using polyvinylpyrrolidone to replace the middle coating material causes the microcapsules to dissolve too quickly in the front digestive fluid environment, resulting in uncontrolled premature loss of the middle layer load and disrupting the preset slow intestinal release rhythm. However, the inner pectin skeleton still retains the colon-targeted delivery capability to the core strain. Test data confirms that the loss of layers or material replacement can lead to uncontrolled dissolution of the embedded material in a specific digestive fluid environment, causing the release position of the internal load to shift.

[0123] Test Example 3:

[0124] Prepare an in vitro static protein digestion simulation solution system. Using purified water as the base solvent, prepare a composite protein matrix solution containing fast-digesting and slow-digesting components. Adjust the pH of the composite protein matrix solution to 1.5 using dilute hydrochloric acid solution, add pepsin, and complete the establishment of a gastric digestion simulation environment.

[0125] Weigh 5.00 g of each of the test powders from Example 1, Comparative Example 3, and Comparative Example 4. Place each of the test powders from Example 1, Comparative Example 3, and Comparative Example 4 into a dialysis bag containing 10 mL of a composite protein matrix solution with the pH value adjusted. Set the molecular weight cutoff of the dialysis bag to 3500 Daltons. Tighten both ends of the dialysis bag for sealing.

[0126] Place the sealed dialysis bags into separate incubation reaction bottles containing 200 mL of in vitro release medium. Place the incubation reaction bottles in a constant temperature water bath shaker set to 37°C, set the shaking speed to 100 rpm, and start the continuous incubation program.

[0127] When the continuous incubation program reaches the 1.0, 2.0 and 3.0 hour nodes, 2 mL of in vitro release medium is extracted from each incubation reaction bottle as the test sample, and 2 mL of fresh in vitro release medium at 37°C is added to the corresponding incubation reaction bottle at the same time.

[0128] After the sample extraction operation at the 3.0 hour node is completed, the pH value of the in vitro release medium inside the incubation reaction bottle and the complex protein matrix solution inside the dialysis bag is simultaneously adjusted to 6.8 using sodium bicarbonate solution. The dialysis bag is then removed and briefly unsealed. Specific pancreatic enzyme powder is added to the dialysis bag, and it is then resealed and placed back into the incubation reaction bottle to complete the establishment of the intestinal digestion simulation environment. The original temperature and oscillation speed are maintained to continue the continuous incubation program.

[0129] When the continuous incubation program reaches the 4.0, 6.0 and 8.0 hour nodes, 2 mL of in vitro release medium is extracted from each incubation reaction bottle as the test sample, and 2 mL of fresh in vitro release medium is added to the corresponding incubation reaction bottle at the same time.

[0130] The collected samples were centrifuged and filtered using a microporous membrane. The filtrates at each time point were separated using high performance liquid chromatography. After pre-column derivatization, the peak area of ​​the free leucine derivative was measured using a UV or fluorescence detector. The cumulative concentration of free leucine was calculated based on the standard working curve. The total cumulative concentration of free amino acids in the filtrate was determined at a specific absorption wavelength using the ninhydrin colorimetric method.

[0131] The test results are shown in Table 3.

[0132] Table 3. Concentration test records of Examples 1, 3, and 4 in an in vitro static protein digestion model:

[0133] Example 1 Cumulative concentration of free leucine (mg / L) 17.84 27.61 31.05 35.28 39.42 42.17 Example 1 Cumulative concentration of total free amino acids (mg / L) 108.45 162.33 188.71 215.62 248.91 271.34 Comparative Example 3 Cumulative concentration of free leucine (mg / L) 29.56 31.14 32.89 38.45 42.12 45.68 Comparative Example 3 Cumulative concentration of total free amino acids (mg / L) 182.17 194.52 203.11 207.84 235.84 268.35 Comparative Example 4 Cumulative concentration of free leucine (mg / L) 9.21 13.45 16.78 21.34 26.55 29.83 Comparative Example 4 Cumulative concentration of total free amino acids (mg / L) 64.38 92.15 115.64 141.22 172.84 195.46

[0134] According to Table 3 and appendix Figure 5 and attached Figure 6 The data shows that Example 1 exhibits phased synergistic release characteristics in an in vitro static protein digestion model. Based on the evolution of Example 1 under different simulated environments, after 1.0 hour of continuous incubation, the cumulative concentration of free leucine in Example 1 was 17.84 mg / L and the cumulative concentration of total free amino acids was 108.45 mg / L.

[0135] At the end of the gastric digestion simulation phase, i.e., after 3.0 hours of continuous incubation, the cumulative concentration of free leucine in Example 1 reached 31.05 mg / L. The measured value reflects that Example 1, in the early stage of simulated digestion, cooperated with the degradation mechanism of the complex protein matrix to establish the initial substrate system supply. After switching to the intestinal digestion simulation phase, at the end of 8.0 hours of continuous incubation, the cumulative concentration of free leucine in Example 1 steadily increased to 42.17 mg / L. The test data confirms that the stepwise release distribution characteristics maintain the system concentration of leucine and the overall amino acids in the intestinal digestion simulation phase.

[0136] Comparative Example 3 showed a cumulative free leucine concentration of 29.56 mg / L after 1.0 hour of continuous incubation, and a cumulative free leucine concentration of 45.68 mg / L at the end of 8.0 hours. The test results showed that Comparative Example 3 caused the coating structure to degrade too quickly, resulting in a concentrated release of the contents during the simulated gastric digestion stage, leading to a high early concentration accumulation and disrupting the supply balance of the substrate release. Comparative Example 4 showed a cumulative free leucine concentration of 16.78 mg / L and a total free amino acid concentration of 115.64 mg / L after 3.0 hours of continuous incubation, which were lower than the test benchmark of Example 1.

[0137] Distribution records revealed that in Comparative Example 4, since all bacterial cells were locked in the pectin inner layer, the strain could not be released during the gastrointestinal digestion simulation stage in the absence of specific colonic enzymes. The lack of drug release assistance resulted in low substrate free levels in the initial and subsequent entire cycle stages. Comparative analysis confirmed that the lack of multi-layer barrier and programmed response structure led to an uneven release state of the test subjects during the digestion simulation stage.

[0138] The filtrate extracted at the 8.0-hour mark was used to culture C2C12 myoblasts. Detection revealed that the phosphorylation level of mTOR protein in the treatment group of Example 1 was upregulated, and the diameter of myotubes increased in the microscopic field of view. Cell-level test records demonstrated the activating effect of free amino acids on the protein synthesis pathway of myoblasts.

[0139] Test Example 4:

[0140] Prepare a simulated gastrointestinal digestion system. Weigh 5.00g of each of the test powders from Example 1, Comparative Example 2, and Comparative Example 5. Place the test powders from Example 1, Comparative Example 2, and Comparative Example 5 into sterile simulated gastric juice and sterile simulated intestinal juice, respectively, and perform isothermal shaking digestion treatment in sequence. Collect the corresponding digestion residues for later use.

[0141] An in vitro simulated colonic fermentation system was established. Fresh fecal samples were collected from healthy volunteers. A 10% fecal suspension was prepared using anaerobic phosphate buffer. A sterile basal fermentation medium containing various carbon sources, nitrogen sources, and trace elements was prepared. The fecal suspension was inoculated into the sterile basal fermentation medium at a volume ratio of 10%. High-purity nitrogen was continuously introduced to remove oxygen, thus establishing an anaerobic fermentation environment.

[0142] The digestion residues collected from Example 1, Comparative Example 2, and Comparative Example 5 were respectively connected to independent in vitro simulated colon fermentation systems. Each in vitro simulated colon fermentation system was placed in an anaerobic constant temperature incubator set at 37°C, and a continuous fermentation culture program was started.

[0143] At the 0, 6, 12, 24 and 48 hour points of the continuous fermentation culture program, 3 mL of culture medium was extracted from each in vitro simulated colon fermentation system as a test sample.

[0144] To perform a viable count test, 1 mL of test sample was serially diluted with sterile physiological saline and spread onto a Bacillus coagulans selective agar plate. After incubation under specific anaerobic conditions for 48 hours, the colony-forming units on the plate were counted and converted into logarithmic values.

[0145] To test the concentration of short-chain fatty acids, the remaining 2 mL of test sample was centrifuged at 10,000 rpm for 15 minutes, and the supernatant was extracted. Metaphosphoric acid was added to the supernatant for acidification, and the solution was filtered through a 0.22 μm microporous membrane. The filtrate at each time point was separated using a gas chromatograph equipped with a flame ionization detector, and the concentrations of acetic acid, butyric acid, and propionic acid were determined. The total concentration of short-chain fatty acids was calculated by summing the results.

[0146] The test results are shown in Table 4.

[0147] Table 4. Test data record of Example 1, Comparative Example 2 and Comparative Example 5 in the in vitro simulated colonic fermentation system:

[0148] Example 1 viable bacteria count (Log 10 CFU / mL) 6.12 7.38 8.41 8.94 8.67 Example 1 Total short-chain fatty acid concentration (mmol / L) 15.31 26.84 41.27 59.45 66.82 Comparative Example 2 viable bacteria count (Log 10 CFU / mL) 4.08 4.17 4.35 4.29 4.02 Comparative Example 2 Total short-chain fatty acid concentration (mmol / L) 14.82 17.56 20.31 22.14 23.05 Comparative Example 5 viable bacteria count (Log 10 CFU / mL) 6.05 6.74 7.18 6.88 6.42 Comparative Example 5 Total short-chain fatty acid concentration (mmol / L) 15.14 21.22 27.65 31.84 33.47

[0149] According to Table 4 and appendix Figure 7 The data shows that Example 1 exhibits a positive synergistic trend of viable cell proliferation and metabolite accumulation in the in vitro simulated colon fermentation system. After the continuous fermentation culture program reached 0 hours, the viable cell count in Example 1 was 6.12 Log. 10 The CFU / mL concentration and total short-chain fatty acid concentration were 15.31 mmol / L; when fermentation reached the 24-hour mark, the viable cell count in Example 1 reached 8.94 Log. 10 The CFU / mL concentration corresponds to a total short-chain fatty acid concentration of 59.45 mmol / L.

[0150] The test results confirmed that the pectin skeleton of the inner layer in Example 1 was targeted for degradation in the colonic enzyme simulated environment, which caused the encapsulated Bacillus coagulans and prebiotics to be released simultaneously. The simultaneous release mechanism enabled Bacillus coagulans to preferentially utilize the coexisting prebiotic carbon source, gain an initial advantage in the nutritional competition of the colonic micro-ecosystem, and thus accelerate the proliferation of live bacteria and generate a large amount of short-chain fatty acid metabolites.

[0151] Comparative Example 2 showed a viable cell count of only 4.29 Log at the 24-hour mark of continuous fermentation. 10 The CFU / mL and total short-chain fatty acid concentration were 22.14 mmol / L. The test data showed that the inner layer material of Comparative Example 2 was replaced with hydroxypropyl methylcellulose, which does not have colon-targeting properties. This caused a large amount of premature release of hydroxypropyl methylcellulose in the simulated gastrointestinal fluid environment. After being treated with the front digestive fluid, the number of internal bacteria remaining in the simulated colon fermentation system was reduced, and it was impossible to establish a sufficient initial bacterial community dominance. As a result, the viable bacterial cells were in a restricted state, and the proliferation and metabolic pathways were inhibited.

[0152] Comparative Example 5 showed a viable cell count of 6.88 Log at the 24-hour fermentation stage. 10The CFU / mL and total short-chain fatty acid concentration were 31.84 mmol / L, both of which were lower than those in Example 1. The test results revealed that, in the absence of prebiotic pre-set components, Bacillus coagulans, which lost the co-release of prebiotics, still faced competition for survival resources from the gut microbiota. A single bacterial cell could not establish a sufficient colonization scale in the complex microecology, resulting in low efficiency of short-chain fatty acid conversion. Comprehensive evaluation confirmed that the inner enzyme response framework combined with the prebiotic co-carrying system can ensure the targeted delivery of microorganisms and the enhanced metabolism at the back end.

[0153] The supernatant obtained by centrifugation after 24 hours of fermentation was used in a TNF-α-induced C2C12 myotube atrophy cell model. The test showed that the expression levels of muscle atrophy-specific genes such as MuRF1 and Atrogin-1 were downregulated in the group of Example 1. The test results reflect that the metabolites generated by fermentation inhibit inflammation-mediated muscle protein degradation, which corresponds to the physiological effect of alleviating the atrophy process of sarcopenia.

[0154] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A layered sustained-release BC39 protein powder compound, characterized in that, The preparation process of the layered sustained-release BC39 protein powder complex includes the following steps: Corn starch is made into starch milk and the pH is adjusted. Porous starch is obtained by isothermal reaction with composite amylase, enzyme inactivation treatment and drying treatment. The porous starch is mixed with silica powder to obtain a lightweight porous composite carrier. A portion of Bacillus coagulans BC39 was prepared into an inner layer bacterial solution, which was then added to the lightweight porous composite carrier and subjected to negative pressure adsorption and vacuum drying to obtain the bacterial core. An inner layer bottom spray solution containing pectin and fructooligosaccharides, and an inner layer top spray solution containing ethyl cellulose were prepared separately. The inner layer bottom spray solution and the inner layer top spray solution were sequentially sprayed onto the surface of the bacterial core in a fluidized bed and continuously dried to obtain inner layer microparticles. Hydroxypropyl cellulose, sodium carboxymethyl starch, and a portion of Bacillus coagulans BC39 were formulated into a middle layer coating solution; the middle layer coating solution was sprayed onto the surface of the inner layer particles in a fluidized bed and continuously dried to obtain middle layer coated particles; Hydroxypropyl methylcellulose, mannitol, and the remaining Bacillus coagulans BC39 were formulated into an outer coating solution; the outer coating solution was sprayed onto the surface of the middle coating particles in a fluidized bed and continuously dried to obtain a three-layer sustained-release microcapsule; The three-layered sustained-release microcapsules, whey protein, and soy protein isolate are physically mixed to obtain the layered sustained-release BC39 protein powder complex.

2. The layered sustained-release BC39 protein powder compound according to claim 1, characterized in that, The steps for obtaining porous starch through a compound amylase isothermal reaction, enzyme inactivation treatment, and drying treatment include: adjusting the pH of the starch milk to 4.8 to 5.2 using citric acid solution, and carrying out the isothermal reaction at 45 to 55°C for 12 to 24 hours; subsequently adding hydrochloric acid solution to adjust the pH to 2.0 to 2.4 and maintaining it for 10 to 20 minutes for enzyme inactivation treatment; then neutralizing the pH to 7.0 using sodium hydroxide solution; and finally performing centrifugation, washing, and drying treatment in sequence. The step of mixing the porous starch with silica powder includes: putting it into a powder mixer and mixing it at a speed of 900 to 1100 r / min for 25 to 35 minutes.

3. The layered sustained-release BC39 protein powder compound according to claim 1, characterized in that, The steps of performing negative pressure adsorption and vacuum drying include: The negative pressure adsorption is carried out for 20 to 30 minutes under a vacuum of 0.04 to 0.08 MPa; then the vacuum drying process is carried out at 35 to 40°C until the moisture content is less than 5 wt%.

4. The layered sustained-release BC39 protein powder compound according to claim 1, characterized in that, The steps of preparing the inner bottom spray liquid containing pectin and fructooligosaccharides, and the inner top coat liquid containing ethyl cellulose, respectively, include: After mixing pectin and fructooligosaccharides in dry powder form, the mixture is slowly dispersed in purified water and mechanically stirred at 350 to 450 r / min at 55 to 65°C for 40 to 45 minutes until completely dissolved. The mixture is then naturally cooled to 20 to 25°C to obtain the inner bottom spray liquid.

5. The layered sustained-release BC39 protein powder compound according to claim 1, characterized in that, The continuous drying step includes: After spraying the inner base spray liquid and the inner top coat liquid, control the air inlet temperature to 35 to 45°C and continue drying for 10 to 15 minutes; After spraying the intermediate coating liquid, control the air inlet temperature at 35 to 40°C and continue drying for 10 to 15 minutes; After spraying the outer coating liquid, control the air inlet temperature to 34 to 38°C and continue drying for 15 to 20 minutes.

6. The layered sustained-release BC39 protein powder compound according to claim 1, characterized in that, The step of preparing the intermediate coating solution from hydroxypropyl cellulose, sodium carboxymethyl starch and a portion of Bacillus coagulans BC39 includes: mixing and dissolving hydroxypropyl cellulose and sodium carboxymethyl starch at a mass ratio of 5:2; The step of preparing the outer coating solution from hydroxypropyl methylcellulose, mannitol and the remaining Bacillus coagulans BC39 includes: mixing and dissolving hydroxypropyl methylcellulose and mannitol at a mass ratio of 3:

1.

7. The layered sustained-release BC39 protein powder compound according to claim 1, characterized in that, Based on the total mass of Bacillus coagulans BC39 contained in the compound: The step of preparing an inner layer bacterial solution from a portion of Bacillus coagulans BC39 includes: preparing the inner layer bacterial solution using 50% to 60% of Bacillus coagulans BC39 by weight; The step of preparing the intermediate coating solution from hydroxypropyl cellulose, sodium carboxymethyl starch and a portion of Bacillus coagulans BC39 includes: preparing the intermediate coating solution using 25% to 30% of Bacillus coagulans BC39 by mass. The step of preparing the outer coating solution from hydroxypropyl methylcellulose, mannitol and the remaining Bacillus coagulans BC39 includes: preparing the outer coating solution using 15% to 20% of the total mass of Bacillus coagulans BC39. Furthermore, the sum of the mass percentages of Bacillus coagulans BC39 used in the inner, middle, and outer layers is 100%.

8. The layered sustained-release BC39 protein powder compound according to claim 1, characterized in that, The steps of preparing the inner bottom spray liquid containing pectin and fructooligosaccharides, and the inner top coat liquid containing ethyl cellulose, respectively, further include: Pectin and fructooligosaccharides are mixed at a mass ratio of 4 to 5:5 to 8; wherein the fructooligosaccharides serve as prebiotics for probiotic colonization, and the pectin, together with ethyl cellulose in the inner coating liquid, serves as a colon-targeting material.

9. The layered sustained-release BC39 protein powder compound according to claim 1, characterized in that, The steps of physically mixing the three-layer sustained-release microcapsules, whey protein, and soy protein isolate include: Mix 10 to 20 parts by weight of the three-layer sustained-release microcapsules, 50 to 70 parts by weight of the whey protein, and 30 to 50 parts by weight of the soy protein isolate at 25°C and a rotation speed of 300 to 500 r / min for 10 to 20 minutes.

10. The use of the layered sustained-release BC39 protein powder compound as described in any one of claims 1 to 9 in the preparation of nutritional foods or health foods for promoting muscle protein synthesis-related nutrient metabolism.

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